Fish 12

455
CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
12
Enteric Redmouth Disease
(Yersinia ruckeri)
M.T. Horne
1
and A.C. Barnes
2
1
Aqua Health (Europe) Ltd, Unit 31, Enterprise House, Springkerse Business
Park, Stirling FK7 7UF, UK;
2
FRS Marine Laboratory, PO Box 101, Victoria
Road, Aberdeen AB11 9DB, UK.
INTRODUCTION
Enteric redmouth (ERM) is an acute or chronic infection of fish caused by
Yersinia ruckeri, a Gram-negative, enteric bacterium. It is present in a carrier
state in many species of fish and remains undetected until stress, particularly
associated with intensive culture and poor water quality, may result in heavy
losses requiring immediate intervention.
Yersinia ruckeri was first isolated in the Hagerman Valley, Idaho, USA,
in the early 1950s and was described fully by Ross et al. (1966) and Rucker
(1966). The early terminology used to describe the disease, such as
redmouth, redthroat or bacterial septicaemia, was derived from the gross
clinical signs, which in many cases were caused by mixed infections of
Gram-negative organisms. In other cases, where Y. ruckeri was isolated and
almost certainly the causative agent, they were absent. Standardization as
enteric redmouth was made by the Fish Health Section of the American
Fisheries Society in 1975 and this, with yersiniosis, remains the
commonest name.
The placement of the causative agent in the genus Yersinia (Ewing et al.,
1978) has also proved problematic, but this remains the accepted identifier.
Since its isolation in the Hagerman Valley, ERM has either spread or been
recognized in most areas of Europe and the USA where freshwater fish are
cultured intensively. It may be controlled to acceptable levels by good
husbandry, particularly high water quality, and it responds well to antibiotics and
vaccines. Even so, it presents a constant threat, requiring management
procedures and treatments which erode the profitability of the farms on which it
is endemic. Losses of 1015% over a growth cycle are not uncommon and
individual outbreaks may result in much higher mortalities. In small scale,
artificial challenge trials, where no intervention takes place, over 90% mortality
may occur.
456
M.T. Horne and A.C. Barnes
THE DISEASE AND AGENT
Species of fish affected and geographical distribution
The initial isolation and description of ERM was from farmed rainbow trout
Oncorhynchus mykiss (Ross et al., 1966; Rucker, 1966), but has since been
isolated from many species of fish, marine and freshwater, and from other
animals (Table 12.1). Environmental samples from river water, from sewage and
from dairy situations have also been found to be positive (Pritchard et al., 1995).
The majority of clinical disease conditions occur in intensively cultured
salmonids and it is considered primarily a disease of salmonids (Busch, 1982),
but a number of other families of fish have also been found to be infected. In
those species where isolations have been made without the presence of clinical
signs, it is probable that such species would succumb to infection if sufficient
husbandry stress were applied.
Following the first recognition of Y. ruckeri in Idaho, other workers began to
identify the pathogen elsewhere. McDaniel (1971) recorded its presence in sites
covering most of the Rocky Mountain area of the western USA, including
Alaska; Wobeser (1973) showed its presence in Canada. Much of this early
apparent spread was probably genuine and resulted from the dissemination of
fish throughout this area, in the absence of strict controls and monitoring
schemes. Its introduction to Europe in 1983 and its subsequent spread probably
occurred in the same way, and it is now known to be present over large areas of
the USA and Europe (Denmark, France, Germany, Italy, Norway and UK). It is
also present in Australia (Bullock et al., 1978; Green and Austin, 1982), South
Africa (Bragg and Henton, 1986) and Chile, all of which received imported
salmonid eggs and fry from these areas. However, there is some variation in the
strains and serotypes within the genus associated with geographical areas and it
cannot be ruled out that it is of a more widespread occurrence in a natural
manner than was at first thought.
The disease
The early signs of yersiniosis in the acute phases of infection are typical of many
other Gram-negative bacterial septicaemias, and anorexia, darkening of the skin
and lethargy almost always precede the disease, except in small fry, where
virtually asymptomatic deaths may occur (Kawula et al., 1996). The reddening
of the throat and mouth, caused by subcutaneous haemorrhaging and from which
the disease received its name, are commonly, but not invariably, present. If the
disease progresses without treatment, erosion of the jaw and palate may occur.
Haemorrhage occurs on the body surface, at the gill tips, at the base of the
fins and around the lateral line. Rarely, these may progress to ulcers. The vent
area may also become inflamed, both externally and internally, at the distal end
of the intestine. Exophthalmia has been reported in some cases in the later stages
of infection, with haemorrhaging of the ocular cavity and iris (Fuhrmann et al.,
1983). This has been referred to as a separate condition in Australian trout
457 Enteric Redmouth Disease
(Llewellyn, 1980). Internally, there is congestion of the blood-vessels
throughout the peritoneum, and petechial haemorrhages, affecting liver,
pancreas, swim-bladder, lateral muscles and adipose tissues associated with the
pyloric caecae (Wobeser, 1973). The kidney and spleen may be swollen and
there may be fluid in both the stomach and the intestine, where it has a yellowish,
opaque, mucoid appearance (Busch, 1982). Light microscopy shows bacteria in
Table 12.1. Species from which Yersinia ruckeri has been isolated.
