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Anthology of Works
Undisclosed Reasons
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Foreword
An anthology is defined as a collection of works chosen by a compiler. The
compiler arranges the works in a deliberate way as he or she fits.
This anthology is no different than any other anthology, ecept that the works
collected here were collected for a reason! a set of reasons that the compilers
ref"se to disclose. The reader is free to read and interpret this anthology in any
manner or mode. #ost importantly, the lack of intent and p"rpose of this
anthology also gi$es the readers the freedom to not read it, th"s gi$ing priority to
the agency of the reader in the process of reading o$er the a"thors% claims of
literary or c"ratorial self&importance.
2
Table of Contents
'The (ederal )o"rt *atabase+ ,ew -esearch Opport"nities...............................1
by Terence *"ngworth
'/ositi$e 0n$ironmental 0ternalities of 1i$estock in
#ied (arming Systems of 2ndia..............................................................11
by A.3. *ikshit and /.S. 4irthal
'The ,eed for A"diologic 5abitation...................................................................20
by 0.6. 5ardick and S.A. 1esner
'The Signat"re of 1ine 7raphs and /ower Trees.
by 1ong 8ang et al....................................................................................90
'0stimation of the 0:"ilibri"m 0change -eal
0change -ate for So"th Africa................................................................9;
by -onald #ac*onald and 1"cca -icci
'The 0fficient #ethod for Sim"ltaneo"s #onitoring of
the )"lt"rable as 8ell as ,onc"lt"rable
Airborne #icroorganisms.
by 4arbara 5"bad.....................................................................................<9
'Attachment Styles at 5ogwarts+ (rom 2nfancy
to Ad"lthood.
by 8ind 7oodfriend...................................................................................=2
'0ndocrine and metabolic manifestations in
inflammatory bowel disease
by Stelios Tigas and Agathocles Tsatso"lis...............................................>>
3
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25
36
47
58
69
7 10
8 11
9 12
10 13
Agricultural Economics Research Review
Vol. 26(No.1) January-June 2013 pp 21-30
Positive Environmental Externalities of Livestock in
Mixed Farming Systems of India
A.K. Dikshit
a*
and P.S. Birthal
b
a
Central Institute for Research on Goats, Makhdoom - 281 122, Uttar Pradesh
b
National Centre for Agricultural Economics and Policy Research, New Delhi - 110 012
Abstract
Livestock are often criticized for their negative externalities to environment. However, in the mixed
farming systems followed in India, the livestock help in saving natural resources through their synergistic
relationship with cropping activities. This paper has quantified the positive environmental externalities
associated with livestock production in Indias mixed farming systems. These include: land saving due to
recycling of agricultural by-products as animal feed and also due to use of dung- cake as domestic fuel;
saving of chemical fertilizers due to use of dung as manure and prevention of carbon dioxide emission
due to use of animal energy in agriculture. Land saving from livestock production system due to recycling
of crop by-products as animal feed and use of dung as domestic fuel has been estimated as 42 Mha. The
use of dung as manure saves 1.2 Mt of soil nutrients. Likewise, use of animal energy as a substitute for
mechanical energy has potential to save diesel consumption to the extent of 13 Mt and prevents greenhouse
gas emission due to burning of diesel.
Key words: Livestock, environment, mixed farming system, India
JEL Classification: Q51, Q20, Q0
*Author for correspondence
Email: akdixit@cirg.res.in
Introduction
Livestock, despite their significant contributions
towards enhancing food and nutritional security and
reducing poverty, are often criticized for the negative
externalities they cause to environment through
emission of greenhouse gases, overgrazing/
deforestation and water pollution (Steinfeld et al.,
2006). Impacts of livestock on environment, however,
differ across production systems. Industrial livestock
production systems cause more harm to environment,
while mixed crop-livestock systems are benign to
environment (Sere and Steinfeld, 1996). In the mixed
farming systems, animals draw their energy
requirements from environment in the form of feed
from by-products of crops, from cultivated green fodder
and from grazing, and in turn, give back that energy in
the form of food (milk, meat, and eggs), draught power,
fuel, and manure. With this process of energy exchange
are associated a number of environmental externalities,
negative as well as positive. Negative externalities of
livestock to environment are well documented and
quantified (Steinfeld et al., 2006); but their positive
contributions have remained less documented and non-
quantified. The prominent positive environmental
contributions include prevention of carbon di-oxide
emission due to use of animal energy in place of fossil
fuel, saving of natural resources mainly land as a result
of recycling of agricultural by-products and residues
as animal feed, and dung in place of firewood as
domestic fuel and as a substitute for chemical fertilizers.
Evidence also suggests that managed grazing helps in
improving biodiversity (Pasha, 2005). In this paper,
we have made an attempt to quantify some of the
11 14
22 Agricultural Economics Research Review Vol. 26(No.1) January-June 2013
positive contributions of livestock to environment in
India where livestock are largely raised in the mixed
farming systems.
Analytical Approach
Information on feed consumption rates, by species,
is an important requirement in estimating the positive
environmental effects of livestock production. To our
knowledge, there is little information available on feed
consumption rates in India, except some localized
information generated through surveys undertaken by
the Indian Agricultural Statistics Research Institute
(IASRI) during 1960s to early-1980s. This information
is quite aged now and also there are problems in pooling
of the data from surveys spread over such a long period.
In this paper, we have used data on feed
consumption and several other attributes of livestock,
viz. body size, grazing practices, dung production and
its utilization, etc. from a nationally representative
survey undertaken as part of a larger project, Indias
Livestock feed balance and its environmental
implications, funded by the Indian Council of
Agricultural Research (ICAR) under the National
Agricultural Technology Project (NATP), and carried
out jointly by the National Centre for Agricultural
Economics and Policy Research (NCAP) and the
Society (now Centre) for Economic and Social
Research (SESR), Delhi. The survey was conducted
in 2001-02 in different agro-climatic regions of India.
A brief description of delineation of regions, survey
design, data collection procedure, feed consumption
estimation procedure and estimation of positive
contributions of Indias livestock production system
have been given in the following sections.
Sampling Design
India has considerable heterogeneity in
topography, soils, rainfall, irrigation, temperature,
cropping pattern and livestock production systems.
Hence, for any survey to qualify as a nationally
representative sample, it must take into account this
heterogeneity. To ensure that survey estimates were
representative of the national situation, a multistage
sampling framework was adopted to generate the
required information. The National Bureau of Soil
Survey and Land Use Planning (NBSS&LUP) an
offshoot of the Indian Council of Agricultural Research,
has mapped Indias territorial space into 20 agro-
ecological zones with their further classification into
60 sub-zones. However, for implementation of the
survey, we have taken into consideration topography,
climatic conditions and cropping pattern of 60 sub-
zones, and re-organized these sub-zone into 11 broad
regions, which we have termed as livestock regions.
In doing so, it was ensured that a livestock region was
contiguous. These regions were: Western Himalayas,
North-West Plains, Eastern Plains, Central Highlands,
Eastern Plateau and Highlands, Deccan Plateau and
Hills, Rajasthan-Gujarat Plains, Eastern Ghats, Western
Ghats, Assam-Bengal Plains and North-Eastern
Highlands.
The survey was conducted in 10 livestock regions,
excluding North-Eastern Highlands. The stratified
multistage random sampling approach was adopted in
the study. From each livestock region, two districts (one
from some regions) were selected at random; and from
each selected district, two villages were selected again
at random. A livestock census was conducted in each
selected village to know the ownership pattern of
different livestock species. Having enumerated
livestock-keeping households, a random sample of 20-
25 livestock-keeping households was drawn from each
village to make up the total sample size of around 1000
households. Excluding the un-surveyed zone, we
collected information from 864 households. The data
were collected during the years 2001 and 2002.
Information related to the households and livestock
holdings was collected from the heads of sample
households. Information that required measurement,
e.g. amount of different types of feed to be fed to
different categories of animals by age-group, sex and
function; and animal characteristics, e.g. body weight
was generated by the field investigators at the
household premises. Investigators were required to
weigh and record the types of feed every day in the
morning and evening, for complete one year to capture
seasonality in feed consumption rates and their
composition which was likely because of the
seasonality in production of different types feed and
also because of seasonal differences in the uses of
livestock or their outputs. Considering that it was
difficult to weigh and record different feeds every day,
each household was revisited every fortnight for one
year to collect this information.
12 15
Dikshit and Birthal : Positive Environmental Externalities of Livestock in India 23
Estimation of Feed Consumption Rates
Household level feed consumption rates serve as
a base to estimate feed consumption rates at the national
level. These rates were estimated applying scale-up
factors at the levels of village, district and region. From
the survey, we gathered information on (i) number of
sample households having livestock, say buffalo in-
milk and (ii) number of buffaloes in-milk observed,
and (iii) amount of feed fed per day to these buffaloes
in-milk. We then scaled-up information (ii) and (iii) to
the successive higher levels, that is to village, district,
region and country.
From the livestock census of each village, we had
the total number of households having buffaloes in-
milk. We obtained a scale-up factor for each village by
dividing the total number of households having an
animal species say buffaloe in-milk by the number
of sample households having buffaloes in-milk. We
applied this factor to its sample estimates of (ii) and
(iii) for each village.
Scaling-up factor for a district was obtained by
dividing the total number of villages in that district by
the number of sample villages from that district. Let
us consider any of the sample districts in a region. For
sample villages falling within it, we had already
generated aggregate estimates of (ii) and (iii),
respectively. We summed-up estimates of (ii) for the
sample villages and multiplied this sum by the scale-
up factor of that district to get district level aggregate
of (ii). In the same way, we obtained district level
aggregate of (iii). Likewise, we worked out aggregate
estimates of (ii) and (iii) for the other sample districts
in the region.
The scale-up factor for a region was obtained by
dividing the number of districts in the region by the
number of sample districts from that region. To obtain
region-level aggregates of (ii) and (iii) we followed
the same procedure as described for the district-level
aggregation. The district-level estimates of (ii) for the
sample districts were summed up; and this sum was
multiplied by the scale-up factor to obtain the region-
level estimates of (ii). Likewise, by multiplying the
sum of (iii) by the scale-up factor, we obtained regional
estimates of (iii).
Having estimated feed consumption rate for a
livestock category at the regional level, the national
level feed consumption rate was obtained as the
weighted average of the regional feed consumption
rates; the weight being regions population of that
livestock category. The regional populations of
different animal categories are aggregates of their
district level populations for 2007 obtained from the
18
th
Livestock Census. The estimated consumption
rates of different types of feeds and their total
consumption are given in Table 1.
Quantification of Positive Externalities of
Livestock to Environment
Land Saving due to Recycling of Crop Residues as
Animal Feed
Using the feed consumption rates reported in Table
1, we quantified the positive contributions of Indias
livestock production systems to the environment
following the Environmental Model of Livestock
Production System of Mishra and Dikshit (2004). The
estimated positive effects included: resource (land)
saving due to crop by-product recycled as animal feed,
and due to use of dung as a domestic fuel; saving in
chemical fertilizers due to dung-use as a manure; saving
in fossil fuel (diesel) due to use of animal energy in
agricultural operations. The model has been described
below.
The gross energy intake per animal per day from
by-product feed was estimated by summing up the
energy values of by-product feed on dry matter basis.
Similarly, the energy value of green fodder fed to the
animals was also calculated on dry matter basis. Then,
the annual quantity of green fodder required, in terms
of energy to replace gross energy from by-product feed,
was estimated as per Equation (1):
(1)
where, Gf is the quantity of green fodder required to
replace the by-product feed, gei is the gross energy
intake from by-product feed (dry fodder and
concentrates), e stands for the energy (million calories)
per unit of Gf; and d is the dry matter fraction of green
fodder. We then estimated the land area required to
produce Gf. Let y be the yield of green fodder per
hectare of land, then the area L required to produce Gf
may be given by Equation (2):
13 16
(2)
Land Saving due to Use of Dung as Substitute for
Fire-wood in Domestic Fuel
In rural households, fire-wood is used as a domestic
fuel; for cultivation or perennial fire-wood trees cover
land and deprive its use for farming. The use of dung-
cake as fire-wood would result in the saving of this
land, which can be used for crop-cultivation. We,
therefore, estimated the land saved due to use of dung
cake as a domestic fuel. The dung-cake output on dry
matter basis was worked out using dung evacuation
rate and its dry matter fraction. Supposing as the rate
of substitution of dry fuel-wood for dung cake (fuel-
wood: dry dung cake) in terms of thermal energy, the
total quantity of dry fuel-wood required to replace the
supply (output) of dung cake was calculated by
Equation (3):
(3)
where, Fw is the quantity of fuel-wood required to
replace dung cake, and dc is the quantity of dung cake
output. Now, let us suppose that fuel-wood is produced
and used within the year, and its yield per hectare on
dry matter basis is y, then the total land area that would
be required for producing Fw can be calculated by
Equation (4):
(4)
The effect of gestation lag in the production of fuel-
wood can be described as follows: The model assumes
that fuel-wood is produced and used within the same
year. This is apparently an unrealistic assumption. In
reality, more than one year is required to cover the
whole process of fuel-wood tree plantation, growth and
logging of trees, drying and use of cut-out wood as
fuel. Suppose it takes 3 years to complete the process
before dry wood is made available for use at the end of
the 3rd or the beginning of 4th year. This means that
whereas the fuel wood made available can replace
equivalent amount of dung cake only in the fourth year,
the necessary land area required for growing and
harvesting of trees for making the fuel-wood available
will have to be kept locked up during the preceding 3
years. This implies that around 3-times as much land
will be required or saved if one years dung cake output
was used in place of fuel wood as energy source.
Table 1. Feed consumption rates and dung evacuation rates, 2001-02
Livestock Type of feed consumed Dry matter Gross energy Wet dung
category (kg/animal/ day) intake per intake, production
animal per animal per per animal
Green fodder* Dry fodder Concentrates day (kg) day per day
(MJ) (kg)
Cattle
In-milk 4.75 5.50 0.64 7.01 108.44 6.63
Dry 3.40 4.02 0.40 5.15 77.82 6.58
Adult male 4.06 6.03 0.33 7.51 107.56 4.46
Young stock 2.18 2.13 0.18 3.07 42.42 4.43
Buffalo
In-milk 5.96 6.34 1.05 8.88 132.34 8.35
Dry 5.44 4.95 0.52 7.35 101.96 8.49
Adult male 4.04 7.47 0.36 8.83 127.95 6.65
Young stock 2.29 2.22 0.19 3.69 44.33 4.43
Goat 1.50 0.20 0.06 0.61 10.58 0.30
Sheep 1.66 0.20 0.04 0.63 10.97 0.80
Others** 15.62 6.72 0.49 10.39 172.37 6.10
Notes: * includes grazing also
** includes horses and camels
14 17
Saving of Chemical Fertilizers due to Use of Dung
as Manure
The extent to which dung manure substitutes the
chemical fertilizers, is a saving of chemical fertilizers.
Fresh cattle dung on an average contains 0.30-0.40 per
cent nitrogen (N), 0.10-0.20 per cent phosphorus
(P
2
O
5
), and 0.10-0.30 per cent potassium oxide (K
2
O)
(Anonymous, 1997). According to the recent estimates
of Ghosh et al. (2004), dung manure contains 0.71 per
cent N, 0.18 per cent P
2
O
5
and 0.71 per cent K
2
O. In
this study, we have used the fraction of soil nutrients
(N,P and K) in dung-manure as estimated by Ghosh et
al. (2004), and the total quantity of N, P and K was
worked out for the proportion of bovine dung used as
manure.
Saving of Fossil Fuel due to Animal Energy-use in
Agriculture
If the working animals were to be replaced by
agricultural machinery, it would require additional fuel
and lead to emission of CO
2
from burning of the fossil-
fuel. It is this emission that is prevented by the
livestock. It has been assumed in the study that tractors
are the only machine used and are of similar power;
and also the working animals do not differ in their work
capacity.
In order to determine the saving of fossil fuel and
prevention of greenhouse gasses due to animal energy
use, we need to know (i) the rate of substitution between
tractors and working animals, (ii) the amount of diesel
required for running a tractor to perform agricultural
operations, and (iii) the amount of CO
2
that will be
emitted from the burning of a unit of diesel. On the
basis of economic substitution rate, 10 working males
are required to replace a tractor. The number of tractors
required for replacing countrys bovine working stock,
diesel use, and the associated emission of greenhouse
gasses have been estimated.
Results and Discussion
India has one of the largest livestock populations
in the world, and therefore its livestock sector has come
under critical scrutiny of the international
environmental monitoring agencies such as IPCC.
According to the estimates of the Indian Network for
Climate Change Assessment, the agricultural sector
emitted 334 Mt of CO
2
in 2007, to which livestock
contributed about 63 per cent. A number of studies in
the past have quantified methane emission from
livestock production. These studies have shown wide
variations, ranging from 8.5 Mt to 10.5 Mt, depending
on the methods and data used and also the year for
which the gas emission was estimated (Lerner et al.,
1988; Ahuja, 1990; Bhattacharya and Mitra, 1998;
Mishra and Dikshit, 2004; Singhal et al., 2005; Swamy
and Bhattacharya, 2006; Singh et al., 2012).
Notwithstanding their negative externalities,
livestock in the mixed farming systems also help
conserve natural resources and improve quality of
environment. The estimates of the positive
contributions of livestock to the environment are
discussed below:
Land Saving due to Recycling of Crop Residues
as Animal Feed
Livestock production in the mixed farming systems
saves land by utilizing or recycling crop by-products,
viz. dry fodder and concentrates as animal feed. If the
by-product feeds were to be replaced by feed grains or
cultivated green fodder, vast additional land will be
required to produce that much feed and fodder. This
land saving is a positive environmental effect. The
problem here can be posed as follows. How much
cultivated green fodder or feedgrains will be required
if the gross energy intake of the ruminant population
made available from the by-product feed, concentrates
and dry fodder, were to be replaced by the energy from
either of the former feeds. We have considered
cultivated green fodder as the alternative source of feed
energy.
The gross energy intake by the ruminant population
from by-product feed was estimated by summing-up
the energy values of the by-product feed on dry matter
basis. Similarly, the energy value of green fodder fed
to the animals was also calculated on dry matter basis.
The energy value of dry fodder and feed concentrate
(on dry matter basis) is 3.69 Mcal/kg and 4.38 Mcal/
kg of feed. Using feed consumption rates given in Table
1, we have estimated that the by-product feed provides
169 million calories of energy per day to livestock. If
this much energy were to be obtained from the
cultivated fodder, India would require 1701 million
tonnes of green fodder annually, and with an average
fodder yield of 42.5 t/ha, the total area required to
15 18
produce this amount of fodder would be as large as 40
million hectares (Table 2).
Land Saving due to Use of Dung as a Substitute
for Fire-wood in Domestic Fuel
Another way that livestock production saves land
is through supplying of dung as a domestic fuel. Of
the total wet dung produced (635 Mt), about 37 per
cent (235 Mt) is used as domestic fuel. Considering 80
per cent moisture in the fresh dung, the total dry dung-
cake production was estimated to be 47 Mt. At a
replacement rate of 3.54 in terms of thermal energy, if
this amount of dung-cake were to be replaced by fuel
wood, India will require 13 Mt of fuel wood in addition
to whatever quantity is produced otherwise. To produce
this much amount of fuel wood, about 1.62 Mha of
land will have to be constantly put to cultivation of
fuel wood plants with 4.5 years of gestation lag (Table
3).
Saving of Chemical Fertilizers due to Use of Dung
as Manure
After meeting its demand as fuel, the remaining
dung is used as manure to fertilize crops, which
indicates the savings in use of chemical fertilizers.
About 76 Mt of dung (on dry matter basis) is used as
manure. The total availability of soil nutrients from
manure was worked out to be of 1.22 Mt comprising
0.54 Mt of N, 0.14 Mt of P and 0.54 Mt of K; it is
equivalent to about 6 per cent of the total nutrients
used in the country in 2007. These nutrients from
manure can replace 2.63 Mt of ammonium sulphate,
Table 2. Land saving due to use of crop by-products as animal feed
Parameters Parameter value Source
Energy value of by-product feed on dry matter basis (Mcal/kg) Krishna et al. (1978)
Dry fodder 3.69
Concentrate 4.38
Consumption of by-product feed and crop residues in terms of 168.8 Estimated by authors
energy (Mcal)
Green fodder to dry matter ratio 1.0:0.25 Sen et al. (1978)
Yield of green fodder (t/ha) 42.5 Anonymous (1997)
Total green fodder required to replace by-products feed (Mt) 1701 Estimated by authors
Land area required to produce substituted quantity of green fodder (Mha) 40.0 Estimated by authors
Table 3. Parameter values for land saving due to use of dung cake as domestic fuel: 2007
Parameters Parameter Source
values
Production of wet dung (Mt)* 635.0 Estimated by authors
Utilized as domestic fuel (%) 37 CSO, GoI (1996)
Proportion of moisture in wet dung (%) 80 Flote (2011)
Fuel wood yield (t/ha) 36.8 Chaturvedi (1993)
Replacement rate of fuel wood for dung cake in terms of thermal energy 1:3.54 KVIC (1983)
Gestation lag between planting and harvesting of fuel wood saplings /trees (years) 4.5 GoO (2007)
Production of dung cake (Mt) 46.99 Estimated by authors
Fuel wood required to replace dung cake (Mt) 13.3
Land required to produce fuel wood :
With 1 year gestation lag (Mha) 0.36
With 4.5 year gestation lag (Mha) 1.62
*Bovine dung production
16 19
Table 4. Saving of chemical fertilizers due to use of dung as manure: 2007
Parameters Parameter values Source
Proportion of wet dung utilized as manure (%) 60 CSO, GoI (1996)
Proportion of moisture in wet dung (%) 80 Flote (2011)
Fraction of plant nutrients in dung manure Ghosh et al. (2004)
Nitrogen 0.0071
Phosphorus 0.0018
Potash 0.0071
Fraction of N, P and K in chemical fertilizers
Ammonium sulphate (N) 0.206 www.indiaagronet.com
Super phosphate (P) 0.444
Murate of potash (K) 0.660
Production of wet dung (Mt) 635.0 Estimated by authors
Dung Utilized as manure (Mt) 381.0
Dung manure on dry matter basis (Mt) 76.2
Saving of soil nutrients (Mt)
Nitrogen (N) 0.541
Phosphorus (P) 0.137
Potash (K) 0.541
Total (N, P & K) 1.219
Saving of chemical fertilizers (Mt)
Ammonium sulphate 2.63
Super phosphate 0.31
Murate of Potash 0.82
0.31 Mt of super phosphate and 0.82 Mt of murate of
Potash (Table 4). The available amount of these
nutrients is apparently small, but its value in monetary
terms could be substantial. Further, its environmental
value can be gauged if we consider the associated
emission in the production and transportation of the
equivalent amount of chemical fertilizers at the farm-
gate.
Saving of Fossil Fuel due to Use of Animal Energy
in Agriculture
To estimate the contribution of animals towards
saving of fossil fuel we need (i) substitution or
replacement rate between working animals and tractors,
and (ii) fossil-fuel (diesel) required per tractor per year
to do the work of replaced animals. On an average, a
bullock is rated at 0.4-0.5 HP (horse power). A 35 HP
tractor is, therefore, supposed to replace at least 70
bullocks. It is a purely engineering rate of substitution
between working animals and tractors. Some farm-
level studies carried out during 1970s and 1980s in the
north-western states of Punjab, Haryana and western
Uttar Pradesh the sheet of green revolution in India
have reported the replacement rates of three to four
bullocks per tractor (Binswanger 1978; Sharma 1987;
Mishra and Sharma 1990). Dikshit and Birthal
(2010a;b) using time series data on the number of
bullocks and tractors have arrived at a substitution rate
of 10, that is, a tractor can replace 10 working animals.
We have used this rate of substitution, and accordingly
the country will require 5.5 million tractors to replace
55 million working animals. To use the services of
required number of tractor stock, approximately 13.13
Mt of diesel would be required annually. Burning of
diesel will emit about 4.17 Mt of carbon di-oxide or
0.2 Mt of methane, which is the methane emission
prevented by the working animals.
Conclusions
Livestock have been singled out as one of the
largest sources of methane emission after rice.
Nevertheless, livestock also help conserve natural
17 20
resources, particularly in the mixed farming systems
where there is a considerable synergy between crop
and livestock activities. In this paper, we have identified
and quantified the positive environmental contributions
of livestock production system. Some of the positive
contributions identified include: saving of land due to
recycling of agricultural by-products as animal feed
and also due to use of dung-cake as domestic fuel;
saving in use of chemical fertilizers due to use of dung
as manure; and prevention of carbon dioxide emission
due to use of animal energy in Indian agriculture.
The study has found that there is enormous saving
of land in the mixed farming system on account of use
of agricultural by-products as feed and use of dung as
domestic fuel. If the by-product feed were to be
replaced by green fodder, as much as 1701 Mt of green
fodder will be required to supply the equivalent amount
of energy. To produce the required amount of green
fodder, about 40 Mha of land will be needed. Further,
to replace the quantity of dung cake used as domestic
fuel by fuel wood, the required amount of fuel wood
in terms of thermal energy would be 13.3 Mt. The land
resource required with three year of gestation lag would
be 1.62 Mha. The total land saving from the livestock
production system thus has been worked out to be 41.62
Mha.
The saving of soil nutrients due to use of dung as
manure has been estimated to the tune of 0.541 Mt of
nitrogen, 0.137 Mt of phosphorus and 0.541 Mt of
potash. If these quantities of soil nutrients are to be
replaced by the equivalent amount of chemical
fertilizers, then 2.63 Mt of ammonium sulphate, 0.31
Mt of super phosphate and 0.82 Mt of murate of potash
would be required. A tractor can replace about 10
working animals and at this rate, approximately 5.5
million tractors would be required to replace the
existing stock of working animals, that will consume
about 13 Mt of diesel annually. Burning of this much
diesel would emit about 4.17 Mt of CO
2
, which is
equivalent to 0.199 Mt of methane emission.
References
Ahuja, D. (1990) United State Environmental Agency
(USEPA), Inventory of U.S. Greenhouse Gas Emissions
and Sink, 1990-98.
Anonymous (1997) Hand Book of Agriculture, Indian
Council of Agriculture Research (ICAR), New Delhi.
Bhattacharya, S. and Mitra, A.P. (1998) Greenhouse Gas
Emission in India for the Base Year 1990. Scientific
Report, No.11, Centre for Global Change, National
Physical Laboratory, New Delhi.
Chaturvedi, P. (1993) Bioenergy Production and Utilization
in India Expert Consultation on Biofuels for
Sustainable Development and their Potential as
Suitable to Fossil Fuels and CO
2
Emission Reduction,
Food and Agriculture Organization, Rome.
Dikshit, A.K. and Birthal, P.S. (2010a) Indias livestock feed
demand: Estimates and projections, Agricultural
Economics Research Review, 23: 15-28.
Dikshit, A.K. and Birthal, P.S. (2010b) Environmental value
of draught animals: Saving of fossil-fuel and prevention
of greenhouse gas emission, Agricultural Economics
Research Review, 23: 227-232.
Floate, K.D. (2011) Arthropods in cattle dung on Canadas
grasslands In: Arthropods of Canadian Grasslands,
Inhabitants of a Changing Landscape, Edited by K.D.
Floate, in Biological Survey of Canada, 2: 71-88.
Table 5. Quantity of diesel saved due to use of animal energy: 2007
Parameter Parameter Source
values
Number of draught animals (millions) 55 GoI (2007)
Replacement rate 10.0 Dikshit and Birthal (2010)
Number of tractors required to replace existing stock of working 5.463 Estimated by authors
animals (millions)
Number of operating hours (hours/year) 801 Stephane et al. (2009)
Consumption of diesel by required No. of tractors (Mt) 13.13 Mishra and Dikshit (2004)
Total carbon released from burning of estimated diesel use (Mt) 11.38 Estimated by authors
Emission of carbon dioxide prevented (Mt) 4.17
Emission prevention equivalent to methane (Mt) 0.198 IPCC (1995)
18 21
Ghosh, P.K. et al. (2004) Comparative effectiveness of cattle
manure, poultry manure, phosphor compost and
fertilizer-NPK on three cropping systems in Vertisols
of semi-arid tropics, Crop Yields and System
Performance, 95: 77-83.
GoI (Government of India) (2007) 18th Livestock Census,
2007, Department of Animal Husbandry and Dairying,
New Delhi.
GoI (1996) National Accounts Statistics, 1993 and 1996,
Central Statistical Organisation (CSO).
GoO (Government of Odisha) (2007) Policy Guidelines for
Raising of Energy Plantations and Bio-diesel
Production, Science & Technology Department, Orissa
Renewable Energy Development Agency (OREDA),
Bhubaneswar.
IPCC (Inter-Governmental Panel on Climate Change) (1995)
Revised IPCC Guidelines for National Greenhouse Gas
Inventories, OECD, Paris.
KVIC (Khadi and Village Industries Commission) (1983)
Gobar Gas: Why and How, Directorate of Gobar Gas
Scheme, Bombay.
Krishna, G., Razdar, M.N. and Ray, S.N. (1978) Effect of
nutritional and seasonal variations on heat and methane
production in BosIndicus, Indian Journal of Animal
Science, 48(5): 366-370.
Lerner, J., Mathews, E. and Fung, I. (1988) Methane
emission from animals, a global high resolution data,
Global Biogeochemical Cycles, 2: 139-156.
Mishra, S.N. and Dikshit, A.K. (2004) Environment and
Livestock in India : With a Comparative Study of the
Indian and US Dairy Systems, Manohar Publishers and
Distributors, New Delhi.
Pasha, S.A.(2005) Livestock-Environment Interaction:
Issues, Problems and Prospects, Institute for Social and
Economic Change (ISEC), Bangalore.
Sen, K.C., Ray, S.N. and Ranjhan, S.K. (1978) Nutritive
Value of Cattle Feed and Feeding of Animals, Indian
Council of Agricultural Research, New Delhi.
Sere, C. and Steinfeld, H. (1996) World Livestock Production
System: Current Status, Issues and Trends. FAO Animal
Production and Health Paper 127. Food and Agriculture
Organization of the United Nations, Rome.
Singh, G.P. (1997) Effect of greenhouse gases on climate
change and Indian ruminant livestock, Current Science,
72: 7.
Singh, S., Kushwaha, B.P., Nag, S.K., Bhattacharya, S.,
Gupta, P.K., Mishra, A.K. and Singh, A. (2012)
Assessment of enteric methane emission of Indian
livestock in different agro-ecological regions, Current
Science, 102(7): 1017-1027.
Singhal, K.K., Mohini, M., Jha, A. K. and Gupta, P.K. (2005)
Methane emission estimates from enteric fermentation
in Indian livestock: Dry matter intake approach, Current
Science, 88: 119-127.
Steinfeld, H., Gerber, P., Wassennar, T., Castel, V., Rosales,
M. and de Haan, C. (2006) Livestocks Long Shadow:
Environmental Issues and Options, Food and
Agriculture Organization of the United Nations, Rome.
Stephane, de la Rue du Can, McNeil, Michael and Sathaye,
Jayant (2009) India Energy Outlook: End Use Demand
in India to 2020, Ernest Orlando Lawrence Berkeley
National Laboratory, LBNL-XXXX, U.S. Department
of Energy National Laboratory Operated by the
University of California.
Swamy, M. and Bhattacharya, S. (2006) Budgeting
anthropogenic greenhouse gas emission from Indian
livestock using country-specific emission coefficients,
Current Science, 91: 1340-1353.
www.indiaagronet.com
Received: January 2013; Accepted: March 2013
19 22
20 23
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25 28
26 29
27 30
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29 32
a
r
X
i
v
:
1
3
1
0
.
1
0
0
3
v
1

[
m
a
t
h
.
C
O
]

3

O
c
t

2
0
1
3
The signature of line graphs and power trees

Long Wang, Yi-Zheng Fan
School of Mathematics Sciences, Anhui University, Hefei 230601, P.R. China

Abstract: Let G be a graph and let A(G) be the adjacency matrix of G. The signature s(G)
of G is the dierence between the positive inertia index and the negative inertia index of A(G).
Ma et al. [Positive and negative inertia index of a graph, Linear Algebra and its Applications
438(2013)331-341] conjectured that c
3
(G) s(G) c
5
(G), where c
3
(G) and c
5
(G) respectively
denote the number of cycles in G which have length 4k + 3 and 4k + 5 for some integers k 0,
and proved the conjecture holds for trees, unicyclic or bicyclic graphs.
It is known that s(G) = 0 if G is bipartite, and the signature is closely related to the odd
cycles or nonbipartiteness of a graph from the existed results. In this paper we show that the
conjecture holds for the line graph and power trees.
AMS subject classication: 05C50
Keywords: Line graph; power graph; inertia; signature
1 Introduction
Throughout this paper we consider only simple graphs. The adjacency matrix A(G) = [a
ij
]
of a graph G with vertex set V (G) = {v
1
, v
2
, . . . , v
n
} and edge set E(G) is dened to be a
symmetric matrix of order n such that a
ij
= 1 if v
i
is adjacent to v
j
, and a
ij
= 0 otherwise.
The positive inertia index p(G), the negative inertia index n(G) and the nullity (G) of G are
respectively dened to be the number of positive eigenvalues, negative eigenvalues and zero
eigenvalues of A(G). The rank of G, written as r(G), is dened to be the rank of A(G).
The signature of G, denoted by s(G), is dened to be the dierence p(G) n(G). Obviously,
p(G) +n(G) +(G) = |V (G)|, p(G) +n(G) = r(G) and p(G) n(G) = s(G).
Motivated by the discovery that the nullity of a graph is related to the stability of the
molecular represented by the graph [1] and the open problem of characterizing all singular
graphs posed by Collatz [2], many authors discuss the nullity of a graph and obtain a lot of
interesting results. Here we particularly mention the results involved with the nullity of line
graphs. Sciriha [11] proved that all trees whose line graph is singular must have an even order.
Supported by National Natural Science Foundation of China (11071002, 11371028), Program for New Cen-
tury Excellent Talents in University (NCET-10-0001), Key Project of Chinese Ministry of Education (210091),
Specialized Research Fund for the Doctoral Program of Higher Education (20103401110002), Scientic Research
Fund for Fostering Distinguished Young Scholars of Anhui University(KJJQ1001).
Corresponding author. E-mail address: fanyz@ahu.edu.cn (Y.-Z. Fan), wanglongxuzhou@126.com (L. Wang)
1
30 33
Gutman and Sciriha [5] showed that the nullity of the line graph of a tree is at most one. Li et
al. [7] proved that the nullity of the line graph of a unicyclic graph with depth one is at most
two. Gong et al. [4] improved the above results as: the nullity of the line graph of a connected
graph with k induced cycles is at most k + 1.
Recently some authors discuss a more general problem, that is, describing the positive or
negative inertia index of graphs or weighted graphs, especially of trees or their line graphs,
unicyclic or bicyclic graphs; see Ma et al. [9], Li et al. [8] and Yu et al. [12, 13]. In the paper [9]
the authors posed a conjecture as follows, and proved the conjecture holds for trees, unicyclic
or bicyclic graphs.
Conjecture 1.1. [9] The inequality c
3
(G) s(G) c
5
(G) possibly holds for any simple
graph G, where c
3
(G) and c
5
(G) denote respectively the number of cycles having length 4k + 3
(or length 3 modulo 4) and the number of cycles having length 4k + 5 for some integers k 0
(or length 1 modulo 4).
Theorem 1.2. [9] Let G be a tree, or a unicyclic graph, or a bicyclic graph. Then c
3
(G)
s(G) c
5
(G).
A weaker result was also given by Ma et al. [9] that |s(G)| c
1
(G) for any graph G, where
c
1
(G) denotes the number of odd cycles of G, or c
1
(G) = c
3
(G) +c
5
(G).
When G is bipartite, surely s(G) = 0 and the conjecture holds in this case. So, from Theorem
1.2 or Conjecture 1.1 (if it was true), we nd that the signature is closely related to the odd
cycles or nonbipartiteness of a graph. In this paper we prove that the conjecture holds for the
line graphs and power trees.
2 Preliminaries
We rst introduce some notations. Let G be a graph and let W V (G). Denote by GW the
subgraph of G obtained by deleting the vertices in W together with all edges incident to them.
If G
1
is a subgraph of G, we sometimes write G G
1
instead of G V (G
1
). In particular, if
W = {x}, we simply write GW as Gx. If G
1
is an induced subgraph of G and x is a vertex
of G outside G
1
, denote by G
1
+x the subgraph of G induced by the the vertices of G
1
and x.
Lemma 2.1. [9] Let G be a graph containing path with four vertices of degree 2 as shown in
Fig. 2.1. Let H be the graph obtained from G by replacing this path with an edge. Then
p(G) = p(H) + 2, n(G) = n(H) + 2, (G) = (H), and hence s(G) = s(H).
G H
Fig. 2.1. The graphs G and H in Lemma 2.1
2
31 34
Lemma 2.2. [7] Let C
n
1
,n
2
,...,nt
be the graph obtained from a cycle C
t
by attaching n
i
pendent
edges to each vertex v
i
of C
t
, where n
i
0. Let G be the line graph of C
n
1
,n
2
,...,nt
, and let
m = |{i|n
i
> 0}|. Then the following results hold, where a zero chain of nite integer sequence
is dened to be a zero subsequence whose (cyclic) predecessor and successor are both nonzero,
and the length of the zero chain is dened to be the number of integers in that zero subsequence.
(1) (G) = 2 if and only if m = 0 and t 0 mod 4.
(2) (G) = 1 if and only if m 1 and either n
i
{0, 1} for i = 1, 2, . . . , t, the length of
any zero chain of (n
1
, n
2
, . . . , n
t
) is even, and t + m 0 mod 4; or t 0 mod 4 and one of
n
1
= n
3
= = n
t1
= 0 and n
2
= n
4
= = n
t
= 0 must hold.
(3) (G) = 0 otherwise.
Lemma 2.3. [4] Let x be a cut vertex of a graph G and G
1
be a component of Gx. If r(G
1
+x) =
r(G
1
) + 2, then r(G) = r(Gx) + 2. If r(G
1
+x) = r(G
1
), then r(G) = r(G
1
) +r(GG
1
).
Lemma 2.4. Let G be a graph and let x be a vertex of G. Then |s(G) s(G x)| 1. In
particular, if r(Gx) = r(G) or r(Gx) = r(G) 2, then s(Gx) = s(G).
Proof. By the eigenvalues interlacing property of real symmetric matrices (or see [3]), we have
p(G) 1 p(Gx) p(G), and n(G) 1 n(Gx) n(G), which yields the required results
immediately.
Corollary 2.5. Let H be an induced subgraph of a graph G. If r(H) = r(G), then s(H) = s(G).
Proof. Note that H can be viewed as one obtained from G by sequently deleting some of its
vertices. The condition r(H) = r(G) implies that in each step the rank, and hence the signature
of the resulting graph keeps invariant by Lemma 2.4, which yields s(H) = s(G).
Corollary 2.6. Let x be a cut vertex of a graph G and let G
1
be a component of G x. If
r(G
1
+x) = r(G
1
) + 2, then s(G) = s(Gx).
Proof. If r(G
1
+ x) = r(G
1
) + 2, then r(G) = r(G x) + 2 by Lemma 2.3, and hence
s(G) = s(Gx) by Lemma 2.4.
Lemma 2.7. Let x be a cut vertex of a graph G and let G
1
, G
2
, . . . , G
k
be all components of
Gx. If r(G
1
) = r(G
1
+x), then s(G) = s(G
1
) +s(GG
1
). In particular, if r(G
i
) = r(G
i
+x)
for all i, then s(G) = s(Gx).
Proof. Let =
k
i=2
G
k
. Write the adjacency matrix of G as follows,
A(G) =
A(G
1
) 0
T
0
0
T
A()
,
where the middle 0 corresponds to the cut vertex x. As r(G
1
) = r(G
1
+x), the matrix equation
A(G
1
)X = has a solution, say , such that
T
= 0. Now, take Q as the following matrix
3
32 35
with the same partition as A(G),
Q =
I 0
0 1 0
0 0 I
,
Then
Q
T
A(G)Q =
A(G
1
) 0 0
0 0
0
T
A()
.
So we have s(G) = s(G
1
) +s(GG
1
).
If r(G
i
) = r(G
i
+x) for all i, by induction on the number of components of Gx, we have
s(G) =
k1
i=1
s(G
i
) +s(G
k
+x). The result follows as s(G
k
+x) = s(G
k
) by Lemma 2.4.
Lemma 2.8. Let x be a cut vertex of a graph G and G
1
, G
2
, . . . , G
k
be all components of Gx.
If s(G) = s(Gx)+1, then s(G
l
+x) = s(G
l
)+1 for some l, and s(G) = s(G
l
+x)+
j=l
s(G
j
).
Proof. Note that r(G
i
+ x) r(G
i
) + 2 for each i. If r(G
i
+ x) = r(G
i
) + 2 for some i or
r(G
i
+x) = r(G
i
) for all is, then s(G) = s(Gx) by Corollary 2.6 or Lemma 2.7; a contradiction.
So r(G
i
+x) r(G
i
) + 1 for all is, with equality for at least one i.
Write the adjacency matrix of G as
A(G) =
0
T
1

T
2

T
k
1
A(G
1
) 0 0
2
0 A(G
2
) 0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
k
0 0 A(G
k
)
,
where the left upper 0 corresponds to the cut vertex x. Observe that for each i the equaiton
A(G
i
)X =
i
has a solution
i
; otherwise r(G
i
+ x) = r(G
i
) + 2; a contradiction. Taking Q as
the following matrix with the same partition as A(G),
Q =
1 0 0 0
1
I 0 0
2
0 I 0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
k
0 0 I
,
we have Q
T
A(G)Q = aA(G
1
)A(G
2
) A(G
k
), where a =
k
i=1
i
. The assumption
s(G) = s(G x) + 1 implies that a > 0. In particular, their exists some l such that
l
l
< 0.
So, A(G
l
+x) is congruent to (
l
l
) A(G
1
)), which implies s(G
l
+x) = s(G
l
) +1. Therefore
s(G) = s(G
l
+x) +
j=l
s(G
j
).
Corollary 2.9. Let x be a cut vertex of a graph G and let G
1
, G
2
, . . . , G
k
be all components
of Gx. If s(G
i
) c
5
(G
i
) and s(G
i
+x) c
5
(G
i
+x) for all is, then s(G) c
5
(G).
4
33 36
Proof. By Lemma 2.4, s(G) s(G x) + 1. If s(G) s(G x), noting that s(G x) =
k
i=1
s(G
i
) and s(G
i
) c
5
(G
i
) for all is, so we have s(G)
k
i=1
c
5
(G
i
) c
5
(G). If s(G) =
s(Gx) + 1, by Lemma 2.8, s(G) = s(G
l
+x) +
j=l
s(G
j
) for some l. By the assumption for
each G
i
and G
i
+x, we have s(G) c
5
(G
l
+x) +
j=l
c
5
(G
j
) c
5
(G).