Species Common name Reference
Salmonids
Oncorhynchus kisutch Coho salmon McDaniel (1971)
Oncorhynchus mykiss Rainbow trout Rucker (1966)
Oncorhynchus nerka Sockeye salmon Dulin et al. (1976)
Oncorhynchus tschawytscha Chinook salmon McDaniel (1971)
Salmo clarkii Cutthroat trout McDaniel (1971)
Salmo salar Atlantic salmon McDaniel (1971)
Salmo trutta Brown trout McDaniel (1971)
Salvelinus alpinus Arctic char McDaniel (1971)
Salvelinus fontinalis Brook trout McDaniel (1971)
Non-salmonids
Acipenser baeri Sturgeon Vuillaume et al. (1987)
Anguilla anguilla Eel Fuhrmann et al
(1984)
Aristichthys nobilis Bighead carp Xu et al. (1991)
Carassius auratus Goldfish McArdle and
Dooley-Martin (1985)
Coregonus artedii Cisco Stevenson and Daly
(1982)
Coregonus peled Whitefish Rintamaki et al. (1986)
Coregonus muksun Whitefish Rintamaki et al. (1986)
Cyprinus carpio Common carp Fuhrmann et al
(1984)
Hypophthalmichthys molitrix Silver carp Xu et al. (1991)
Ictalurus punctatus Channel catfish Lewis (1981)
Lota lota Burbot Dwilow et al. (1987)
Notemigonus atherinoides Emerald shiner Mitchum (1981)
Pimephales promelas Minnow Michel et al. (1986)
Scophthalmus maximus Turbot Michel et al. (1986)
Solea solea Sole Michel et al. (1986)
Other animals
Human Farmer et al. (1985)
Otter Collins et al. (1996)
Ondatra zibethica Muskrat Stevenson and Daly
(1982)
Kestrel Bangert et al. (1988)
Turkey vulture Bangert et al. (1988)
Eisenia foetida Earthworm Dales and Kalac
(1992)
Gulls Willumsen (1989)
458
virtually all tissues, particularly the kidney, heart, liver and gills. There may be
severe necrosis of the haemopoietic tissues of the kidney.
Wobeser (1973), Quentel and Aldrin (1986) and Lehman et al. (1987)
observed acute anaemia, with an average haematocrit as low as 23% and total
serum-protein values of 2.8 g 100 ml
1
. Miller (1983) attributed this to the effects
of endotoxin on coagulation ultimately producing thrombosis in the capillaries
and generalized haemorrhaging.
Many potentially pathogenic Gram-negative organisms persist in small
numbers in the intestine of apparently healthy fish and remain undetected in
standard health checks. Viable bacteria detected in the lymphoid tissues of fish,
however, indicate that the health of the fish has been compromised, even though
no external indicators of poor condition may be noticeable. Yersinia ruckeri
behaves in this manner and is sometimes described as establishing a carrier
state although, in the strictest definition of this phrase, this is arguable.
Throughout the farm cycle, periodic stress factors may move the balance of
advantage towards the pathogen, as the performance of the immune system and
other physiological factors are reduced to suboptimal levels. Fish carrying
Y. ruckeri become sluggish in behaviour, reduce diet intake and may have a
darkening of the skin. Factors known to promote subclinical and clinical
infection by Y. ruckeri are, predictably, handling, grading and excessive stocking
densities. When infection occurs in the absence of these factors, suspended
organic matter in the water, coupled with high temperatures and consequentially
low oxygen, along with exposure to low levels of chemicals in the water-supply,
may promote clinical disease (Bullock and Snieszko, 1975; Knittel, 1981). In
conditions where Y. ruckeri is endemic, therefore, the farm cycle is characterized
by rapid changes in the external appearance of the stock and persistent low
mortalities from ERM. Correction of the environmental factor(s) responsible
may restore the fish to health, although an antibiotic may be needed to reduce the
level of loss if condition has deteriorated too far. Acute infections, if not rapidly
checked, can vary between 30 and 70% of the stock. As the fish become larger,
chronic, slower infections are more characteristic, but these too may reach
epizootic proportions if mishandled.
The causative agent
Placement of the causative organism in the genus Yersinia has always been
controversial (Austin and Austin, 1993). Ross et al. (1966) initially placed it in
the enterococci based on traditional morphological and biochemical attributes,
but within this group its allocation to either Yersinia or Serratia was uncertain.