3 Signature of line graphs
The line graph of a graph G, denoted by L
G
, is the graph whose vertex set is E(G), where two
vertices of L
G
are adjacent if and only if the corresponding edges are incident in G.
Lemma 3.1. If G is one of the following graphs: a cycle with two pendant edges, two cycles
sharing a common vertex, two cycles sharing a common path of length at least 1, where all
cycles have length 2 modulo 4, then s(L
G
) c
5
(L
G
).
Proof. First suppose G is a cycle C of length 2 modulo 4 with two pendant edges e
1
= x
1
y
1
and e
2
= x
2
y
2
, where y
1
, y
2
are pendant vertices of G. If x
1
= x
2
or x
1
, x
2
are connected by
paths on C of even length, by Lemma 2.2, L
G
is nonsingular. Note that L
G
has an even order
so that s(L
G
) is an even number. By Theorem 1.2, s(L
C
+e
1
) 0 as L
C
+e
1
is bicyclic. Now
by Lemma 2.4, s(L
G
) s(L
C
+e
1
) + 1 1. So, s(L
G
) 0 = c
5
(L
G
).
If x
1
, x
2
are connected by paths on C of odd length, then (L
G
) = 1 by Lemma 2.2. Note
that Cx
1
x
2
consists of two disjoint paths P
1
, P
2
both with order 0 or 2 modulo 4. By Lemma
2.1, it suces to consider the line graphs G
1
, G
2
in Fig. 3.1. We have s(G
1
) = s(G
2
) = 1 by
using Mathematica.
Next we consider the case that G is two cycles sharing a common vertex. Also by Lemma 2.1
it suces to consider the line graph G
3
in Fig. 3.1. By a direct calculation, we have s(G
3
) = 1.
Finally we consider the case that G is two cycles sharing a common path P of length at least
1. We stress all cycles have length 2 modulo 4. If the path P has length 1, then by Lemma 2.1 it
suces to consider the line graph G
4
in Fig. 3.1. By a direct calculation, we have s(G
4
) = 1.
If P has length greater than 1, then by Lemma 2.1 it suces to consider the line graphs G
5
, G
6
in Fig. 3.1. Also by calculation, we get s(G
5
) = s(G
6
) = 1.
5
34 37
1
G
2
G
3
G
4
G
5
G
6
G
Fig. 3.1. The graphs in the proof of Lemma 3.1
Theorem 3.2. Let T be a tree with at least one edge, then s(L
T
) c
5
(L
T
).
Proof. We use induction on the number of internal edges (i.e. non-pendant edges) of T to
prove the result. If T has no internal edges, then T = K
1,m
(i.e. a star), and hence L
T
= K
m
,
a complete graph. The result holds in this case by a simple verication.
Suppose the result holds for all trees with k ( 0) internal edges. Let T be a tree with k +1
internal edges and let e be one of the internal edges of T. Then T e consists of two subtrees
T
1
, T
2
of T. Obviously, each T
i
and each T
i
+ e has fewer internal edges than that of T. By
induction we have s(L
T
i
) c
5
(L
T
i
) and s(L
T
i
+ e) c
5
(L
T
i
+e) for each i = 1, 2. Noting that
e is a cut vertex of L
T
, so s(L
T
) c
5
(L
T
) by Corollary 2.9.
Theorem 3.3. Let G be a graph without isolated vertices. Then c
3
(L
G
) s(L
G
) c
5
(L
G
).
Proof. Without loss of generality we may assume G is connected. Let (G) be the set of edges
of G with at least one endpoint having degree greater than 2, and let (G) := |(G)|. We will
use induction on (G) to prove the left inequality. If (G) = 0, namely each vertex of G has
degree 1 or 2, then G is the disjoint union of paths and/or cycles. Thus, L
G
is the disjoint union
of paths and/or cycles. By Theorem 1.2, we have c
3
(L
G
) s(L
G
).
Assume that c
3
(L
H
) s(L
H
) for all graphs H with (H) k, where k 0. Let G be a
graph with (G) = k +1 and let x be a vertex of G with degree at least 3. Suppose e is an edge
incident to x. Then the vertex e of L
G
is contained in one triangle. So c
3
(L
Ge
) = c
3
(L
G
e)
c
3
(L
G
) 1. By Lemma 2.4 and by induction,
s(L
G
) s(L
G
e) 1 = s(L
Ge
) 1 c
3
(L
Ge
) 1 c
3
(L
G
).
Next, set o(G) := |E(G| |V (G)| + 1, the dimension of G. We also use induction on o(G)
to prove the right inequality. If o(G) = 0, then G is a tree, and the result holds in this case by
Theorem 3.2. Assume the result holds for all connected graphs G with o(G) k, where k 0.
6
35 38
Let G be a connected graph with o(G) = k +1. Note that G must contain cycles. A cycle C of
G is said of type l if there are exactly l edges between C and GC.
Case 1: If G contains a cycle C of type l with l 3, letting m be the length of C and
letting e
1
, e
2
, e
3
be three edges joining C and G C, then the line graphs L
C
, L
C
+ e
1
, L
C
+
e
1
+ e
2
, L
C
+ e
1
+ e
2
+ e
3
contain cycles of length m, m + 1, m + 2, m + 3 respectively. Surely
one cycle among them must have length 1 modulo 4. Deleting an arbitrary edge, say e on the
cycle C, will break the cycle of length 1 modulo 4 and decrease the dimension of G. That is,
c
5
(L
G
e) c
5
(L
G
) 1, and o(Ge) < o(G). Now by Lemma 2.4 and by induction,
s(L
G
) s(L
G
e) + 1 = s(L
Ge
) + 1 c
5
(L
Ge
) + 1 c
5
(L
G
).
Case 2: If G contains a cycle of type 1, say C, then C is connected to GC by an edge, say
e = xy, where x V (C) and y V (GC). Surely e is a cut edge of G. If G = C +y, then L
G
is bicyclic and the result holds by Theorem 1.2. If G = C+y, then e is a cut vertex of L
G
, Ge
has two components: C and another subgraph say D, where o(D) < o(G) and o(D+x) < o(G).
So, by induction, s(L
D
) c
5
(L
D
) and s(L
D
+ e) c
5
(L
D
+ e). Observe that s(L
C
) c
5
(L
C
)
and s(L
C
+e) c
5
(L
C
+e) by Theorem 1.2. The result now follows by Corollary 2.9.
Case 3: If all cycles of G are of type 2, then G is either (i) one obtained from a cycle with
two pendant edges (denoted by H) by possibly attaching trees at the pendant vertices of H,
or (ii) two cycle sharing a common vertex or a common path of length at least 2, or (iii) G is
obtained from a tree by replacing some vertices of degree 2 by cycles.
If G is one of graphs in (i) and (ii), and in addition if one cycle has odd length or length 0
modulo 4, then we will nd a cycle in G of length 1 modulo 4 containing the edges of the cycle.
Similar to Case 1, deleting an arbitrary edge on the cycle will break the cycle of length 1 modulo
4 and decrease the dimension of G. The result will follows by Lemma 2.4 and by induction.
Now assume G is one of graphs in (i) and (ii), and all cycles have length 2 modulo 4. If G
is exactly the graph H (a special case of (i)) or a graph in (ii), we get the result by Lemma 3.1.
If G is a graph in (i) obtained from H by attaching exactly one tree T at the pendant vertex
of a pendant edge say e, then G contains a cut edge say e such that Ge has two components:
G
1
, T, where G
1
is the cycle together with a pendant edge. Note that e is a cut vertex of L
G
, and
s(L
T
) c
5
(L
T
), s(L
T
+e) c
5
(L
T
+e) by Theorem 3.2. Also s(L
G
1
) c
5
(L
G
1
) by Theorem 1.2
as L
G
1
is bicyclic, s(L
G
1
+e) c
5
(L
G
1
+e) by Lemma 3.1. The result now follows by Corollary
2.9.
If G is a graph in (i) obtained from H by attaching two trees at the pendant vertices of
two pendant edge say e
1
, e
2
respectively, Then G e
2
has two components: G
1
, G
2
, where G
1
contains the cycle and G
2
is a tree. Note that in the graph G
1
the cycle is of type 1, and hence
s(L
G
1
) c
5
(L
G
1
) by the result in Case 2. Also s(L
G
1
+ e) c
5
(L
G
1
+ e) by what we have
proved in this case. So the result also follows by Corollary 2.9.
If G is a graph in (iii), then there exists a cut edge e of G such that Ge has two components:
G
1
, G
2
, where G
1
, G
2
both contain cycles. So o(G
i
) < o(G), o(G
i
+e) < o(G), and by induction
7
36 39
s(L
G
i
) < c
5
(L
G
i
), s(L
G
i
+e
) < c
5
(L
G
i
+e
) for i = 1, 2. Note that e is a cut vertex of L
G
. The
result also follows by Corollary 2.9.
Case 4: If G contains a cycle of type 0 and contains no cycles of type 1 or type l with l 3,
then G itself is the cycle or the cycle with a chord (an edge with two endpoints on the cycle).
Clearly the result holds if G is a cycle. If G is a cycle with a chord, letting C
1
, C
2
be two smaller
cycles containing the chord, if one cycle has odd length or length 0 modulo 4, then the result
follows by a similar discussion as in Case 3. Otherwise, C
1
, C
2
, and hence C all have length 2
modulo 4. In this case, we can get the result by Lemma 3.1.
4 Signature of power trees
Recall that the k-th power G
k
of a graph G is obtained from G by adding edges between all
pairs of vertices within distance at most k. In particular G
1
is exactly the graph G, and G
2
is
called the square of G.
Lemma 4.1. Let G be a graph on at least 5 vertices. If k 2, then in the graph G
k
every vertex
v is contained in at least one C
3
and one C
5
. That is to say, c
3
(G
k
v) c
3
(G
k
) 1 and
c
5
(G
k
v) c
5
(G
k
) 1.
Proof. Let H be an arbitrary connected graph induced by ve vertices of G. Then H contains
one of H
1
, H
2
, H
3
as a subgraph; see Fig. 4.1. Thus G
2
, and hence G
k
contains H
2
1
as a subgraph
by considering the squares of H
1
, H
2
, H
3
. Note that in H
2
1
each vertex is contained in at least
one C
3
and one C
5
. The result follows.
1
H
2
H
3
H
2
1
H
Fig. 4.1. The graphs in the proof of Lemma 4.1
Theorem 4.2. If G is a tree, then c
3
(G
k
) s(G
k
) c
5
(G
k
) for k 2.
Proof. If |V (G)| 4, the result follows by a direct calculation. Assume the result holds
for all trees on n vertices, where n 4. Let G be a tree on n + 1 vertices. By Lemma 4.1,
c
3
(G
k
v) c
3
(G
k
) 1 and c
5
(G
k
v) c
5
(G
k
) 1 for an arbitrary vertex v of G. Let u be a
pendant vertex of G. Then G
k
u = (Gu)
k
. So Lemma 2.4 and by induction,
s(G
k
) s(G
k
u)+1 = s((Gu)
k
)+1 c
5
((Gu)
k
)+1 = c
5
(G
k
u)+1 c
5
(G
k
)1+1 = c
5
(G
k
).
Similarly,
s(G
k
) s((Gu)
k
) 1 (c
3
(G
k
u) + 1) c
3
(G
k
).
The result follows.
8
37 40
Recall that the total graph T
G
of G is the graph with vertex set corresponding to union of
vertex and edge sets of G, with two vertices of T
G
adjacent if and only if the corresponding
elements in G are adjacent or incident. It is known that T
G
= S(G)
2
(or see [6]), where S(G) is
the subdivision of G. If G is a tree, then S(G) is also a tree. So we have the following corollary.
Corollary 4.3. If G is a tree, then c
3
(T
G
) s(T
G
) c
5
(T
G
).
References
[1] P. W. Atkins, J. de Paula, Physical Chemistry, eighth ed., Oxford University Press, 2006.
[2] L. Collatz, U. Sinogowitz, Spektren endlicher Grafen, Abh. Math. Sem. Univ. Hamburg, 21 (1957)
63-77.
[3] D. Cvetkovc, M. Doob, H. Sachs, Spectra of Graphs - Theory and Application, Academic Press, New
York, 1980.
[4] S. C. Gong, G. H. Xu, On the nullity of a graph with cut-points, Linear Algebra Appl., 436 (2012)
135-142.
[5] I. Gutman, I. Sciriha, On the nullity of line graphs of trees, Discrete Math., 232 (2001) 35-45.
[6] F. Harary, Graph Theory, Addison-Wesley, Reading, 1969.
[7] H.-H. Li, Y.-Z. Fan, L. Su, On the nullity of the line graph of unicyclic graph with depth one, Linear
Algebra Appl., 437 (2012) 2038-2055.
[8] S. Li, F. Song, On the positive and negative inertia of weighted graphs, arXiv: 1307.5110.
[9] H. Ma, W. Yang, S. Li, Positive and negative inertia index of a graph, Linear Algebra Appl., 438
(2013) 331-341.
[10] X. Ma, D. Wong, M. Zhu, The positive and the negative inertia index of line graphs of trees, Linear
Algebra Appl. (2013), http://dx.doi.org/10.1016/j.laa.2013.08.024.
[11] I. Sciriha, On singular line graphs of trees, Congr. Numer., 135 (1998) 73-91.
[12] G. Yu, L. Feng, Q. Wang, Bicyclic graphs with small positive index of inertia, Linear Algebra Appl.,
438 (2013) 2036-2045.
[13] G. Yu, X.-D. Zhang, L. Feng, The inertia of weighted unicyclic graphs, arXiv: 1307.0059.
9
38 41
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40 43
41 44
42 45
43 46
44 47
45 48
46 49
47 50
48 51
49 52
50 53
51 54
52 55
53 56
54 57
55 58
56 59
57 60
58 61
59 62
60 63
61 64
62 65
The Efficient Method for Simultaneous Monitoring of the
Culturable as Well as Nonculturable Airborne
Microorganisms
Barbara Hubad, Ales Lapanje*
Institute of Microbial Sciences and Technologies, Domzale, Slovenia
Abstract
Cultivation-based microbiological methods are a gold standard for monitoring of airborne micro-organisms to determine
the occupational exposure levels or transmission paths of a particular infectious agent. Some highly contagious
microorganisms are not easily culturable but it is becoming evident that cultivation and molecular methods are
complementary and in these cases highly relevant. We report a simple and efficient method for sampling and analyzing
airborne bacteria with an impactor-type high-flow-rate portable air sampler, currently used for monitoring culturable
bacteria or fungi. A method is reported for extraction of nucleic acids from impacted cells without prior cultivation and
using agarose as a sampling matrix. The DNA extraction efficiency was determined in spiked samples and, samples taken
from a wastewater treatment plant and an alpine area. The abundance, diversity and quantity of total bacteria were
analysed by a quantitative polymerase chain reaction, and by construction and analysis of clone libraries. The method does
not interfere with downstream PCR analysis and can cover the gap between traditional culture and molecular techniques of
bioaerosol monitoring.
Citation: Hubad B, Lapanje A (2013) The Efficient Method for Simultaneous Monitoring of the Culturable as Well as Nonculturable Airborne Microorganisms. PLoS
ONE 8(12): e82186. doi:10.1371/journal.pone.0082186
Editor: Martin Rottman, Harvard Medical School, United States of America
Received May 10, 2013; Accepted October 22, 2013; Published December 20, 2013
Copyright: 2013 Hubad, Lapanje. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the European Union Social Fund (grant no. P-MR-07/55) and the European Commission Seventh Framework program,
projects MULTISENSE CHIP (grant no. 261810), KillSpill (grant no. 312139) and Patch (grant no. A-1087-RT-GC). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journals policy and have the following conflicts: Patent number SI23434 Procedure for concentrating cells
and extraction of nucleic acids from polymer matrix for analysis of cells from the air was granted by The Slovenian Intellectual Property Office on 31.1.2012. Both
authors confirm that this does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: ales.lapanje@imst.si
Introduction
Effective monitoring of bioaerosols requires efficient collection
of microorganisms and an appropriate technique for their analysis
[1,2]. There is no standard method for collecting bioaerosols, but
culture dependent methods are generally recognized as the gold
standard in monitoring clean rooms (e.g. pharmaceutical and
medical instrumentation production facilities, operating rooms
and hospital indoor air), since isolation and cultivation of a specific
organism is currently the only validated approach to link causative
agents to a particular disease. However, some bacteria, including
pathogens such as Legionella pneumophila, can be in a viable but
nonculturable physiological state and others, such as Mycobacte-
rium tuberculosis are initially hard to cultivate. Although cultivation
techniques can be used to isolate most of the microorganisms that
are of concern to humans, a majority of bacteria, which arguably
are the most environmentally relevant, cannot be cultivated at all
[37]. This suggests the need to improve current methods for
bioaerosol analysis. Introduction of molecular methods based on
DNA isolated directly from environmental samples of culturable
and non-culturable bacteria, is expected to provide more
information than each one separately [1,7].
Methods currently used to collect airborne bacteria include
sampling with filters, liquid impingement, impaction on solid agar
or passive sedimentation. However, when both culturable and
non-culturable fractions of bacteria are desired, liquid impinge-
ment is most frequently used [7,8]. The impingement samplers are
less robust which results in several disadvantages such as rapid
evaporation of sampling liquid, samplers are typically not battery
driven and can be used only in vertical position. In these samplers
the evaporation of sampling liquid limits sampling time and lowers
collection efficiency. Moreover, additional handling of liquid, such
as inoculation onto growth media, is needed. Impactor samplers
can overcome these obstacles, but are currently used mainly for
collection and analysis of airborne microorganisms, which can be
grown on agar growth media [9,10]. In favor of impactor based
sampling method, diversity of culturable bacteria was reported to
be higher then by air filtration method as well as by impingement
[9]. Despite the advantages of impactors used for collection and
characterization of culturable bacteria, only three studies have
been published that extend their use in molecular approaches
based merely on isolated DNA from collected airborne bacteria
without prior cultivation [9,11,12]. In each case, solid gelatin or
liquid mineral oil were used as an impactor matrix, which were
chosen based on low melting point or low evaporation rate,
respectively. Accordingly, mineral oil enables longer sampling
times, but it cannot provide solid support during impaction. This
results in uneven distribution of oil in impaction holders and liquid
loss during handling of the sampling liquid, which presumably
influences DNA extraction efficiency [12]. Gelatin however, has a
PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e82186
63 66
solid structure at room temperature and low melting point (in a
range of 3037uC), which is beneficial for DNA extraction, since it
simplifies dissolution of the solid matrix [13]. Accordingly, the
solid matrix is the most preferable for sampling. However,
according to our knowledge the poorly defined chemical
characteristics of gelatin, which is composed of mixed size and
differentially branched polymeric matrix, as well as inhibition of
PCR due to high protein content, is especially pronounced in
samples with low numbers of cells [14]. If needed to use cultivation
in parallel to molecular methods, the low melting point of gelatin
limits its use at temperatures of 37uC and above, which is
especially problematic for incubation of pathogenic bacteria.
Additionally, gelatin can be degraded by many bacteria especially
eutrophic ones resulting in liquefied medium [15]. Therefore it is
of certain need to use alternative matrix with very similar
properties as gelatin but without drawbacks described above.
An ultimate approach to bioaerosol monitoring would be
simultaneous analysis of air samples by classical culturing methods
and by molecular methods without additional handling. One
favorable approach would be to use an impactor sampler with
classic growth media, from which in parallel one part of the sample
would be used in cultivation approach and the other for direct
DNA extraction. However, agar used to solidify nutrient media is
not molecular biology grade product and as such is inappropriate
to be used in DNA based analysis. Since this methodology is
currently unavailable, we have sought to develop and evaluate a
new method for monitoring total and culturable airborne bacteria,
adapted to the portable impaction based air sampler. The method
reported here is capable of speeding up the detection of the source
and identification of a particular microorganism.
Materials and Methods
Preparation of agarose matrix
RCS High Flow plastic strips (impaction holders) (Biotest,
Germany) were used for agarose impaction matrix. The impaction
holders are comprised of two rows of 17 wells, each containing
250 mL of matrix. The impaction matrix was prepared with
analytical grade low melting point (LMP) agarose (Promega) in
DNase- and RNase-free 16TAE buffer (Sigma-Aldrich) and was
sterilized by autoclaving for 20 min at 121uC. According to our
preliminary studies, 0.7% (w/v) of agarose matrix was the most
appropriate concentration, which provided a stable solid gel
structure that withstanded air velocity during sampling. This
concentration of agarose also minimized the interference with
downstream methods since the large amounts of agarose in DNA
extracts can inhibit PCR [16,17]. Autoclaved agarose was kept
liquid by incubation at 60uC and then applied to the empty, sterile
impaction holders. A total of 8 mL of agarose was applied to each
holder and the holder was then closed with a sterile cover and the
contents were left to solidify at room temperature.
Extraction of DNA from agarose matrices spiked with
bacterial cells
To determine the most efficient protocol for extraction of DNA
from agarose matrices, a random amount of Escherichia coli (ATCC
15597) and Staphylococcus aureus (ATCC 9144) in a range between
10
7
and 10
8
CFU were used. Overnight cultures grown in Luria-
Bertani medium (Sigma-Aldrich) were applied to solid sterile
agarose matrices and left for 1 hour at 22uC to soak completely
into the matrix. The efficiency and yield of DNA extraction was
determined by comparison of the obtained total DNA mass
extracted from spiked agarose matrices with the amount of DNA
extracted directly from bacterial cultures by the SmartHelixH
Complex samples Kit (Sekvenator Ltd., Slovenia) DNA extraction
protocol.
Development and optimisation of DNA extraction
protocol
Dissolution of the agarose matrix. To extract DNA from
the captured cells, it is (i) necessary to collect cells from the matrix,
(ii) to efficiently lyse the cells and (iii) to extract, purify and
concentrate the DNA. The most appropriate way to obtain
bacterial cells from the matrix is to completely dissolve and then
degrade the agarose into its monomeric units. However, in our
procedure, the amount of agarose that had to be dissolved
exceeded the volumes typically used (0.5 mL) for direct DNA
extraction. Therefore, adjustment of the reagent volumes and
times for incubation at elevated temperatures to completely melt
the agarose had to be determined from sample to sample. 0.7%
LMP agarose with spiked bacterial cells was transferred from
impaction holders into DNase- and RNase-free 10 mL tubes
(Sarstedt). Acid and enzymatic hydrolysis of agarose was tested in
combination with different temperatures to dissolve the agarose
completely and at the same time to minimize prolonged exposure
of sampled cells to elevated temperatures. Accordingly, for acid
hydrolysis, 1 mL of 5.5 M guanidinium thiocyanate (GuSCN,
pH 7) (Sigma-Aldrich) per 1 mL of agarose was added and then
incubated at 65uC for 10 min, 15 min or 30 min. For enzymatic
hydrolysis, agarose was first incubated at 65uC for 30 min or at
100uC for 1 min, 1.5 min or 5 min to dissolve and then left for
10 min to cool to 40uC. 7 U of b-agarase (Fermentas-Thermo
Scientific) per 1 mL of dissolved agarose was then added and the
mixture was incubated for 1 h or 1.5 h at 42uC.
Concentrating bacterial cells from dissolved
agarose. The initial volume of agarose matrix expands to more
than 8 times the volume that is normally used in most
commercially available DNA extraction kits, and it was necessary
to concentrate bacterial cells and cell debris by filtration prior to
the DNA extraction. Accordingly, dissolved agarose samples from
bacterial spiking experiments were passed through PES membrane
filters (25 mm, pores 0.22 mm, Milipore). DNA was extracted from
the filters by SmartHelixH Complex samples Kit (Sekvenator Ltd.,
Slovenia), since PES is completely dissolved in phenol/chloro-
form/isoamyl alcohol, the solvent in this DNA extraction kit.
Concentrating DNA in filtrates. Since microbial cells
collected on the agarose matrix were exposed to elevated
temperatures during the agarose melting, it was expected that a
fraction of cells will have been lysed. Thus, DNA released from
lysed cells freely passes through the 0.22 mm pore size PES filters
during the cell concentration procedure and is found in the filtrate.
Concentration of the DNA in the filtrate was achieved by
centrifugation of samples in ultrafiltration columns (10,000 Mw
cut-off, Vivaspin) at 9000 rpm for variable times, from 20 min to
60 min, until 100 mL of final DNA concentrate was obtained. The
DNA concentration was determined in all samples by Quant-iT
TM
High-Sensitivity DNA Assay Kit Assay Kit using QubitTM
fluorometer (Invitrogen) and the total extracted DNA mass was
calculated.
Statistical analysis. Linear regression method was used to
model the total DNA mass extracted from spiked agarose matrices
as a function of total DNA mass extracted directly from bacterial
cells. The suitability of linear regression line was evaluated based
on random distribution of residual errors around the fitted values,
normal distribution of residuals errors and absence of laverage
points and outliers. The significance of correlation of was tested
based on Pearsons and Spearman correlation coefficients. All
statistical calculations were performed with the R software version
Method for Monitoring Airborne Microorganisms
64 67
2.14.1 according to the function lm from the R software package
[18]. Descriptive statistics was performed in R by deducer package
and graphs were visualised by ggplot2 package.
Evaluation of the procedure on environmental samples
Sampling. To evaluate our procedure on actual samples,
outdoor sampling was performed at two locations, a wastewater
treatment plant (WWTP) which was predicted to have a higher
bacterial load [19,20], and an alpine mountainous area, which was
expected to have a lower bacterial load [21].
Impaction holders with sterile 0.7% LMP agarose as an
impaction matrix were inserted into portable RCS High Flow
air samplers (Biotest, Germany). For analysis based on culture-
independent methods, two air samples, designated Air1 and Air2,
each of 2 m
3
were collected in the alpine mountain area
(Slovenian Alps, 46u2497.020N, 13u36926.540E, 760 a.s.l) and nine
air samples, each of 2 m
3
were collected at the WWTP (200,000
population units). In parallel, 0.5 m
3
of air was sampled in
triplicate on the R2A growth media for alpine air and on nutrient
agar (NA) for samples taken at three locations in the WWTP to
permit characterisation of culturable bacteria from both locations
(Table 1).
Quantification. In samples from the WWTP, quantification
of total DNA concentrations and genes was performed by qPCR.
Two types of qPCR quantification were performed, a more
general method based on 16S rRNA genes and another specific
method targeting the Mycobacterium avium spp. hominisuis mtbA gene -
because the WWTP is located next to a pig farm. DNA
concentrations were measured with Quanti-It
TM
dsDNA HS
Assay Kit using QubitTM fluorometer (Invitrogen). In the same
DNA samples 16S rRNA genes were quantified in triplicate by
qPCR (7500 Real-Time PCR System, Applied Biosystems). The
20 mL volume reaction mixtures contained 200 nM primers U968
and L1401 (Table 1, [22]), 10 mL of 26SYBR Green PCR master
mix (Applied Biosystems), 8.6 mL DNAse-free water and 1 mL of
genomic DNA. Cycling conditions for real-time PCR were 2 min
at 50uC for prevention of DNA carryover, 10 min at 95uC for
enzyme activation and initial denaturation, which was followed by
50 cycles of 15 s at 95uC for denaturation and 60 s at 60uC for
annealing, extension and data acquisition. A final dissociation step
was added to exclude dimer interferences with the quantification.
A qPCR standard curve was determined with a series of 10-fold
dilutions of pCR2.1 plasmid with inserted partial 16S rRNA gene
of E.coli, amplified with the same primers. The detection limit was
set at a Ct value of 38 that corresponded to 15 copy numbers. 16S
rRNA gene copy numbers were calculated from triplicates of up to
500-fold dilutions of DNA samples.
For mbtA gene qPCR quantification the 20 mL reaction mixtures
contained 600 nM primers mbtAPH_F and mbtAHA1_R
(Table 1), 10 mL of 26SYBR Green PCR master mix (Applied
Biosystems), 7.8 mL DNAse-free water and 1 mL of genomic DNA.
Cycling conditions for real-time PCR were 2 min at 50uC for
DNA carryover prevention, 10 min at 95uC for enzyme activation
and initial denaturation, followed by 50 cycles of: 15 s at 95uC for
denaturation and 60 s at 61uC for annealing, extension and data
acquisition. A final dissociation step was added in order to assess
the potential occurrence of dimers. A qPCR standard curve was
determined with a series of 10-fold genomic DNA of Mycobacterium
avium spp. hominisuis, amplified with the same primers. The
detection limit was set at a Ct value of 36 that corresponded to 6
copy numbers. mbtA gene copy numbers were calculated from
triplicates of up to 100-fold dilutions of DNA samples.
Diversity. For culture-dependent analysis bacteria sampled
on R2A solid growth media, taken at the alpine mountain area,
were incubated at room temperature for 48 h. Colonies of bacteria
were enumerated and pure cultures from all grown colonies were
isolated. The culturable bacteria from one of three strips were
characterized by sequencing of 16S rRNA genes. The restriction
fragment length polymorphism (RFLP) analysis of 16S rRNA
genes was used on 33 and 39 colonies from Air1 and Air2,
respectively and the 16S rRNA genes from representatives of each
RFLP group were sequenced. Shannon-Wiener (H) index and
rarefaction analysis were calculated using Mothur software with a
97% OTU threshold [23].
For culture-independent analysis, DNA was extracted from
agarose according to the established protocol (see final protocol)
and a clone library was constructed based on PCR-amplified and -
cloned 16S rRNA genes in the pCR 2.1 vector. Accordingly,
25 mL of a PCR mixture consisting of 106 PCR buffer,
31.25 nmol MgCl
2
, 2.5 nmol dNTPs, 1.5 U of Ampli Taq
polymerase (Applied Biosystems), 10 pmol of each primer U968
and L1401 (Table 1, [22]), DNAse-free water and 1 mL of
genomic DNA were used in PCR amplification. During PCR
amplification the following cycling conditions were used: 3 min of
denaturation at 92uC, followed by 35 cycles of 30 s at 92uC, 30 s
for primer annealing at 54uC, 1 min at 68uC for extension, and a
final cycle at 72uC for 7 min on TProfessional temperature cycler
(Biometra, Goettingen, Germany). PCR products of 433 bp were
separated on the 1% agarose gels, excised and purified through
silica columns according to the manufacturers instructions
(Wizard SV Gel and PCR Cleaning System, Promega). Clone
libraries were constructed by ligation of purified PCR products in
TOPO TA vector pCR2.1 and transformed in electrocompetent
E. coli Top10 strain (Invitrogen) according to the manufacturers
instructions. From each constructed clone library, 60 clones were
randomly selected and 16S rRNA gene inserts in pCR2.1 plasmids
were sequenced by using M13f primer (Macrogen, Korea). The
closest relatives for given sequences were determined by using the
BLAST tool in the GenBank sequences databank and SeqMatch
in the RDPII database [24]. The sequences were grouped into
operational taxonomic units (OTUs) using a threshold of $97%
sequence similarity. Shannon-Wiener (H) index, rarefaction curves
and Libshuff analysis were calculated using Mothur software [23].
Table 1. Primers and amplification conditions for the detection of mbtA or 16S rRNA gene.
Oligonucleotide Name Sequence (59R39) Target gene Amplicon size
Forward primer U968 AACGCGAAGAACCTTAC 16S rRNA 433 bp
Reverse primer L1401 CGGTGTGTACAAGACCC 16S rRNA 433 bp
Forward primer mbtAPH_F CGACGACGCCCGTGTGATC mbtA 65 bp
Reverse primer mbtAHA1_R GCCATCCCGAACACCTGCT mbtA 65 bp
doi:10.1371/journal.pone.0082186.t001
65 68
Negative controls
Negative controls on DNA contamination of 0.7% LMP
agarose, membrane PES filters and all chemicals, were done
simultaneously for bacterial spiking and outdoor sampling. These
controls were treated in the same way as samples, total extracted
DNA concentrations were measured and 16S rRNA genes
detection was performed by PCR and qPCRs.
Results
Optimization of DNA extraction from agarose matrices
To sufficiently dissolve 8 mL of 0.7% LMP agarose at least
30 min at 65uC or 1.5 min at 100uC was required, as determined
by the disappearance of agarose residuals, visible on the
membrane filters after the filtration procedure. Acidic and
enzymatic hydrolysis both prevented reverse gelling of agarose.
However, addition of GuSCN to the acid hydrolysis reaction
significantly increased the final sample volume by a factor of at
least 2 of the initial sample volume, to approximately 16 mL.
Increase in the overall volume after the addition of agarase was
,110 mL and was deemed to be negligible. Incubation with
agarase for 1.5 h showed greater agarose degradation than 1 h
incubation, as determined by the higher viscosity in less degraded
Figure 1. Ratio of total DNA extracted either from retentate or filtrate. Ratio of total DNA extracted either from PES membrane filter
(retentate) (
N
) or concentrated with ultrafiltration (filtrate) (m), after bacterial spiking of agarose followed by enzymatic hydrolysis in relation to
the sum of DNA extracted from each individual sample (abbreviated as total extracted DNA mass). The sum of the
N
and m percentages in each
vertical line is 100%, which is represented on the total extracted DNA mass axis. Black lines represent linear trend: filtrate (R
2
=0.331, slope coefficient
20.03 and intercept 89.7) and retentate (R
2
=0.331, slope coefficient 0.03 and intercept 10.3). 95% confidence intervals for both fitted lines are
presented with grey area.
doi:10.1371/journal.pone.0082186.g001
Figure 2. Total DNA mass extracted after bacterial spiking.
Linear regression between total DNA mass extracted with developed
protocol from spiked agarose matrices and total DNA mass extracted
directly from bacterial cells (R2 =0.76, slope coeficient 0.68, intercept
38.2). 95% confidence interval for fitted line is presented with grey area.
66 69
samples. After both acid and enzymatic hydrolyses, on average
32.5%611.3% and 19.5%68.5% (Figure 1) of total DNA mass
were isolated from retentates, which showed that most of the cells
were lysed during the agarose melting procedure and the released
DNA successfully passed through the filter. The DNA in the
filtrate had to be extracted, and this was the most conveniently
achieved by ultrafiltration. Since the DNA in the filtrate was above
5 kbp, it was determined that ultrafiltration would be at least 50%
efficient. In samples treated by enzymatic hydrolysis in which
1.5 min at 100uC was used to dissolve agarose followed by 1.5 h
incubation with agarase, the minimum centrifugation time needed
to concentrate the sample to a final volume 100 mL, was 20 min.
All other samples had centrifugation times between 30 min and
60 min, especially in samples treated with GuSCN. In these cases,
the centrifugation time was in excess of 45 min since the overall
volume was up to twice that developed in the enzymatic hydrolysis
procedure.
Performance evaluation
Yield. A positive linear regression (R
2
=0.76, slope coefficient
0.68, intercept 38.2) was determined between the total DNA mass
extracted from spiked agarose matrix and total DNA mass
extracted directly from bacterial cells (Figure 2) as well as
significant positive correlation based on Pearsons and Spearman
coefficient (p,0.0001 for both). Residuals were randomly and
normally distributed (Figure S1). Since the coefficient of the
regression line is below 1, at higher values of total amount of DNA
the extraction yield is lower (Figure 2), while the ratio of DNA
mass extracted from retentate was higher (Figure 1). The
extraction efficiency was higher than 50% in all tested samples,
with an average value of 79.2%618.0%.
PCR amplification efficiency. The PCR amplification
efficiency was assessed in spiked as well as actual samples obtained
from air with low (alpine region) and high (WWTP area) amounts
of cells. The conventional amplification of 16S rRNA genes was
successful from spiked samples as well as samples collected
outdoors. Approximately 10
3
CFU/m
3
were determined at
WWTP and 10
1
CFU/m
3
at the alpine area (Table 2, Table 3).
For efficient amplification, dilutions of at least 10-fold had to be
applied to samples taken at the WWTP, whereas in samples
obtained from the alpine area or spiked, the PCR amplification
could be detected in non-diluted samples.
In all nine samples from the WWTP we extracted enough DNA
to measure fluorometrically the total DNA mass which was found
to be between 12.1 and 37.7 ng/m
3
(Table 2). With real time
PCR, we detected 10
6
10
9
copy numbers of partial 16S rRNA
gene per m
3
. Amplification was achieved within a linear range
from 50- to 500-fold dilution, with a dynamic range of Ct 2238,
extending from 330 to 1260 pg total DNA/m
3
at a maximum to
24.2 to 75.5 pg of total DNA/m
3
at a minimum.
By M. avium spp. hominisuis-specific qPCR we detected 215 to
628 copy numbers of mbtA gene per m
3
(Table 2). Amplification
was achieved within a linear range from 50- to 100-fold dilution,
with a dynamic range of Ct 3136, extending from a maximum of
330 to 1260 pg total DNA/m
3
to a minimum of 36.1 to 24.6 pg of
total DNA/m
3
.
Bacterial diversity. The diversity of culturable bacteria was
compared to the 16S rRNA gene diversity in the clone library.
According to the calculated rarefaction curves and H index, the
bacterial diversity was higher in both clone libraries than was
determined by cultivation (Figure 3, Table 3). The taxonomic
assignment of clones and isolates showed sequences in common
Table 2. Total DNA mass, copy numbers of 16S rRNA gene and mbtA gene per m
3
of sampled air at three locations inside WWTP.
16S rRNA gene
Mycobacterium avium
spp. hominisuis (mbtA)
Sample
name Air sampling location and sampling time CFU/m
3
Total DNA mass
(ng/m
3
) Copy numbers/m
3
Copy numbers/m
3
AGC-1 Aerated grid chamber Day 1 - 37.7 2.1E+0967.3E+08 -
AGC-2 Aerated grid chamber Day 2 17136677 33.2 2.1E+0968.7E+08 215664
AGC-3 Aerated grid chamber Day 3 - 26.5 2.3E+0861.3E+08 -
AB-1 Aeration basin Day 1 - 14.6 3.8E+0763.7E+07 -
AB-2 Aeration basin Day 2 16296437 36.1 2.1E+0861.1E+08 3376134
AB-3 Aeration basin Day 3 - 27.3 6.3E+0765.7E+07 -
E-1 Entrance to the management building Day 1 - 12.1 4.2E+0763.7E+07 -
E-2 Entrance to the management building Day 2 859659 24.6 4.3E+0762.1E+07 8846115
E-3 Entrance to the management building Day 3 - 26.5 1.7E+0769.3E+06 -
Table 3. CFU per 2 m
3
air sample size (CFU6SD) and H index determined in clones and isolates from alpine area. Values in
brackets are high and low 95% confidence interval.
H index
Sample name CFU/2 m
3
isolates sequenced clones sequenced isolates clones
Air1 89622 31 50 1.63 (1.90, 1.37) 2.63 (2.26, 2.99)
Air2 96649 39 56 2.47 (2.75, 2.16) 3.57 (3.80, 3.35)
67 70
with the obtained isolates only among Methylobacterium, Acinetobacter
and Brevundimonas in Air1 and Air2, respectively (Figure S2, S3, S4,
S5). All other clones were found to be exclusive to clone libraries
or among isolates. Bacterial diversity of culturable bacteria and
clones were significantly different in each location (p,0.05) as
determined by Cramer-von Mises test.
False positives and false negatives
Since air samples contain relatively low quantities of bacterial
cells it was necessary to determine the purity of the reagents. After
DNA isolation from PES as well as from chemical reagents, the
total DNA concentrations were below the detection limit
(,0.5 pg/mL of DNA extract).
Optimized protocol for isolation of DNA from cells
sampled from air by impactor on an agarose matrix
According to our optimisation of (i) the volume of a melted
agarose solution, (ii) sufficient degradation of polysaccharides and
(iii) recovery of DNA from filtrate as well as retentate, the final
DNA extraction protocol is described here (see Figure 4). LMP
agarose (0.7%) from the impactor holder must be completely
dissolved in a water bath for 1.5 min at 100uC in DNase- and
RNase-free 10 ml tubes (Sarstedt). Dissolved agarose (,8 mL) is
then cooled to 40uC for 10 min and degraded into oligosaccha-
rides by treatment for 1.5 h at 42uC with 7 U of b-agarase
(Fermentas - Thermo scientific) per 1 mL of agarose. Degraded
agarose was filtered through polyethersulphone (PES) membrane
filters with 0.22 mm pore size (25 mm, Milipore) the filtrate being
directly collected into ultrafiltration columns (Vivaspin 4, MWCO
10 000, Sartorius). Parallel extraction of DNA from retentate and
filtrate is performed to extract DNA from intact bacterial cells and
cell debris. DNA from the filter is extracted with phenol/
chloroform/isoamyl alcohol according to the protocol of
SmartHelixH Complex samples Kit (Sekvenator Ltd., Slovenia).
The method combines dissolution of PES filters and lysis of
bacterial cells by bead beating (MillMix 20, Tehtnica, Slovenia).
The dried DNA pellet isolated from the retentate is resuspended in
DNA solution from the filtrate (approx. 100 mL). The filtrate is
concentrated beforehand by centrifugation in the ultrafiltration
columns at 9000 rpm for approximately 20 min. (Figure 4). Such
DNA solutions can be used directly in further downstream
analyses.
Discussion
In clinical microbiology, the Koch postulates restrict the use of
methods based merely only on isolation and identification of
nucleic acids since disease-causing agents must be isolated and
confirmed. However, in some cases molecular methods may be the
only feasible choice for investigation of microorganisms which are
difficult or impossible to cultivate. Cultivation and molecular
methods are not mutually exclusive but rather are complementary.
For instance, after molecular detection of highly contagious
microorganisms that are hard to isolate, the cultivation methods
must be utilized finally to establish the cause of the disease.
Methods which utilize both approaches are in such cases highly
relevant. Our current understanding of human microbiomes has
revealed many previously unknown aspects of microbial interfer-
ence with the human body and progressively more attention is
being paid to environmental microorganisms [8,25], most of
which, unfortunately are currently not culturable. Accordingly, we
have developed a method which can be used by both molecular
and cultivation-based approaches, and is at the same time highly
efficient, does not interfere with downstream applications and can
be applied for analysis of quantity and diversity of airborne
microorganisms. Since both approaches can be used in parallel
this method speeds up the identification of microorganisms and
the detection of the source of a particular microorganism by
applying fast molecular methods.
The efficiencies of both the bioaerosol collection and
nucleic acids extraction, are the most important factors in air
Figure 3. Rarefaction curves from clone libraries and from isolates. Rarefaction analysis of 16S rRNA genes from clone libraries and from
isolates, both obtained from alpine air samples. 95% confidence intervals are shown.
68 71
investigations, since amount of bacterial cells in air (10
3
to 10
6
/m
3
)
are low in comparison to other environmental samples (in rivers
for example, 10
12
to 10
14
of bacteria per m
3
of water is common).
Since impactor type air sampler has a predefined efficiencies of
aerosol collection [26,27], only the structure of impaction matrix
and DNA extraction efficiency can be optimized. Higher diversity
of culturable bacteria was reported after impaction of bioaerosols
onto solid nutrient agar than when impacted on solid gelatin as
well as when air was impinged into water or filtrated through
cellulose acetate or gelatin filters [9]. The developed method that
use solid agarose as a support for impaction, which is very similar
to nutrient agar support, showed higher DNA extraction
efficiencies (79% efficiency, see Figure 2) than it was reported
for DNA extraction efficiencies from filters (50% efficiency) [28],
currently the preferential method used in culture independent
studies. There are no published data concerning DNA extraction
efficiencies after direct bacterial spiking in gelatin or mineral oil.
However, in the studies in which researchers used these two
matrices for bioaerosol sampling and where they compared the
estimated number of microorganisms from qPCR to the number
of culturable bacteria it resulted in between 1 to 2 [29] and
between 2 to 3 [12] orders of magnitude more bacteria determined
by molecular approach than they were able to cultivate when
collected in gelatin or mineral oil, respectively. If we use the same
comparison approach based on our method in which we sampled
outdoor air, we determined 3 orders of magnitude more overall
bacteria than can be obtained only by cultivation. Comparable
results were obtained when samples were collected into liquid by
cyclone or glass impingers [9,30]. Although these results cannot be
directly compared to the gelatin and mineral oil based sampling
method, one can speculate that agarose based method developed
in our study is very efficient in obtaining bacterial DNA, since we
determined high ratio between unculturable and culturable
bacteria.
Although our method resulted in relatively high DNA
extraction efficiencies in spiking experiments, some substantial
losses were still observed. Since the same phenol/chloroform/
isoamyl alcohol DNA extraction method was used to extract DNA
from agarose as well as from bacterial cell suspension, we were
able to exclude the influence of the DNA extraction method on the
overall DNA loss. We speculate that DNA can be lost (i) during
removal of impaction matrix from impaction holders, as has been
observed [12] for a mineral oil matrix, (ii) during the ultrafiltration
step by passing smaller fragments through filter membrane and (iii)
by the attachment of DNA to plastic tubes that were used for
agarose melting.
In addition to DNA extraction efficiency, the quality and purity
of extracted DNA is important for downstream metagenomic
analysis, especially in relation to inhibition of PCR-based
amplifications. Such inhibition can be attributed to environmental
impurities as well as impurities introduced during the procedure. It
is known that agarose can inhibit PCR reactions [7,31] and
therefore, in our procedure the most critical step is the elimination
of residual agarose and its degradation products. Agarose must be
completely melted so that agarase can access the polysacharide
and produce complete hydrolysis after a certain incubation time.
Since we did not observe inhibition in spiked samples or in samples
taken in the alpine mountain area, the agarose contamination was
assumed to be below the PCR interference threshold but in
samples collected at the WWTP we observe PCR inhibition which
is most likely attributable to inhibitory substances present in the
DNA samples [32]. In such cases it is necessary to purify the DNA
samples further.
It is known that culturable bacteria represent only a fraction of
total bacteria present in air [33] and that by observing only
culturable bacteria the actual quantity and diversity of total
bacteria will be underestimated. In this regard, the results of our
taxonomic assignment of clones obtained from impaction of
bioaerosols on agarose matrices show that indeed not all cultured
bacteria are reflected in bacteria determined from total extracted
DNA. Moreover, the majority of closest representatives of
obtained clones belonged to uncultured bacteria. It has to be
noted that by molecular methods we observed presence of some
pathogens (e.g. Streptococcus pneumoniae) or opportunistic pathogens
(e.g. Mycobacterium avium spp. hominisuis) which were absent from
culturable bacteria. This can be of extreme importance, since
immunosuppressed individuals can be infected, an event which
was actually noted by the workers employed at the sampled
WWTP and which was severally reported by other researchers (see
[34]). Therefore, the method reported here can be of particular
interest for monitoring occupational exposures to bioaerosols.
In conclusion, the DNA extraction efficiency of the developed
protocol was on average 79.2%618.0%. We were able to
successfully extract enough DNA with good quality to proceed
with downstream analysis such as PCR and the method was
applied to quantify and to determine diversity of bacteria in air
from the WWTP and from air from an alpine environment, which
have a high and low bacterial load, respectively. Compared to
culturable fractions, culture-independent fractions showed (i)
higher number of calculated bacteria, (ii) higher diversity and
(iii) large proportion of closest relatives to uncultured bacteria. In
Figure 4. Scheme of the developed protocol.
69 72
future, our DNA extraction protocol should be implemented in air
monitoring to link the gap between traditional culture and
molecular techniques, which both together deliver highly relevant
and complementary information concerning bioaerosols.
Supporting Information
Figure S1 Evaluation of the linear regression model for
total DNA mass extracted with developed protocol from
spiked agarose matrices and total DNA mass extracted
directly from bacterial cells. (A) the residuals errors versus
their fitted values, (B) square root of the standardized residuals as a
function of the fitted values, (C) Q-Q plot of normal distribution of
residuals, (D) standardized residuals as a function of leverage.
(TIF)
Figure S2 Phylogenetic analysis of partial bacterial 16S
rRNA gene sequences of isolates obtained from Air1
sampled at the alpine mountain area. The tree based on
Kimura two-parametric distances was constructed by a neighbour-
joining algorithm. Aquifex pyrophilus was used as the outgroup.
Bootstrap values (1000 replicates) greater than 20% are indicated
above the branches. The scale bar represents the 0.05 nucleotide
substitution per base.
(TIF)
rRNA gene sequences of clones obtained from Air1
sampled at the alpine mountain area. The tree was
constructed using the neighbour-joining algorithm. Aquifex pyr-
ophilus was used as outgroup. Bootstrap values (1000 replicates)
greater than 20% are indicated above the branches. The scale bar
represents the 0.05 nucleotide substitution per base.
(TIF)
rRNA gene sequences of isolates obtained from Air2
ophilus was used as the outgroup. Bootstrap values (1000 replicates)
(TIF)
rRNA gene sequences of clones obtained from Air2
ophilus was used as the outgroup. Bootstrap values (1000 replicates)
(TIF)
Acknowledgments
The authors wish to thank Fani Oven for technical assistance and Rok
Kopinc, both from the Institute of Microbial Sciences and Technologies,
for helpful comments on this manuscript.
Author Contributions
Conceived and designed the experiments: BH AL. Performed the
experiments: BH. Analyzed the data: BH AL. Contributed reagents/
materials/analysis tools: BH AL. Wrote the paper: BH AL.
References
1. Alvarez AJ, Buttner MP, Stetzenbach LD (1995) PCR for bioaerosol monitoring:
sensitivity and environmental interference. Appl Environ Microbiol 61: 3639
3644.
2. DArcy N, Canales M, Spratt DA, man Lai K (2012) Healthy schools:
standardisation of culturing methods for seeking airborne pathogens in
bioaerosols emitted from human sources. Aerobiologia 28: 413422.
3. Angenent LT, Kelley ST, Amand AS, Pace NR, Hernandez MT (2005)
Molecular identification of potential pathogens in water and air of a hospital
therapy pool. Proc Natl Acad Sci U S A 102: 48604865.
4. Delgado-Viscogliosi P, Simonart T, Parent V, Marchand G, Dobbelaere M, et
al. (2005) Rapid method for enumeration of viable Legionella pneumophila and
other Legionella spp. in water. Appl Environ Microbiol 71: 40864096.
5. Chang C-W, Hung P-Y (2012) Evaluation of sampling techniques for detection
and quantification of airborne legionellae at biological aeration basins and
shower rooms. J Aerosol Sci 48: 6374.
6. Chang C-W, Hung P-Y (2012) Methods for Detection and Quantification of
Airborne Legionellae Around Cooling Towers. Aerosol Sci Technol 46: 369
379.
7. Rinsoz T, Duquenne P, Greff-Mirguet G, Oppliger A (2008) Application of real-
time PCR for total airborne bacterial assessment: Comparison with epifluores-
cence microscopy and culture-dependent methods. Atmos Environ 42: 6767
6774.
8. Han Y, Li L, Liu J (2013) Characterization of the airborne bacteria community
at different distances from the rotating brushes in a wastewater treatment plant
by 16S rRNA gene clone libraries. J Environ Sci 25: 515.
9. Li K (2011) Molecular comparison of the sampling efficiency of four types of
airborne bacterial samplers. Sci Total Environ 409: 54935498.
10. Carducci A, Verani M, Lombardi R, Casini B, Privitera G (2011)
Environmental survey to assess viral contamination of air and surfaces in
hospital settings. J Hosp Infect 77: 242247.
11. Nehme B, Letourneau V, Forster RJ, Veillette M, Duchaine C (2008) Culture-
independent approach of the bacterial bioaerosol diversity in the standard swine
confinement buildings, and assessment of the seasonal effect. Environ Microbiol
10: 665675.
12. He Q, Yao M (2011) Integration of high volume portable aerosol-to-hydrosol
sampling and qPCR in monitoring bioaerosols. J Environ Monit 13: 706712.
13. Joly-Duhamel C, Hellio D, Djabourov M (2002) All gelatin networks: 1.
Biodiversity and physical chemistry. Langmuir 18: 72087217.
14. Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl
Environ Microbiol 63: 3741.
15. Smith HL, Goodner K (1958) Detection of bacterial gelatinases by gelatin-agar
plate methods. J Bacteriol 76: 662665.
16. Gibb AP, Wong S (1998) Inhibition of PCR by agar from bacteriological
transport media. J Clin Microbiol 36: 275276.
17. Reading F (2002) Inhibitory effects of agarose gel and LB medium on DNA
sequencing. BioTechniques 33: 282284.
18. Team RDC (2005) R: A language and environment for statistical computing.
ISBN 3-900051-07-0. R Foundation for Statistical Computing. Vienna, Austria,
2013. url: http://www. R-project. org.
19. Karra S, Katsivela E (2007) Microorganisms in bioaerosol emissions from
wastewater treatment plants during summer at a Mediterranean site. Water Res
41: 13551365.
20. Bauer H, Fuerhacker M, Zibuschka F, Schmid H, Puxbaum H (2002) Bacteria
and fungi in aerosols generated by two different types of wastewater treatment
plants. Water Res 36: 39653970.
21. Bauer H, Kasper-Giebl A, Loflund M, Giebl H, Hitzenberger R, et al. (2002)
The contribution of bacteria and fungal spores to the organic carbon content of
cloud water, precipitation and aerosols. Atmospheric Res 64: 109119.
22. Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, et al. (1996) Sequence
heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa
detected by temperature gradient gel electrophoresis. J Bacteriol 178: 5636
5643.
23. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009)
Introducing mothur: open-source, platform-independent, community-supported
software for describing and comparing microbial communities. Appl Environ
Microbiol 75: 75377541.
24. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al. (2009) The Ribosomal
Database Project: improved alignments and new tools for rRNA analysis.
Nucleic Acids Res 37: D141D145.
25. Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, et al. (2009)
Metagenomic analysis of respiratory tract DNA viral communities in cystic
fibrosis and non-cystic fibrosis individuals. Plos One 4: e7370.
26. An HR, Mainelis G, Yao M (2004) Evaluation of a high-volume portable
bioaerosol sampler in laboratory and field environments. Indoor Air 14: 385
393.
27. Yao M, Mainelis G (2006) Investigation of cut-off sizes and collection efficiencies
of portable microbial samplers. Aerosol Sci Technol 40: 595606.
28. Hospodsky D, Yamamoto N, Peccia J (2010) Accuracy, precision, and method
detection limits of quantitative PCR for airborne bacteria and fungi. Appl
Environ Microbiol 76: 70047012.
70 73
29. Yamamoto N, Kimura M, Matsuki H, Yanagisawa Y (2010) Optimization of a
real-time PCR assay to quantitate airborne fungi collected on a gelatin filter.
J Biosci Bioeng 109: 8388.30.
30. Le Goff O, Godon J-J, Milferstedt K, Bacheley H, Steyer J-P, et al. (2012) A new
combination of microbial indicators for monitoring composting bioaerosols.
Atmos Environ.
31. Ziros PG, Kokkinos PA, Legaki E, Vantarakis A (2011) Development of an
optimized method for the detection of airborne viruses with real-time PCR
analysis. Virol J 8: 16.
32. Shannon KE, Lee D-Y, Trevors JT, Beaudette LA (2007) Application of real-
time quantitative PCR for the detection of selected bacterial pathogens during
municipal wastewater treatment. Sci Total Environ 382: 121129.
33. Brodie EL, DeSantis TZ, Parker JPM, Zubietta IX, Piceno YM, et al. (2007)
Urban aerosols harbor diverse and dynamic bacterial populations. Proc Natl
Acad Sci 104: 299304.
34. Lee JA, Thorne PS, Reynolds SJ, OShaughnessy PT (2007) Monitoring risks in
association with exposure levels among wastewater treatment plant workers.
J Occup Environ Med 49: 12351248.
71 74
Goodfriend is a bit worried about Harry. She describes the three
styles of psychological attachment, which are ways that we learn
to relate to others as children. These tend to stick with us through-
out our lives. She shows how Rawling has given each Harry, Ron,
and Hermione one of these different styles and speculates what this
might mean for each of their futures.
WIND GOODFRIEND, PH.D.
Attachment S1yles at Hogwarts
From Infancy to Adulthood
PICTURE LONDON DURING the height of World War II. The
city was constantly threatened by bombings from above. Parents had
only one greater fear than the fear of their own death: the death of
their children. To keep its young ones safe, the B:ritish government
evacuated children to the countryside. After the war, families were
reunited. _Physically, the children were fine. However, an unforeseen
side effect had occurred. The children had been separated from their
parents at a crucial time in their development, and, as young adults,
they began displaying a variety of psychological disorders. For many
of these adolescents, a key problem among these issues was the in-
ability to form strong, loving ties with other people. From this phe-
nomenon, psychology researchers noticed and created. attachment
theory. Attachment theory focuses on how the familial environment
during one's formative years affects one's ability to begin and main-
tain normal, adult relationships-including romantic relationships.
Psychologists have now identified tl;lree distinct attachment styles,
75
72 75
76 THE PSYCHOLOGY OF HARRY POTTER
or patterns of behavior relevant to how one interacts with potential
romantic partners. Perhaps it is coincidental, but these three attach-
ment styles can be seen in almost perfect form in the three main char-
acters in the Harry Potter series: Hermione Granger, Ron Weasley,
and Harry himself.
A BRIEF HISTORY OF ATTACHMENT THEORY
Inspired by the emotional troubles caused by mass child evacuations
in London, British psychologist John Bowlby began a long line of
research into child-parent bonds. Bowlby observed infants with their
primary caregivers (usually their mothers) and noted that the in-
fants grew upset when their mothers would leave, even temporarily.
Bowlby began writing about the bond that exists between children
and their parents and how disruptions of this bond can lead to emo-
tional problems later in the child's life.
It was one of John Bowlby's students, Mary Ainsworth, who pio-
neered research showing that different people can have different at-
tachment styles. In other words, one child will respond to his or her
parents in a very different way than another child. Ainsworth's re-
search, as well as the research of hundreds of people after her, showed
that, for the most part, there are three main attachment styles: secure,
anxious, and avoidant. Although modem researchers argue constantly
about what to call these styles and how to measure them, there is
a high level of agreement about what each style means in terms of
behaviors. These behaviors are linked to two key times in life: (l)
early childhood experiences with one's family, and (2) beginning
and-maybe-maintaining adult relationships. Because romantic
relationships are so important to our adult lives, the concentration
of research has been in applying the attachment style formed in a
person's early childhood to how that affects his or her romantic rela-
tionships (e.g., Hazan & Shaver).
A good way to understand the different styles of attachment is to
see how research psychologists measure them. Although many dif-
ferent measures exist, one of the most popular measures today is a
scale called "Experiences in Close Relationships" (Brennan, Clark, &
Shaver). Below is a brief version with ten sentences (the actual survey
is much longer). How much do you agree with each sentence?
73 76
Attachment Styles at Hogwarts 'C/(J 7 7
Anxiety I terns
l. I'm afraid that I will lose my partner's love.
2. I often worry that my partner will not want to stay with me.
3. I often worry that my partner doesn't really love me.
4. I often wish my partner's feelings for me were as strong as my
feelings for him/her.
5. When my partner is gone, I worry that he/she might become
interested in someone else.
Avoidance Items
l. I prefer not to show a partner how I feel, deep down.
2. I find it difficult to depend on romantic partners.
3. I don't feel comfortable opening up to romantic partners.
4. I get uncomfortable when a partner wants to be very close.
5. I prefer not to be too close.
The more sentences you agree with, the less secure you are. In
other words, if you agree with items l-5, you would be classified
as anxious. If you agree with items 6-10, you would be classified as
avoidant. If you don't agree with any of the items, you can rest as-
sured you are secure.
Although some people might argue that the children in Harry
Potter are too young to use as examples of people in romantic rela-
tionships, I believe they are a perfect opportunity to see young adults
attempting romance for the first time. How will they approach each
other? How do they attempt to form emotional bonds? How will they
end relationships? Fortunately for us, each of the three main charac-
ters is quite different, and each is an example of one of the three main
styles of attachment.
HERMIONE GRANGER: A CASE STUDY IN SECURITY
All three attachment styles follow the same principle: the behaviors,
self-esteem, and trust of others established in someone as a young
child will follow that person for the rest of his or her life. The first
attachment style identified by researchers is secure. Secure is, by far,
the most common of the three styles, emerging in about two-thirds
of children (Berscheid & Regan). This pattern or style is first estab-
74 77
78 "Cfb THE PSY-CHOLOGY ,.OF HARRY POTTER
lished when a child's parents provide reliable, steady, sensitive care-
parents who are consistently responsible and loving (Berscheid &:
Regan). Securely attached children are typically more socially gifted
and more competent in social situations. Secure children have high
self-esteem and are likely to tfl1St others.
Hermione is a great example of a secure individual. Admittedly,
we know the least about her early and family life, compared with the
other two main characters. We never see her at home, and she spends
little time at home past the age of eleven. However, what we do see is
a stable, normal, supportive relationship with both parents. We know
they are dentists who are not part of the magical world. However,
when Hermione receives her invitation to attend Hogwarts School
of Witchcraft and Wizardry, they recognize the opportunity for their
daughter and trust that she will be all right entering this new and
exciting world. They regularly accompany her to Diagon Alley for
school supplies and attempt to get to know her friends' parents. They
plan elaborate family trips during school breaks, such as to France
and a skiing resort.
We also see Hermione's love for her parents. Even though she is
often away, she regularly sends letters home and always thinks of
her parents in times of joy and sadness. For example, one of her first
thoughts after she is named prefect of Gryffindor is to send a message
to her parents. She is considerate of their feelings and knows they
will be proud: '"They'll be really pleased-! mean, prefect is some-
thing they can understand'" (Harry Potter and the Order of the Phoenix
165). Hermione is understanding of their basic ignorance of the mag-
ical world but confident in their support. Another example is seen
later in the same year. Hermione was planning to spend Christmas
skiing with her parents, but she decides to stay at Sirius Black's house
over the holidays after Mr. Weasley is injured. She explains to Harry,
"'Mum and Dad are a bit disappointed, but I've told them that every-
one who's serious about the [O.WL.] exams is staying at Hogwarts
to study. They want me to do well, they'll understand"' (Order of the
Phoenix 498). Clearly, they are willing to support and trust Hermione
in everything she does, while still showing her that they love her and
want to spend time with her. When her parents finally get to see her
at the end of the year on the train platform, we witness them "taking
it in turns to hug" her (Order of the Phoenix 868).
75 78
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Attachment Styles at Hogwarts 7 9
This is the parental pattern that results in a secure attachment
style: loving and supportive, while encouraging exploration of one's
own interests. As a result, Hermione displays classic secure behav-
iors. A nice comparison of Hermione, Ron, and Harry is seen at their
first meeting: the Hogwarts Express. Harry attempts to find a com-
partment by himself. Ron sits with him but complains about his fam-
ily and is embarrassed about being poor. What is Hermione doing?
She is helping Neville find his lost toad, confident and generous with
someone who clearly needs help. Just hours later, Harry and Ron are
both completely scared to try on the Sorting Hat that will decide their
House assignment, but Hermione is eager and confident, practically
running to the stool. And who can forget her secure confidence in all
of her classes, always the first person to raise her hand?
Hermione's general confidence is pretty clear. She does have mo-
ments of doubt (for example, waiting for her O.WL results), but her
"doubt" is over not receiving excellent grades in every class-includ-
ing Divination, which she quit! For attachment theory, however, we
must focus specifically on her romantic relationships and examine if
this security follows her into that area.
We see Professor Lockhart as Hermione's first crush. Like any
twelve-year-old girl with a crush, she exhibits typical girlish behav-
iors regarding the object of her affection. When Ron asks if she's out-
lined her class schedule with little hearts, she blushes. She is also
constantly defending him to Ron and Harry, who don't understand
the attraction. When she receives a get-well card from Lockhart, she
keeps it under her pillow. Later it is clear that she sent Lockhart a
valentine; when Ron asks her about it, she blushes and tries to avoid
the question. It is also in this book (Harry Potter and the Chamber of
Secrets) that we see the first seeds of a growing relationship between
Ron and Hermione, as Ron shows boyish jealousy over Hermione's
attentions to Lockhart. This jealous tendency will be a recurrent pat-
tern in Rons life (see below).
It is not until a couple of years later, however, that we see Hermione's
first official romantic relationship begin. (Note: this is two years ahead
of her friends Ron and Harry.) In Harry Potter and the Goblet of Fire, the
entire school is excited about the Yule Ball. It is clear that Hermione
would like Ron to ask her to the dance. However, secure people do not
wait around forever, pining after people who show no interest. When
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80 'Cfb THE PSYCHOLOGY OF HARRY POTTER
Ron doesn't ask her, Hermione moves on to someone who does appre-
ciate her: the famous and talented Viktor Krum. They spend the entire
year running into each other in the library, and originally Hermione
is annoyed by his presence. Eventually, however, she learns that they
have a lot in common and she easily strikes up a good relationship
with him. She has a great time at the ball until her fight with Ron.
The fight with Ron also shows us Hermione's character and at-
tachment style. Ron has spent the entire evening being rude and ob-
sessive (again, see below for a detailed analysis of Ron). At the end
of the evening, Hermione calls him on his cowardice and true feel-
ings: "'Well, if you don't like it, you know what the solution is, don't
you? ... Next time there's a ball, ask me before someone else does, and
not as a last resort!"' (Goblet of Fire 432). Hermione has feelings just
like any healthy person would. She is hurt by Ron's lack of attention
but brave enough to call 'him on it, and she has enough self-worth
to value a relationship partner who will value her equally. Hermione
continues to have a with Krum, mostly through letters,
but it is clear that their relationship is based on mutual interests and
respect for each other.
Throughout the next two years, Hermione's relationship with Ron
is mostly characterized by petty fighting. However, their fights are
obviously driven by feelings that Ron is not willing to admit and that
Hermione is not willing to push. She understands feelings much better
than either of her male friends and knows how to get to them. When
Ron begins his sickening relationship with Lavender, Hermione goes
on a date with McLaggen, Ron's competition for the Keeper posi-
tion on the Quidditch team. Predictably, Ron is driven crazy by this.
However, Hermione ends the relationship with McLaggen within a
few hours of their date; she is not willing to use another person for
her own selfish purposes, again showing her healthy relationship
patterns and understanding of emotional bonds between people. At
the end of Harry Potter and the Half-Blood Prince, she and Ron finally
seem to have resolved their issues, realizing that time is short in the
reflection of Dumbledore's funeral. However, their relationship was
much more stalled because of Ron's stupidity than because of any-
thing Hermione did. Ron's way of interacting with potential partners
is much different from Hermione's, and it is a perfect example of the
second major attachment style.
77 80
Attachment Styles at Hogwarts ~ 81
RON WEASLEY: A CASE STUDY IN ANXIETY
The second attachment style identified by researchers is called anx-
ious. Anxiety, in terms of attachment, has origins in parents who are
inconsistent in their caregiving-sometimes supportive and loving
and sometimes absent, distracted, or simply unnoticing of the needs
of their child (Berscheid & Regan). Children raised in this environ-
ment often have low self-esteem, with no confidence that the ones
they love will reciprocate. They seem to scare easily, meaning they are
often upset when thingsgo wrong, and they find it easier to pretend
to be angry or annoyed with the ones they love, when they desperate-
ly want to reunite and smooth things over. In short, the anxious at-
tachment style is characterized by people who have an extreme need
for closeness and attention from others and, at the same time, have a
constant fear of rejection.
It is easy to see how Ron's home life is characterized by instabil-
ity and distracted parents. With seven children, Mrs. Weasley un-
derstandably can't give each of them the attention a child needs. In
fact, when we see Mrs. Weasley interact with her children, it is only
in one of three modes: (1) warning them not to get into trouble, (2)
yelling at them when they have, inevitably, gotten into trouble, or
(3) crying/fussing over them when the trouble has caused injury or
potential death. Often her style of parenting is simply to berate them.
After Ron steals the flying family car and crashes it, she sends him a
Howler that screams at him in front of the entire school: '"I DON'T
SUPPOSE YOU STOPPED TO THINK WHAT YOUR FATHER AND
I WENT THROUGH ... ! THOUGHT YOUR FATHER WOULD DIE
OF SHAME ... ABSOLUTELY DISGUSTED ... "' (Chamber of Secrets
88). These are not words of loving support, but it's clear h ~ Ron has
her attention.
Ron is aware of his mother's challenges-in fact, when he first
meets Harry he's a bit embarrassed by her, and explains to him, "'[S]he
hasn't got much time ... with five of us [still at home]'" (Sorcerers
Stone 101). It was likely much worse when Ron was younger and all
seven children were home. It is clear that Mrs. Weasley loves her fam-
ily; she is just stretched too thin. For example, she hand-makes (or
wand-makes) sweaters for her children every year. However, she nev-
er notices or remembers that Ron hates maroon, the color he always
78 81
82 '()Z} THE PSYCHOLOGY OF HARRY POTTER
gets. This mixture of loving attention and lack of ability to focus on
detail is the hallmark of a parent with an anxious child. Ron's father
is equally tom in two directions, but we see this in his work life. Mr.
Weasley's entire essence is ambivalence: his job is to bust wizards for
misuse of Muggle objects, but his joy in life comes from doing that
very thing. He tries to manage the entire family but must constantly
be at work to support the nine of them, thus does not have any time
to actually spend with his family except on special occasions.
This lack of attention and quality time with either parent leads
directly to the most central trait of an anxious person: his or her
need for attention and a jealous tendency. We constantly see these
patterns in Ron throughout the series. Ron's ultimate goal is to be
the most popular and well-liked person at Hogwarts. What is the
evidence for this? In the very first book of the series, Ron and Harry
stand before the Mirror of Erised, which shows them their deepest,
darkest desires. Ron's vision is of himself, standing alone, as Head
Boy. He has won both the Quidditch and House cups, implying the
glory and fame that he seeks. Finally, Dumbledore explains to Harry
the importance of these visions: "Ronald Weasley, who has always
been overshadowed by his brothers, sees himself standing alone,
the best of all of them" (Harry Potter and the Sorcerers Stone 213).
Throughout their friendship, Ron is happy for Harry's successes, but
Ron also clearly desires the attention for himself. After Sirius Black
breaks into Hogwarts and seems to have attempted to kill Ron (he
was really trying to kill Ron's rat), Ron becomes an "instant celeb-
rity. For the first time in his life, people were paying more attention
to him than to Harry, and it was clear that Ron was rather enjoy-
ing the experience" (Harry Potter and the Prisoner of Azkaban 270).
Similarly, after Ron finally succeeds at Quidditch and wins the game,
all he can do is give Harry and Hermione (and anyone else nearby) a
blow-by-blow reenactment of the match. Even while trying to study
for O.WL.s, "he was thoroughly enjoying being patted on the back
by Gryffindors walking past his chair, not to mention the occasional
outburst of 'Weasley is our King"' (Order of the Phoenix 703). Ron is
clearly in his version of paradise with all this attention.
Importantly, attachment theory suggests that we should see these
patterns of jealousy and the desperate need for relationships in Ron's
romantic life. Again, attachment theory is supported throughout
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Attachment Styles at Hogwarts '-C/b 8 3
Ron's entire young life. He constantly attempts to get attention from
girls, but at the same time seems too scared to act on important feel-
lings because of his fear of rejection. We see this clearly in the turbu-
lent relationship between Ron and Hermione.
Ron's jealousy toward Hermione becomes obvious in his reaction
to seeing her crush on Lockhart: he's disgusted by her infatuation and
begs her to tell him that she didn't send Lockhart a valentine. We see
his jealousy again when Hermione goes to the ball with Krum. Ron
has no right to be jealous when he waited until the last minute to ask
Hermione (and then in a very rude way), but Ron's focus at the time
is simply getting the prettiest possible date in order to impress others.
He actually asks Fleur out, to everyone's dismay. Ron places no value
on Hermione as a possible relationship partner until someone else
desires her, thus inflating her public value. It drives Ron crazy that
Hermione and Krum continue their relationship after the ball.
What is Ron's way of dealing with this situation? To simply tell
Hermione that he has feelings for her? No-anxious people (accord-
ing to attachment theory) do not have the confidence in themselves
to believe that others will return their affection. As stated above, they
desperately crave loving relationships but don't have the self-esteem
necessary to maintain a trusting partnership. Thus, Ron's solution is
to date a non-threatening alternative: Lavender Brown. Lavender of-
fers no real threat to Ron because she is clearly infatuated with him,
and she is just kind of silly. Therefore, she provides both an easy
romantic relationship and a nice method of passive-aggressive action
toward Hermione, Ron's real object of desire. It becomes clear early
in the relationship, however, that Lavender isn't a good partner for
Ron. Another trait of anxious people, though, is that they cannot end
relationships. Even when in a bad relationship, anxious people are so
afraid of being alone that they cling to others. Ron therefore doesn't
end things-he just waits for Lavender to become so annoyed at his
lack of attention that she breaks things off.
Although his feelings seem obvious to the outside observer, Ron
::doesn't seem to have a grasp on them at all. He wants to be with
. Hermione, but he doesn't understand these cravings. Thus, this anx-
. ious conflict comes out in constant petty squabbles between Ron and
).iermione, over stupid things like Hermione's cat and her surprised
xeaciion to the news that he's been named a prefect. These fights con-
80 83
84 'C/?J THE PSYCHOLOGY OF HARRY POTTER
tinue for years. Finally, an implicit understanding takes place between
Ron and Hermione, but only after two tragedies. Ron is accidentally
poisoned in Half-Blood Prince, and while he's barely conscious in the
hospital ward, the only word he squeaks out is, "'Er-my-nee"' (Half-
Blood Prince 402). At the end of the book, Dumbledore's funeral
pushes them over the edge, and they cling to each other in misery
and grief. It seems that after six years of secret, unspoken yearning,
Ron finally understands his desires and acts upon them.
Importantly, attachment theory would predict that every one of
the Weasley children should have this need for attention. Bill works
in a bank (a seemingly mundane job), but he refuses to conform by
wearing an earring and a long ponytail. Charlie works with danger-
ous dragons. Percy desperately attempts to become the Minister of
Magic himself, or at least be connected to the most powerful people
in the wizarding world of bureaucracy. Percy is also annoyingly proud
of being a prefect and Head Boy. Fred and George's attempts to be the
center of attention are totally obvious. Finally, Ginny's first crush is
on the most popular boy in the school, and she quickly becomes
one of the most popular girls at Hogwarts. It seems the entire family
shares the traits of anxious individuals. Although anxious people cer-
tainly have a rocky road toward relationship bliss, their path seems
easy compared with our third and final attachment style: avoidance.
HARRY POTTER: A CASE STUDY IN AVOIDANCE
The last attachment style identified by researchers is called avoidant.
Avoidant children are often treated as strangers by their parents, and
vice versa. They don't act distressed when parents leave; often, ifs
more of a relief. In short, avoidant children don't attempt to make
strong bonds, they act indifferently about potential romantic partners,
and they consistently shut down their emotional life. Avoidant chil-
dren often don't like themselves much, and they have little or no trust
in others. Why should they? In essence, they have been abandoned
for most of their young lives. They are pessimistic and generally have
a pattern of purposeful isolation. We see all of these characteristics in
abundance in our main character, Harry Potter.
Harry's home life is, in a word, pitiful. When Harry's parents
are murdered when he is only a year old, Harry moves in with the
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Attachment Styles at Hogwarts '(J{) 8 5
Dursleys, his aunt and uncle. However, he is never treated as a family
member (more like a servant or a disobedient dog). In stark contrast
to their neglect of Harry, the Dursleys pamper their own son, Dudley,
to the point of absurdity. Dudley has two televisions, two bedrooms,
thirty-nine birthday presents, etc. Dudley is the face of indulgence
(both figuratively and literally). The direct result of this polarized
treatment of the children is that Harry learns to be resentful and that
others are not trustworthy. We see an example of this in the first
book, during their trip to the zoo. When Harry assures the Dursleys
that he'll behave, "Uncle Vernon didn't believe him. No one ever did"
(Sorcerer:S Stone 24). When it seems that things are going well, we see
the first signs of Harry's chronic pessimism: "Harry felt, afterwards,
that he should have known it was all too good to last" (Sorcerers
Stone 26). Harry simply never believes that good things will come his
way, especially from his interpersonal relationships. His lack of trust
and pessimism follow him like dark shadows in everything he does.
He doesn't believe Hagrid when he's told that he's a wizard. He
doesn't think he'll be able to afford his required school supplies. He
then thinks he'll be the worst in his classes, impossibly behind the
other children. When he's awaiting Sorting, he's sure he'll end up in
Hufflepuff or Slytherin. Finally, when he's actually under the Sorting
Hat, he's afraid that the Hat will decide he shouldn't be in any of the
Houses and simply kick him out of Hogwarts before he's even begun.
Note, all of these examples come before Harry has even gone to his
first day of schooL Ies clear that the Dursley home environment has
taught Harry to feel worthless and that nothing good will ever hap-
pen to him.
Although Harry's first year goes extremely well (i.e., he basically
saves the entire wizarding world by himselD, he still has no confi-
dence in others or in himself. During the first summer vacation after
his return, he quickly believes that Ron and Hermione aren't really
his friends. Later, when he has to pick his third-year school subjects,
Harry assumes he'll be "lousy" -at all of them, so he just picks what-
ever Ron picks (Chamber of Secrets 252). When trying to figure out
how to breathe underwater for the Triwizard Tournament, Harry pro-
crastinates until the last minute, then gives up the night before the
task: "It's over, he told himself. You can't do it" (Goblet of Fire 488).
As he ages, Harrys pessimism takes an ugly turn to resentfulness.
82 85
86 THE PSYCHOLOGY OF HARRY POTTER
This is another hallmark of avoidant people: their emotions are on
a negative track, and they tend to lash out at others, pushing them
away. At the beginning of Order of the Phoenix, Harry has spent his
entire summer glowering over the fact that Ron and Hermione get to
spend time together working against Voldemort, while Harry is home
with the Dursleys. Hany obsesses about how he's being completely
left in the dark, as their letters carefully avoid mentioning details:
He could hardly bear to think of the pair of them having fun at
the Burrow when he was stuck in Privet Drive. ln fact, he was so
angry at them he had thrown both their birthday presents away
unopened .... Hadn't he proven himself capable of handling much
more than they? ... The injustice of it all welled up inside him so that
he wanted to yell with fury ... his.insides writhed with anger (Order of
the Phoenix 8-10).
And Harry certainly does yell with fury, to anyone who will put
up with his whining for more than a few minutes. Harry alternates
between jealousy, laughing indignation, and guilt, but he seems inca-
pable of taking control of his emotions or understanding the perspec-
tives of others.
This leads Harry to the solace typical of avoidant people and the
classic behavior that leads to the name avoidant: purposeful isola-
tion. Even from the beginning, Harry avoids making new friends-
for example, he tries to find a solitary compartment on the Hogwarts
Express on his first day. The first time he tries out his Invisibility
Cloak, he consciously does not include Ron in the adventure, and
he earlier hid it when Fred and George burst into his room because
"[h] e didn't feel like sharing it with anyone else yet" (Sorcerers Stone
202). Before he battles with Quirrell and Voldemort, Hermione hugs
him for luck, but Harry doesn't hug back; he just stands there, un-
comfortable with her reaching out, both emotionally and literally.
Harry could certainly use more friends and support throughout his
time at Hogwarts, but he pushes away people like Colin Creevey, who
clearly worships Harry and would like to get close.
Although Ron is Harry's best friend, Harry doesn't do much to
maintain their relationship. When Harry's name mysteriously comes
out of the Goblet of Fire, Ron believes Harry submitted it, and Harry
83 86
Attachment Styles at -Hogwarts ~ 8 7
reacts to Ron's accusations with anger and insults. The fight contin-
ues for weeks, as Harry snarls and insults Ron at every opportunity.
When Ron accidentally walks in on Harry's secret conversation with
Sirius, causing it to end early, Harry "knew that Ron had no idea
what he'd walked in on, knew he hadn't done it on purpose, but he
didn't care-at this moment he hated everything about Ron" (Goblet
of Fire 335).
The point of attachment theory, however, is that Harry should
show these avoidant characteristics in his romantic relationships. It
is clear over and over again that Harry avoids romantic relationships,
preferring to silently fantasize with no action. Harry has a major, ma-
jor crush on classmate Cho Chang for years. However, all he ever
does is remain passive, never acting on his feelings unless forced.
The perfect opportunity to ask her out arrives with the Yule Ball, but
Harry puts off asking her until the last minute. When she tells him
that she's already agreed to go with someone else, Harry's reaction is
relatively bland-he just kind of mutters "okay" and heads off to be
by himself. He then asks the next person who happens to walk by,
clearly giving up. From that point on, he never initiates anything,
though he still pines for her. After their first D.A. meeting, Cho waits
behind the others, crying. It is she who arranges for them to be alone
together. It is she who points out the mistletoe, she who moves closer
to Harry, she who tells Harry that she really likes him. Finally, they
kiss, but only because (as Harry explains later), '"[S]he just kind of
.came at me'" O r d ~ r of the Phoenix 459). Harry is so stunned that he
does what he always does: nothing.
Again, it is she who makes a move to progress the relationship:
she approaches Harry regarding the trip to Hogsmeade on Valentine's
Day. He actually asks her out, but only accidentally! When they do
finally go out, Harry does nothing to make her feel comfortable or
.happy. She starts to cry again, and he does not comfort her. In fact,
he stupidly tells her that he's going to leave to meet Hermione. She
storms out, and does Harry chase her to explain? No-he only thinks
to himself that Cho closely resembles a hose, considering how much
water comes out of her. This is basically the end of their relationship,
caused by Harry's complete inability to d<? anything to maintain it.
Harrys first, and relatively successful, real romantic relationship
finally comes at the end of his last year at Hogwarts, with Ginny
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88 'CJb THE PSYCHOLOGY OF HARRY POTTER
Weasley. Importantly, although he's harbored secret desires for Ginny
for several months, he has again done absolutely nothing about it.
Again, it she who makes the first move, running at him to kiss him
after a successful Quidditch match. They are happy together for a very
brief time. However, as avoidant people usually do, it is Harry who
quickly ends the relationship. He tells Ginny that he won't be. able to
concentrate on his mission to kill Voldemort if she is there-that he
must do this alone. She accepts this, but note that Harry easily agrees
to be accompanied by Ron and Hermione. It is the romantic relation-
ship that Harry cannot maintain. According to attachment theory,
Harry's dismal early home life has caused him to enter a never-ending
pattern of relationship misery that he cannot escape.
THE FUTURE
Things look bad for Harry. Will he ever be able to initiate and main-
tain a happy romantic relationship? Attachment theory would argue
that this is impossible unless Harry can satisfy his needs for a stable
parental relationship, to fulfill his deep-seated needs for nurturance
and unconditional love. Is there hope for this?
Harry has had borderline parental relationships with three figures.
First, Harry clearly has a loving relationship with Hagrid. However,
Hagrid cannot offer the guidance that Harry needs, because he is
rather childlike himself and has his own parental issues to deal with
(including, most directly, his own mother's abandonment of him as
a child). The next opportunity comes with Sirius Black. When Sirius
and Harry finally meet and he understands that Sirius is not trying
to murder him, Harry instantly bonds to Sirius as his father's best
friend and as his own godfather. Clearly, Sirius could serve as a fa-
ther-figure. When Harry needs advice, he thinks of Sirius: "What he
really wanted ... was someone like-someone like a parent: an adult
wizard whose advice he could ask without feeling stupid, someone
who cared about him .... And then the solution came to him ... Sirius"
(Goblet of Fire 22). However, this does not work out. Sirius cannot
serve as a father substitute for Harry because Sirius is not grown up
himself. Sirius is still the boy who lost his best friend-he acts like a
child, reckless and easily angered, even daring Harry to do dangerous
things just for amusement. Unfortunately, Sirius's reckless tendencies
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Attachment Styles at Hogwarts 8 9
lead to his early death, leaving Harry, again, with no family to offer
loving support.
Harry's last chance at a parental figure comes with his idol and
distant mentor, Dumbledore. Throughout the years that Harry is at
Hogwarts, Dumbledore looks after Harry, guiding him and protecting
him. Whenever Harry is in trouble or his scar hurts, Ron and Hermione
encourage him to seek Dumbledore. However, Harry hesitates, believ-
ing that Dumbledore either won't believe him or that Dumbledore
would be disappointed in Harry for not having more control. It's clear
that Harry does not want to disappoint Dumbledore, just like chil-
dren avoid disappointing beloved parents. Harry has complete faith in
Dumbledore's abilities and that Dumbledore can save him in any situa-
tion. Dumbledore himself realizes that he has come to see Harry as a son
and admits to making mistakes in order to protect Harry. Dumbledore
tells him, '"I cared about you too much .... I cared more for your hap-
piness than your knowing the truth, more for your peace of mind than
my plan, more for your life than the lives that might be lost if the plan
failed"' (Order of the Phoenix 838). Sounds just like any good parent
protecting his child. Again, though, Harry's life is marked for misfor-
tune. The death of Dumbledore seals Harry's fate: He will never have a
long-term parental figure on whom he can draw support.
Attachment theory therefore predicts that Harry will never be able
to resolve this missing piece of his life, and that he will never be able
to maintain a healthy romantic relationship. It is not the ending we
would like. Perhaps Harry will form a successful romantic relation-
ship, perhaps with Ginny. It is possible that by observing other adult
relationships (such as one between Ron and Hermione) that Harry
can learn how to show love and support. It is possible that with a
great partner, Harry will have a relationship that will last a relatively
long time. However, it is the unfortunate prediction we must make
that in the end, Harry will live his life in basic isolation. Harry must,
inevitably, rely on himself for major missions and decisions in life.
Harry is a great wizard. But often, greatness is bred from childhood
strife and results in interpersonal loneliness and separation in adult-
hood. It is a reality of life that is not optimistic, that does not end
with "happily ever after." But, usually happiness is only temporary,
anyway. For Harry, quiet isolation, without the constant threat of
murder and betrayal, may be the best we can hope for.
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WIND GOODFRIEND, Ph.D., is an assistant professor
of psychology at Buena Vista University. She earned her
Ph.D. in social psychology in 2004 from Purdue University
in Indiana. Her areas of research expertise include gender
stereotypes and romantic relationships, focusing specifi-
cally on positive and negative .predictors of relationship
stability over time. In her final year of graduate school, Dr.
Goodfriend received both the "Outstanding Teacher of the
Year" ~ r d and the "Outstanding Graduate Student of the
Year" award for her research.
REFERENCES
Berscheid, E. and P. Regan. The Psychology of Interpersonal Relationships.
Upper Saddle River, NJ: Prentice Hall, 2005.
Brennan, K. A., C. L Clark, and P. R. Shaver. "Self-Report Measurement
of Adult Attachment: An Integrative Review." In ] . A. Simpson and W
S. Rholes (Eds.), Attachment Theory and Close Relationships. New York:
Guilford Press, 1998. 46-76.
C. Hazan and P. R. Shaver, "Romantic Love Conceptualized as an Attachment
Process," journal of Personality and Social Psychology 52 (1987): 511-
524.
Rowling,]. K. Harry Potter and the Sorcerers Stone. New York: Scholastic
Inc., 1998.
--. Harry Potter and the Chamber of Secrets. New York: Scholastic Inc.,
1999.
--. Harry Potter and the Prisoner of Azkaban. New York: Scholastic Inc.,
1999.
--. Harry Potter and the Goblet of Fire. New York: Scholastic Inc., 2000.
--. Harry Potter and the Order of the Phoenix. New York: Scholastic Inc.,
2003.
--. Harry Potter and the Half-Blood Prince. New York: Scholastic Inc.,
2005 ..
87 90
2012 Hellenic Society of Gastroenterology www.annalsgastro.gr
Endocrine and metabolic manifestations
in inflammatory bowel disease
Stelios Tigas, Agathocles Tsatsoulis
University of Ioannina, Greece
Annals of Gastroenterology (2012) 25, 1-8
Introduction
The two main forms of inflammatory bowel disease (IBD) are
ulcerative colitis (UC) and Crohns disease (CD). These disorders
are characterized by chronic inflammation of the gastrointestinal
(GI) tract and are defined by clinical, endoscopic, pathological
and radiographic features [1]. UC and CD commonly follow
a relapsing and remitting course and share a number of clini-
cal features such as diarrhea, rectal bleeding and abdominal
pain. Accumulating evidence shows that IBD results from an
inappropriate inflammatory response to intestinal microbes
in genetically susceptible individuals [2]. The inflammation
in UC is limited to the mucosa of the large intestine, typically
beginning in the rectum and occasionally extending proximally
to the sigmoid colon in a continuous fashion or, less commonly,
to the entire colon (pancolitis). In contrast, the inflammation
in CD involves all layers of the bowel wall and although the
disease most commonly affects the terminal ileum and the
large bowel, any part of the GI tract from mouth to anus may
be affected, not necessarily in a continuous way [1].
Extraintestinal manifestations from nearly every organ
system are frequent in IBD, occurring in 20-40% of patients
[3,4]. Systems commonly involved are the skin, musculoskel-
etal system and eyes, but the hepatopancreatobiliary, nervous,
cardiovascular, renal and respiratory systems may also be
affected [4-8]. This article reviews the endocrine and meta-
bolic manifestations of IBD focusing on their epidemiology,
pathogenesis, as well as their diagnosis and management by
the practicing physician.
Metabolic bone disease
Epidemiology and pathogenesis
The reported prevalence of osteoporosis (defined as T-
score <-2.5) in patients with established IBD varies widely
from 17 to 41% [9-11] with an overall prevalence of 15% [10].
The prevalence of osteopenia (T-score -1 to -2.5) varies from
22 to 67% [9]. The variation in prevalence rates is a result of
differences in population characteristics, age, disease dura-
tion, dual energy x-ray absorptiometry (DXA) methodology
and study design. Patients with IBD have an estimated 40%
higher risk of fracture compared to the general population that
increases further with age [10], or if asymptomatic vertebral
fractures are taken into account [12].
The causes of reduced bone mineral density (BMD) in
IBD are multifactorial; apart from the common risk factors
such as age, smoking and low body mass index (BMI), other
Department of Endocrinology, University of Ioannina, Greece
Confict of Interest: None
Correspondence to: Stelios Tigas, MD, PhD, MRCP,
Dept of Endocrinology, University of Ioannina,
Ioannina 45110, Greece,
Tel.: +30 26510 08089, fax: +30 26510 08098,
e-mail: stigas@cc.uoi.gr
Received 7 September 2011; accepted 5 December 2011
INVITED REVIEW
Abstract Extraintestinal manifestations from nearly every organ system are common in inflammatory
bowel disease (IBD). This review article describes the epidemiology, pathogenesis, diagnosis
and management of the main endocrine and metabolic manifestations in IBD, including
metabolic bone disease, growth retardation, hypogonadism, pubertal delay, lipid abnormalities
and insulin resistance. These clinical problems are commonly interrelated and they share a
common basis, influenced by disease-related inflammation and nutritional status. In addition
to nutritional support, every effort should be made to achieve and maintain disease remission,
thus correcting the underlying chronic inflammation. The criteria for screening and diagnosing
osteoporosis are described and treatment options are discussed (lifestyle advice, vitamin D and
calcium supplementation, use of bisphosphonates or other specific antiosteoporotic agents,
correction of hypogonadism). Chronic glucocorticoid therapy may affect growth as well as
predispose to osteoporosis. The diagnosis and management of growth failure, pubertal delay
and hypogonadism in IBD are discussed.
Keywords Inflammatory bowel disease, osteoporosis, growth failure, hypogonadism, lipids,
insulin resistance
Ann Gastroenterol 2012; 25 (1): 1-8
88 91
2 S. Tigas, A. Tsatsoulis
Annals of Gastroenterology 25
factors are glucocorticoid (GC) use, nutritional deficiencies
including vitamin D and K, malabsorption of calcium, hypo-
gonadism and finally, disease-related chronic inflammation
(Table 1) [13]. In addition to reducing bone formation and
increasing bone resorption, GCs have been shown to reduce
absorption of calcium in the small intestine and increase ex-
cretion of calcium by the kidneys. Vertebral fractures (often
asymptomatic) may affect 30-50% of patients on chronic GC
therapy; the risk of bone loss and fractures is already consider-
able in the first few months of therapy due to a phase of early
accelerated bone resorption which is then followed by a more
progressive phase of impaired osteoblastogenesis. Although
fracture risk increases with larger doses and prolonged length
of GC treatment, fractures may occur at prednisone equivalent
doses as low as 2.5 7.5 mg daily [14,15]. Since patients with
more severe or active disease are more likely to receive GC
treatment, it is difficult to estimate the relative contribution of
(a) GC therapy or (b) disease-related inflammation, on BMD.
Vitamin D levels are lower than those of healthy individuals
in adult and pediatric patients with IBD [16]. The prevalence
of vitamin D deficiency (defined as serum 25-OH vitamin
D concentration of 15 ng/mL) ranges from 22 to 70% for
CD, and up to 45% for UC [16]. The reasons for the reduced
vitamin D levels include impaired absorption (especially in
CD patients who had small bowel resection) [17], reduced
intake [18], lack of exposure to sunlight, altered metabolism
[19] and loss through the GI tract when protein-losing en-
teropathy develops [16]. Serum and bone levels of vitamin
K (a cofactor in the carboxylation of osteocalcin, a protein
essential for calcium binding to bone) are reduced in patients
with IBD and are inversely related to the rate of bone resorp-
tion in CD [20,21]. Vitamin K deficiency in IBD may be
due to malabsorption or alterations in vitamin K-producing
bacterial flora. Finally, calcium absorption may be impaired
in IBD patients with steatorrhea, because of calcium binding
to intraluminal fat. As a result, patients are predisposed to
hyperoxaluria and renal stones, since calcium normally binds
oxalate in the lumen facilitating oxalate extraction. When free
intraluminal calcium is decreased as a result of steatorrhea,
oxalate absorption increases [22].
Although it is clear that all factors described above contrib-
ute to osteoporosis in IBD, there are a number of patients with
reduced BMD or osteoporosis who have adequate vitamin D
levels and were never treated with GCs [23]. These observa-
tions, together with evidence from studies in experimental
animals [24] suggest that disease-related chronic inflammation
may play a role in the development of osteopenia or osteopo-
rosis in adult and pediatric [25] IBD patients. It is possible that
cytokines such as interleukin (IL)-6, IL-1 and tumor necrosis
factor alpha (TNF), directly impair bone metabolism by
increasing bone resorption. Interestingly, treatment with
infliximab, an anti-TNF agent, has been shown to improve
markers of bone metabolism and BMD in IBD patients [26].
Recent evidence suggests that alterations in the RANKL
(receptor activator of nuclear factor kappa-B ligand): OPG
(osteoprotegerin) ratios may be implicated in IBD-related
bone loss [9,27].
Diagnosis and evaluation
Osteoporosis can be diagnosed either when there is a his-
tory or evidence of fragility fractures (regardless of BMD), or
according to the BMD as measured by DXA. The American
Gastroenterological Association recommends DXA screening
of IBD patients with one or more risk factors for osteoporosis
and fracture: history of previous fragility fracture, postmeno-
pausal, male >50 years, corticosteroid therapy for more than 3
months, or hypogonadism [10]. The British Society of Gastro-
enterology (BSG) recommends DXA screening of IBD patients
with continuing active disease, weight loss >10%, body mass
index <20 kg/m
2
, age >70 years, and those <65 years requiring
corticosteroids for >3 months [28]. BMD can be expressed as
the number of standard deviations (SD) above or below either
the mean BMD for young adults (T-score) or the mean BMD
for age-matched controls (Z-score). According to the World
Health Organization (WHO), osteoporosis in peri- and post-
menopausal women or men >50 years of age can be defined as a
T-score at the spine or hip of <-2.5 SD [29]. A T-score of between
-1 and -2.5 is defined as osteopenia. For premenopausal women
or men <50 however, Z-scores, not T-scores should be used;
a Z-score -2.0 should be interpreted as below the expected
range for age [30]. If BMD is within the normal range, DXA
should be repeated in 2-3 years time. In patients with abnormal
BMD measurements, further clinical and laboratory evaluation
is required to exclude secondary causes of osteoporosis (such as
primary hyperparathyroidism, hyperthyroidism, hypogonadism
or Cushings syndrome) and to guide treatment. Investigations
should include measurement of serum calcium, phosphate, al-
bumin, total protein, creatinine, liver enzymes including alkaline
phosphatase, electrolytes, 25-hydroxyvitamin D and complete
blood count. When clinically indicated, thyroid stimulating
hormone (TSH), free T4, testosterone (in men), estradiol (in
women), prolactin, LH and FSH should also be measured.
The FRAX (Fracture Risk Assessment Model) is a web-based
tool that can help physicians to assess the 10-year probability
of major osteoporotic and hip fracture by calculating a score
Table 1 Risk factors of reduced bone mineral density in inflammatory
bowel disease
Age
Smoking
Low body mass index, malnutrition
Previous fragility fracture
Chronic glucocorticoid use
Nutritional defciencies (vitamin D, vitamin K)
Malabsorption of calcium
Hypogonadism
Disease-related chronic infammation
89 92
Endocrine and metabolic manifestations in IBD 3
based on a number of clinical risk factors, and may therefore
assist in making treatment decisions [31,32].
Treatment
The main treatment aim is prevention of fractures. All
patients should receive lifestyle-advice on: prevention of falls,
adequate calcium and vitamin D intake, avoidance of excess
alcohol intake and smoking cessation, regular weight-bearing
exercise. An attempt should be made to control the underly-
ing disease and maintain remission. If necessary, calcium and
vitamin D supplements should be prescribed to ensure a daily
calcium intake of 1000 mg in younger patients and 1200 - 1500
mg in postmenopausal women and men >55 and a daily vita-
min D intake of 400 - 800 IU. Larger amounts of vitamin and
calcium may be necessary in CD patients with extensive small
bowel disease. Hypogonadal patients should receive hormone
replacement. Premenopausal women with IBD and amenorrhea
are usually treated with combined estrogen-progestin replace-
ment (an oral contraceptive pill or transdermal estradiol plus
oral progestin administered cyclically). Because GCs may cause
hypogonadism by suppressing gonadotrophins, men who are
on long-term GC treatment should have periodic assessments
of their morning serum testosterone (and gonadotrophins)
and be put on testosterone replacement should they become
hypogonadal [33]. GC use should be minimized when pos-
sible by using alternative therapies (appropriate/early use of
immunomodulators, budesonide [15], elemental or polymeric
diet, surgery for resistant disease). All IBD patients receiving
GCs should be given calcium and vitamin D supplements.
Bisphosphonate prophylaxis should be used (a) for those >65
years at commencement of GC, or (b) for those <65 who are due
to receive GCs for more than three months and their T score is
<-1.5 [28]. Specific antiosteoporotic treatment is indicated for
IBD patients with: (a) fragility fractures or a T-score of <-2.5 (b)
T-scores of -2.5 to -1.0 plus long-term GC therapy or presence of
other additional risk factors (Fig. 1) [10,28]. Most of the evidence
regarding treatment options comes from studies conducted
in postmenopausal women who did not have IBD or patients
on GC-induced osteoporosis. Patients may be treated with an
oral bisphosphonate (e.g. weekly alendronate or risedronate) or
with 3-monthly intravenous ibandronate or yearly intravenous
zolendronic acid for those unable to take oral bisphosphonates
or if compliance is an issue. If bisphosphonates are poorly tol-
erated or ineffective, alternative agents are strontium ranelate,
raloxifene in postmenopausal women, teriparatide (by daily
subcutaneous injections), calcitonin by intranasal spray and
denosumab [10,28]. These agents have not been specifically
studied in IBD. In young men and in premenopausal women
with osteoporosis (who are not hypogonadal and therefore sex
hormone replacement therapy is not indicated) bisphosphonates
should be used with caution as there are insufficient efficacy and
safety data in this group of patients and there are potential risks
during pregnancy [34] and with long-term use (osteonecrosis
of the jaw, atypical femoral fractures) [35,36].
Growth failure
Pathogenesis - epidemiology
In almost 1 of 4 patients with IBD, the disease presents in
childhood, mostly during puberty [37]. Only 12% of children with
CD have normal height velocity at diagnosis and up to 46% have
Figure 1 Management algorithm of osteoporosis in inflammatory bowel disease (IBD) [adapted from reference 10].
DXA, dual energy x-ray absorptiometry; BMD, bone mineral density
90 93
reduced height velocity before the onset of any symptoms [38]. In
contrast, a significantly smaller percentage of children with UC
(3-10%) have reduced height velocities at the time of diagnosis
[39]. Children with IBD often present with delayed puberty,
weight loss or inability to gain weight, whereas a decrease in
height velocity may occur at a later stage. A significant proportion
(1937%) of patients who had CD during childhood fail to reach
their expected adult height [40,41]. The pathogenesis of growth
failure in children with IBD is multifactorial, the most important
factors being disease-related inflammation, malnutrition, GC use
and hypogonadism [42]. The growth hormone (GH) insulin-
like growth factor (IGF)-1 axis is impaired in IBD resulting in
a state of relative GH resistance, manifested by low IGF-1 and
IGFBP-3 levels and reduced growth [43,44]. This process may
be mediated via proinflammatory cytokines such as IL-6 and
IL-1, although other factors such as under-nutrition or the use
of GCs may also impair GH secretion and growth plate function
[43-45]. Indirect evidence that disease-related inflammation may
affect growth comes from studies in children with resistant CD
who underwent complete surgical resection of affected bowel
segment; following surgery there is an increase in resting energy
expenditure and significant catch up growth [45,46]. Apart from
surgery, significant improvements in linear growth have been
observed following treatment with infliximab [47,48] or exclusive
enteral nutrition [37,49]. There is no doubt that malnutrition is a
major factor for growth failure and that appropriate nutritional
support and management may reverse growth retardation in these
children [50]. Factors contributing to malnourishment include
reduced intake (cytokine induced anorexia, avoidance of food due
to fear of exacerbation of symptoms), increased resting energy
expenditure (REE), malabsorption of fat, protein, vitamins and
micronutrients, gastritis, esophagitis. Chronic GC therapy may
affect growth by impairing a number of processes essential for
normal growth such as endogenous GH secretion and action,
bone and collagen formation, IGF-1 binding in cartilage and
nitrogen retention [41,51]. It is however difficult to estimate the
GC effect on growth since disease severity/activity, anatomic
location and other clinical parameters are potential confound-
ing factors; hence, not all studies have confirmed an association
between GC use and growth failure in IBD [40,52,53].
Diagnosis of growth failure /Assessment of growth and nu-
tritional status
Monitoring at regular intervals of growth and nutritional
status of children with IBD, is essential and should include a
number of anthropometric and other parameters as described
in Table 2 [37,54]. Additionally, in children with growth
failure, measurement of IGF-1, thyroid hormones, TSH and
assessment of bone age are indicated.
Management
Attempts to improve growth rate in children with IBD
should focus on achieving and maintaining disease remission
to minimize disease-related inflammation. Systematic assess-
ment of growth failure is recommended, with the help of a
pediatric endocrinologist. The use of long-term GC therapy
should be minimized, if possible [37]. In addition, all children
should receive appropriate specialist nutritional advice and
support. Children with growth failure have increased energy
requirements (125-150% of the recommended daily allowance
for energy, and 2.4 to 3 g/kg per day of protein) and those with
malabsorption may require supplementation of vitamins and
minerals [54,55]. Supplemental enteral nutrition in the form
of nocturnal feeding via nasogastric tube or exclusive enteral
nutrition may be necessary in more severe cases, especially
those children with intolerable GC-related side effects and/or
growth failure [37,54,56-58]. As already mentioned, several
interventions including surgery and immunomodulators
such as infliximab, have been shown to have a positive effect
on growth in children with IBD [45-48]. In a relatively small
study in children with CD, 6-mercaptopurine (6-MP) (whose
parent drug is azathioprine) was effective and reduced the
need for steroids but had no effect on growth after 18 months
compared with conventional prednisone treatment [59].
Growth hormone therapy should be considered in children
with severe growth failure when other measures fail, as it has
been shown to increase height velocity, improve BMD [60]
Table 2 Assessment of growth and nutritional status in inflammatory
bowel disease
Anthropometric data
Height, weight and body mass index (BMI; weight in kg/height
in m2) measurement*.
Documentation of pre-morbid height and weight if possible.
Calculation of mid-parental height/centiles
Assessment of bone age at diagnosis and annually, if growth delay
Calculation of height velocity at 46-month intervals
Calculation of height and weight Z scores for age and BMI
Assessment of pubertal stage
Tanner staging and assessment of testicular volume (using an
orchidometer) at 6-month intervals
Assessment of osteoporosis
DXA scan to assess BMD
Dietetic assessment
Calorie intake, calcium and vitamin D sources, micronutrients
Monitoring for anemia, malabsorption and nutritional
defciencies
Complete blood count, albumin, 25-OH vitamin D, prothrombin
time, vitamin B12, iron, ferritin, and if indicated folate, calcium,
magnesium, phosphorus, vitamins A and E, zinc, selenium
*It is important that anthropometric data and growth parameters are plotted
and followed longitudinally on appropriate growth charts.
BMI, body mass index; DXA, dual energy x-ray absorptiometry; BMD, bone
mineral density
91 94
and induce anabolic effects even in GC-dependent children
with CD [61]. If GH is to be used, it is generally more effec-
tive when administered in the early rather than in the late
puberty [44].
Hypogonadism, pubertal delay and fertility
issues
Hypogonadism is not uncommon in both male and female
patients with IBD, and may be the result of (a) a direct effect of
inflammation/cytokines on the reproductive axis, ovarian and
testicular function [62-64], (b) undernutrition and reduced
leptin levels [65] and (c) the effect of chronic GC treatment
on gonadotrophin secretion [33] (as already discussed in the
paragraph discussing treatment of osteoporosis).
An association between the bone formation marker osteo-
calcin and testosterone levels in men with CD was reported
by Robinson et al in 1998 [66]. A more recent study con-
firmed lower testosterone and estradiol levels in men with
CD compared to controls, but failed to show any association
with BMD or markers of bone metabolism [67]. Nevertheless,
hormone replacement should be considered in hypogonadal
IBD patients with osteoporosis, especially young men and
premenopausal women. When IBD presents during child-
hood, hypogonadism and pubertal delay are common (in CD
more often than in UC) [68]. These children have reduced
linear growth and delayed accretion of lean body mass com-
pared to their healthy peers. Because children with pubertal
delay usually have delayed bone age, some catch-up growth
is possible after the onset of puberty. Studies report average
delays in puberty onset about 1.5 years for girls and 0.8 years
for boys [69]. Management involves nutritional support and
appropriate treatment of the underlying disease-related in-
flammation. Boys with IBD and pubertal delay may be treated
with testosterone therapy; controlled studies of testosterone
use in IBD are however needed [68].
Fertility is not generally impaired in men and women
with IBD compared to the general population [70]. Sexual
dysfunction appears to be mostly related to depression [71].
However, subfertlity may be encountered under certain
circumstances such as in men who develop impotence fol-
lowing proctocolectomy, men with sulfasalazine-induced
oligospermia (reversible, not observed with 5-ASA agents)
and women who have undergone surgery [72].
Lipid abnormalities and insulin resistance
Although factors associated with atherosclerosis such as
inflammation, carotid intima media thickness (cIMT), homo-
cysteine levels and insulin resistance are commonly increased
in IBD patients, low density lipoprotein cholesterol and total
cholesterol levels are generally low [73]. These changes are
more pronounced in CD compared to UC and they are pres-
ent independently to disease activity, possibly mediated by
cytokine production. LDL cholesterol is lower compared to
healthy individuals, even after bowel resection. This has been
attributed to malabsorption of bile acids leading to parallel
stimulation of cholesterol synthesis, cholesterol degradation
and LDL receptor expression in the liver, the net effect being
a significant reduction in LDL cholesterol [74]. Hypocholes-
terolemia is a common feature of various types of acute illness
such as sepsis, trauma and surgery and has been related to the
severity of the illness. In addition, lipoprotein (a) levels are
increased and apolipoprotein A-1 and apolipoprotein B-100
are reduced in CD, possibly predisposing CD patients to a
higher risk of thrombosis [75].
In a previous study from Italy in patients with IBD during
remission, indirect calorimetry was employed to assess the
basal metabolic rate and substrate oxidation and the euglycemic
hyperinsulinemic clamp technique was used to measure insulin
sensitivity; these parameters were similar in the study group
(inactive CD or UC) compared to healthy controls, whereas
basal lipid oxidation was higher in patients with CD compared
to both UC and healthy controls [76]. In contrast, during ac-
tive CD there is increased insulin resistance as well as insulin
secretion (based on the plasma insulin response during a 75
g oral glucose tolerance test) [77]. Anti-TNF- therapy with
infliximab did not alter insulin resistance in patients with ac-
tive IBD whereas it increased total and HDL cholesterol levels
and apo-A1 [78]. Despite the recent discovery of numerous
shared susceptibility loci/genes, there is no epidemiological
evidence to support an association between IBD and either
type 2 or type 1 diabetes [79]. Insulin resistance, hypergly-
cemia, and overt diabetes may however be a consequence of
GC therapy in IBD. GC-induced hyperglycemia results from
an increased rate of hepatic gluconeogenesis, inhibition of
glucose uptake in adipose tissue, or impairment of insulin
action at the receptor and post-receptor level [80]. The risk of
developing GC-induced diabetes depends on the dose used,
the age, BMI and genetic predisposition. Clinically signifi-
cant hyperglycemia is generally treated in the same way as it
is treated in type 2 diabetes (with dietary modification, oral
anti-diabetic agents and/or insulin if needed).
Conclusion
Recent evidence suggests that changes in the endocrine
milieu play an important role in the pathogenesis of IBD
[81-86]. Adipokines such as leptin, adiponectin and resistin
are involved in a number of processes that characterize IBD
including anorexia, malnutrition, altered body composition
and mesenteric white adipose tissue hypertrophy [83]. Animal
studies suggest that expression of peroxisome proliferator-
activated receptor (PPAR ) in intestinal epithelial cells as
well as in macrophages and T cells of mice with experimental
IBD, exhibit immunoregulatory actions and may be involved
in preventing gut inflammation [84,87]. On the basis of such
observations, thiazolidinediones (drugs that have been widely
92 95
used to treat type 2 diabetes) have recently been tried in IBD
with interesting results [85,88].
It is becoming clear that the endocrine system is involved
in the pathogenesis and clinical manifestations of IBD in
several ways. The main endocrine manifestations of IBD
described in this brief review article (metabolic bone disease,
growth failure, hypogonadism, pubertal delay and changes in
lipid and carbohydrate metabolism) are interrelated and their
multifactorial pathogenesis is influenced by disease-related
inflammation and nutritional status. To achieve best results,
interventions need to target the underlying mechanisms; it is
essential that in addition to nutritional support and specific
therapies for osteoporosis, growth failure and hypogonadism,
a continuous effort is made to control the underlying chronic
inflammation by achieving and maintaining disease remission.
References
1. Xavier RJ, Podolsky DK. Unraveling the pathogenesis of
inflammatory bowel disease. Nature 2007;448:427-434.
2. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med
2009;361:2066-2078.
3. Bernstein CN, Blanchard JF, Rawsthorne P, Yu N. The prevalence
of extraintestinal diseases in inflammatory bowel disease: a
population-based study. Am J Gastroenterol 2001;96:1116-1122.
4. Levine JS, Burakoff R. Extraintestinal manifestations of
inflammatory bowel disease. Gastroenterol Hepatol (NY) 2011;
7:235-241.
5. Rothfuss KS, Stange EF, Herrlinger KR. Extraintestinal
manifestations and complications in inflammatory bowel diseases.
World J Gastroenterol 2006;12:4819-4831.
6. Voulgari PV. Rheumatological manifestations in inflammatory
bowel disease. Ann Gastroenterol 2011;24:173-180.
7. Katsanos KH, Tsianos EV. The kidneys in inflammatory bowel
disease. Ann Gastroenterol 2002;15:41-52.
8. Karamanolis GP. Cutaneous and ocular manifestations of IBD.
Ann Gastroenterol 2006;19:181-183.
9. Ali T, Lam D, Bronze MS, Humphrey MB. Osteoporosis in
inflammatory bowel disease. Am J Med 2009;122:599-604.
10. Bernstein CN, Leslie WD, Leboff MS. AGA technical review
on osteoporosis in gastrointestinal diseases. Gastroenterology
2003;124:795-841.
11. van Hogezand RA, Hamdy NA. Skeletal morbidity in inflammatory
bowel disease. Scand J Gastroenterol Suppl 2006;243:59-64.
12. Siffledeen JS, Siminoski K, Jen H, Fedorak RN. Vertebral fractures
and role of low bone mineral density in Crohns disease. Clin
Gastroenterol Hepatol 2007;5:721-728.
13. Bernstein CN, Leslie WD. The pathophysiology of bone disease in
gastrointestinal disease. Eur J Gastroenterol Hepatol 2003;15:857-
864.
14. Mazziotti G, Angeli A, Bilezikian JP, Canalis E, Giustina A.
Glucocorticoid-induced osteoporosis: an update. Trends
Endocrinol Metab 2006;17:144-149.
15. Schoon EJ, Bollani S, Mills PR, et al. Bone mineral density in
relation to efficacy and side effects of budesonide and prednisolone
in Crohns disease. Clin Gastroenterol Hepatol 2005;3:113-121.
16. Pappa HM, Grand RJ, Gordon CM. Report on the vitamin D
status of adult and pediatric patients with inflammatory bowel
disease and its significance for bone health and disease. Inflamm
Bowel Dis 2006;12:1162-1174.
17. Leichtmann GA, Bengoa JM, Bolt MJ, Sitrin MD. Intestinal
absorption of cholecalciferol and 25-hydroxycholecalciferol in
patients with both Crohns disease and intestinal resection. Am
J Clin Nutr 1991;54:548-552.
18. Jowett SL, Seal CJ, Phillips E, Gregory W, Barton JR, Welfare MR.
Dietary beliefs of people with ulcerative colitis and their effect
on relapse and nutrient intake. Clin Nutr 2004;23:161-170.
19. Liu N, Nguyen L, Chun RF, et al. Altered endocrine and autocrine
metabolism of vitamin D in a mouse model of gastrointestinal
inflammation. Endocrinology 2008;149:4799-4808.
20. Schoon EJ, Mller MC, Vermeer C, Schurgers LJ, Brummer
RJ, Stockbrgger RW. Low serum and bone vitamin K status in
patients with longstanding Crohns disease: another pathogenetic
factor of osteoporosis in Crohns disease? Gut 2001;48:473-477.
21. Duggan P, OBrien M, Kiely M, McCarthy J, Shanahan F, Cashman
KD. Vitamin K status in patients with Crohns disease and
relationship to bone turnover. Am J Gastroenterol 2004;99:2178-
2185.
22. Kappelman MD, Bousvaros A. Nutritional concerns in pediatric
inflammatory bowel disease patients. Mol Nutr Food Res
2008;52:867-874.
23. Bjarnason I, Macpherson A, Mackintosh C, Buxton-Thomas
M, Forgacs I, Moniz C. Reduced bone density in patients with
inflammatory bowel disease. Gut 1997;40:228-233.
24. Lin CL, Moniz C, Chambers TJ, Chow JW. Colitis causes bone loss
in rats through suppression of bone formation. Gastroenterology
1996;111:1263-1271.
25. Paganelli M, Albanese C, Borrelli O, et al. Inflammation is the
main determinant of low bone mineral density in pediatric
inflammatory bowel disease. Inflamm Bowel Dis 2007;13:416-423.
26. Veerappan SG, OMorain CA, Daly JS, Ryan BM. Review article:
the effects of antitumour necrosis factor- on bone metabolism
in inflammatory bowel disease. Aliment Pharmacol Ther
2011;33:1261-1272.
27. Moschen AR, Kaser A, Enrich B, et al. The RANKL/OPG system
is activated in inflammatory bowel disease and relates to the state
of bone loss. Gut 2005;54:479-487.
28. Lewis, NR, Scott, BB. Guidelines for Osteoporosis in Inflammatory
Bowel Disease and Coeliac Disease. London: British Society
of Gastroenterology 2007; Available at: http://www.bsg.org.uk/
clinical-guidelines/ibd/guidelines-for-osteoporosis-in-inflammatory-
bowel-disease-and-coeliac-disease.html
29. Kanis JA, Melton LJ 3rd, Christiansen C, Johnston CC, Khaltaev
N. The diagnosis of osteoporosis. J Bone Miner Res 1994;9:1137-
1141.
30. Lewiecki EM. Premenopausal bone health assessment. Curr
Rheumatol Rep 2005;7:46-52.
31. Kanis JA, Johnell O, Oden A, Johansson H, McCloskey E. FRAX
and the assessment of fracture probability in men and women
from the UK. Osteoporos Int 2008;19:385-397.
32. Goodhand JR, Kamperidis N, Nguyen H, Wahed M, Rampton DS.
Application of the WHO fracture risk assessment tool (FRAX)
to predict need for DEXA scanning and treatment in patients
with inflammatory bowel disease at risk of osteoporosis. Aliment
Pharmacol Ther 2011;33:551-558.
33. Odell WD. Testosterone treatment of men treated with
glucocorticoids. Arch Intern Med 1996;156:1133-1134.
34. Djokanovic N, Klieger-Grossmann C, Koren G. Does treatment
with bisphosphonates endanger the human pregnancy? J Obstet
Gynaecol Can 2008;30:1146-1148.
35. Khosla S, Burr D, Cauley J, et al. Bisphosphonate-associated
osteonecrosis of the jaw: report of a task force of the American
Society for Bone and Mineral Research. J Bone Miner Res
2007;22:1479-1491.
36. Shane E, Burr D, Ebeling PR, et al. Atypical subtrochanteric
and diaphyseal femoral fractures: report of a task force of the
American Society for Bone and Mineral Research. J Bone Miner
93 96
Res 2010;25:2267-2294.
37. Heuschkel R, Salvestrini C, Beattie M, Hildebrand H, Walters T,
Griffiths A. Guidelines for the Management of Growth Failure
in Childhood Inflammatory Bowel Disease. Inflamm Bowel Dis
2008;14:839-849.
38. Kanof ME, Lake AM, Bayless TM. Decreased height velocity in
children and adolescents before the diagnosis of Crohns disease.
Gastroenterology 1988;95:1523-1527.
39. Hildebrand H, Karlberg J, Kristiansson B. Longitudinal growth
in children and adolescents with inflammatory bowel disease. J
Pediatr Gastroenterol Nutr 1994;18:165-173.
40. Sawczenko A, Ballinger AB, Savage MO, Sanderson IR. Clinical
features affecting final adult height in patients with pediatric-
onset Crohns disease. Pediatrics 2006;118:124-129.
41. Markowitz J, Grancher K, Rosa J, Aiges H, Daum F. Growth failure
in pediatric inflammatory bowel disease. J Pediatr Gastroenterol
Nutr 1993;16:373-380.
42. Shamir R, Phillip M, Levine A. Growth retardation in pediatric
Crohns disease: pathogenesis and interventions. Inflamm Bowel
Dis 2007;13:620-628.
43. Katsanos KH, Tsatsoulis A, Christodoulou D, Challa A, Katsaraki
A, Tsianos EV. Reduced serum insulin-like growth factor-1 (IGF-
1) and IGF-binding protein-3 levels in adults with inflammatory
bowel disease. Growth Horm IGF Res 2001;11:364-367.
44. Savage MO. Growth-promoting hormone therapy in inflammatory
bowel disease. JPGN 2010;51:S135-S136.
45. McLain BI, Davidson PM, Stokes KB, Beasley SW. Growth after
gut resection for Crohns disease. Arch Dis Chil 1990;65:760-762.
46. Varille V, Czard JP, de Lagausie P, et al. Resting energy expenditure
before and after surgical resection of gut lesions in pediatric
Crohns disease. J Pediatr Gastroenterol Nutr 1996;23:13-19.
47. Walters TD, Gilman AR, Griffiths AM. Linear growth improves
during infliximab therapy in children with chronically active
severe Crohns disease. Inflamm Bowel Dis 2007;13:424-430.
48. Malik S, Wong SC, Bishop J, et al. Improvement in growth of
children with crohn disease following anti-TNF- therapy can be
independent of pubertal progress and glucocorticoid reduction.
J Pediatr Gastroenterol Nutr 2011;52:31-37.
49. Newby EA, Sawczenko A, Thomas AG, Wilson D. Interventions
for growth failure in childhood Crohns disease. Cochrane Database
Syst Rev 2005;(3):CD003873.
50. Kirschner BS, Klich JR, Kalman SS, deFavaro MV, Rosenberg
IH. Reversal of growth retardation in Crohns disease with
therapy emphasizing oral nutritional restitution. Gastroenterology
1981;80:10-15.
51. Allen DB. Growth suppression by glucocorticoid therapy.
Endocrinol Metab Clin North Am 1996;25:699-717.
52. Griffiths AM, Nguyen P, Smith C, MacMillan JH, Sherman PM.
Growth and clinical course of children with Crohns disease. Gut
1993;34:939-943.
53. Motil KJ, Grand RJ, Davis-Kraft L, Ferlic LL, Smith EO. Growth
failure in children with inflammatory bowel disease: a prospective
study. Gastroenterology 1993;105:681-691.
54. Kleinman RE, Baldassano RN, Caplan A, et al. Nutrition
support for pediatric patients with inflammatory bowel
disease: a clinical report of the North American Society for
Pediatric Gastroenterology, Hepatology And Nutrition. J Pediatr
Gastroenterol Nutr 2004;39:15-27.
55. Oliva MM, Lake AM. Nutritional considerations and management
of the child with inflammatory bowel disease. Nutrition
1996;12:151-158.
56. Wilschanski M, Sherman P, Pencharz P, Davis L, Corey M,
Griffiths A. Supplementary enteral nutrition maintains remission
in paediatric Crohns disease. Gut 1996;38:543-548.
57. Aiges H, Markowitz J, Rosa J, Daum F. Home nocturnal
supplemental nasogastric feedings in growth-retarded adolescents
with Crohns disease. Gastroenterology 1989;97:905-910.
58. Moeeni V, Day AS. Impact of inflammatory bowel disease upon
growth in children and adolescents. ISRN Pediatrics 2011:365712.
59. Markowitz J, Grancher K, Kohn N, Lesser M, Daum F. A multicenter
trial of 6-mercaptopurine and prednisone in children with newly
diagnosed Crohns disease. Gastroenterology 2000;119:895-902.
60. Heyman MB, Garnett EA, Wojcicki J, et al. Growth hormone
treatment for growth failure in pediatric patients with Crohns
disease. J Pediatr 2008;153:651-658.
61. Mauras N, George D, Evans J, et al. Growth hormone has
anabolic effects in glucocorticosteroid-dependent children
with inflammatory bowel disease: a pilot study. Metabolism
2002;51:127-135.
62. Azooz OG, Farthing MJ, Savage MO, Ballinger AB. Delayed
puberty and response to testosterone in a rat model of colitis.
Am J Physiol Regul Integr Comp Physiol 2001;281:R1483-R1491.
63. Hales DB, Diemer T, Hales KH. Role of cytokines in testicular
function. Endocrine 1999;10:201-217.
64. Terranova PF, Rice VM. Review: cytokine involvement in ovarian
processes. Am J Reprod Immunol 1997;37:50-63.
65. Warren MP, Voussoughian F, Geer EB, Hyle EP, Adberg CL, Ramos
RH. Functional hypothalamic amenorrhea: hypoleptinemia and
disordered eating. J Clin Endocrinol Metab 1999;84:873-877.
66. Robinson RJ, Iqbal SJ, Al-Azzawi F, Abrams K, Mayberry JF.
Sex hormone status and bone metabolism in men with Crohns
disease. Aliment Pharmacol Ther 1998;12:21-25.
67. Klaus J, Reinshagen M, Adler G, Boehm B, von Tirpitz C. Bones
and Crohns: estradiol deficiency in men with Crohns disease
is not associated with reduced bone mineral density. BMC
Gastroenterol 2008;8:48.
68. Ballinger AB, Savage MO, Sanderson IR. Delayed puberty
associated with inflammatory bowel disease. Pediatr Res
2003;53:205-210.
69. Brain CE, Savage MO. Growth and puberty in chronic inflammatory
bowel disease. Baillieres Clin Gastroenterol 1994;8:83-100.
70. Hudson M, Flett G, Sinclair TS, Brunt PW, Templeton A, Mowat
NA. Fertility and pregnancy in inflammatory bowel disease. Int
J Gynaecol Obstet 1997;58:229-237.
71. Timmer A, Bauer A, Dignass A, Rogler G. Sexual function in
persons with inflammatory bowel disease: a survey with matched
controls. Clin Gastroenterol Hepatol 2007;5:87-94.
72. Peppercorn MA. Fertility, pregnancy, and nursing in inflammatory
bowel disease. In: UpToDate, Basow, DS (Ed), UpToDate, Waltham,
MA, 2011.
73. Agouridis AP, Elisaf M, Milionis HJ. An overview of lipid
abnormalities in patients with inflammatory bowel disease.
Ann Gastroenterol 2011;24:181-187.
74. Akerlund JE, Reihner E, Angelin B, et al. Hepatic metabolism
of cholesterol in Crohns disease. Effect of partial resection of
ileum. Gastroenterology 1991;100:1046-1053.
75. Koutroubakis IE, Malliaraki N, Vardas E, et al. Increased levels
of lipoprotein (a) in Crohns disease: a relation to thrombosis?
Eur J Gastroenterol Hepatol 2001;13:1415-1419.
76. Capristo E, Mingrone G, Addolorato G, Greco AV, Gasbarrini
G. Glucose metabolism and insulin sensitivity in inactive
inflammatory bowel disease. Aliment Pharmacol Ther 1999;13:209-
217.
77. Bregenzer N, Hartmann A, Strauch U, et al. Increased insulin
resistance and beta cell activity in patients with Crohns disease.
Inflamm Bowel Dis 2006;12:53-56.
78. Koutroubakis IE, Oustamanolakis P, Malliaraki N, et al. Effects
of tumor necrosis factor alpha inhibition with infliximab on lipid
levels and insulin resistance in patients with inflammatory bowel
disease. Eur J Gastroenterol Hepatol 2009;21:283-288.
79. Lees CW, Barrett JC, Parkes M, J Satsangi. New IBD genetics:
common pathways with other diseases. Gut 2011;60:1739-1753.
94 97
80. van Raalte DH, Ouwens DM, Diamant M. Novel insights into
glucocorticoid-mediated diabetogenic effects: towards expansion
of therapeutic options? Eur J Clin Invest 2009;39:81-93.
81. Peyrin-Biroulet L, Chamaillard M, Gonzalez F, et al. Mesenteric
fat in Crohns disease: a pathogenetic hallmark or an innocent
bystander? Gut 2007;56:577-583.
82. Dubuquoy L, Rousseaux C, Thuru X, et al. PPARgamma as a
new therapeutic target in inflammatory bowel diseases. Gut
2006;55:1341-1349.
83. Karmiris K, Koutroubakis IE, Kouroumalis EA. Leptin,
adiponectin, resistin, and ghrelin--implications for inflammatory
bowel disease. Mol Nutr Food Res 2008;52:855-866.
84. Hontecillas R, Horne WT, Climent M, et al. Immunoregulatory
mechanisms of macrophage PPAR- in mice with experimental
inflammatory bowel disease. Mucosal Immunol 2011;4:304-313.
85. Lewis JD, Lichtenstein GR, Deren JJ, et al. Rosiglitazone for
Ulcerative Colitis Study Group. Rosiglitazone for active ulcerative
colitis: a randomized placebo-controlled trial. Gastroenterology
2008;134:688-695.
86. Valentini L, Wirth EK, Schweizer U, et al. Circulating adipokines
and the protective effects of hyperinsulinemia in inflammatory
bowel disease. Nutrition 2009;25:172-181.
87. Mohapatra SK, Guri AJ, Climent M, et al. Immunoregulatory
actions of epithelial cell PPAR gamma at the colonic mucosa of
mice with experimental inflammatory bowel disease. PLoS One
2010;5:e10215.
88. Lund JL, Strmer T, Porter CQ, Sandler RS, Kappelman MD.
Thiazolidinedione use and ulcerative colitis-related flares: an
exploratory analysis of administrative data. Inflamm Bowel Dis
2011;17:787-794.
95 98

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