Serological and biochemical studies had also indicated relationships with
Arizona (= Salmonella) arizonae (Ewing et al., 1978) and Stevenson and Daly
(1982) found serological cross-reactions with Hafnia alvei. Deoxyribonucleic
acid (DNA) homology indicated an approximately equal relationship to Yersinia
enterocolitica (3031%), Yersinia pseudotuberculosis (3031%), Serratia
marcescens (2428%) and Serratia liquefaciens (31%). Whereas the latter two
have been isolated from fish-farm situations and occasionally suspected of
causing disease, neither Yersinia species has been associated with fish health
problems. Green and Austin (1982) showed that there was a greater phenotypic
relationship between Y. ruckeri and S. arizonae than with either Y. enterocolitica
or Y. pseudotuberculosis, and Bercovier and Mollaret (1984) suggested that
Y. ruckeri might be more properly considered a new species of
Enterobacteriaceae. Its elevation to species status and designation as Yersinia
ruckeri was made by Ewing et al. (1978), and its acceptance in this genus was
strengthened by analysis of the DNA base composition (Ewing et al., 1978).
Yersinia ruckeri, at 47.548%, is clearly different from Serratia (5260%) and S.
arizonae (5053%) (de Grandis et al., 1988), whereas it is close to those of the
other yersiniae (4650%). Nevertheless, its placement with the other yersiniae,
on the basis of DNA hybridization, biochemical attributes and lifestyle, still
appears anomalous and a new genus would be perhaps justified (Bercovier and
Mollaret, 1984), except that it exists as a single, relatively homogeneous set of
isolates. The use of more modern, molecular techniques to study the taxonomic
relationships, for example, the study of the 16S ribosomal DNA of Ibrahim et al.
(1993) and the basic genetic analysis by Romalde et al. (1991b, 1993), will
undoubtedly increase knowledge of the detailed genetic relationships of Y.
ruckeri to other taxa. However, as in other areas of bacterial taxonomy, this may
not necessarily give any clearer a picture of its true position.
The basic biochemical attributes have been described by many authors
(Ross et al., 1966; Wobeser, 1973; Bullock et al., 1978; Ewing et al., 1978;
OLeary et al., 1979; Busch, 1982; Stevenson and Daly, 1982; Bercovier and
Mollaret, 1984; Hastings and Bruno, 1985; Ewing, 1986; Davies and Frerichs,
1989; Austin and Austin, 1993).
Yersinia ruckeri cells are Gram-negative, rod-shaped, slightly curved, 1.0
m in diameter and 23 m in length. Cells cultured for 48 h or longer produce
long filamentous cells. Approximately 80% of isolates are motile, using seven or
eight peritrichously arranged flagella but, at 9C or lower, the flagella, although
present, fail to function; at 35C they are absent and the cells are not motile
(OLeary, 1977). Cells cultured longer than 48 h are poorly motile. The common
biochemical attributes considered characteristic are shown in Table 12.2. One of
the most thoroughly reported attributes of Y. ruckeri is the ability to ferment
sorbitol. OLeary (1977) reported that this trait was characteristic of serovar II
and could be used to distinguish it from serovar I. However, subsequent studies
have demonstrated that some sorbitol fermenting isolates cross-react
serologically with serovar I. Indeed, 32% of strains tested ferment sorbitol (de
Grandis et al., 1988), with Austin and Austin (1993) suggesting that this trait
may be plasmid-mediated.
Although the biochemical attributes of isolates from different areas are
mostly uniform, serological variation exists, based upon lipopolysaccharide
(LP) types, O-antigens recognized in rabbit sera or whole-cell serological
reactions. Serological variation is complex, and numerous methods have been
applied, resulting in conflicting nomenclature. A recent review by Stevenson et
al. (1993) provides some clarification. Originally, five major serovars were
recognized, based on whole-cell reactions and described as type I (Hagerman),
the most virulent, type II (OLeary), avirulent; type III (Australian), avirulent;
460
Table 12.2. Biochemical characteristics of Yersinia ruckeri.
Substrate/test Reaction Notes
Adonitol
Arabinose
Cellobiose
Dulcitol
Erythritol
Esculin
Galactose
Glucose + 17% produce gas
Glycerol +(70%); +d (15%) Reversible
Inositol
Lactose
Maltose +
Mannitol +
Mannose +(97%)
Melibiose
Methyl red +(79%)
Mucate
Raffinose
Rhamnose
Salicin
Sorbitol +(25%); +d (75%) See text
Starch
Sucrose
Trehalose +(97%)
Triple sugar iron K/A Alkaline slant, acid
butt
Xylose
Enzymatic reactions
Aesculinase
Arginine dihydrolase +(59%); +d (41%)
Beta-galactosidase (ONPG) +
Caseinase +(51%)
Catalase +
Chitinase
Chondroitin sulphatase
Cytochrome oxidase
Deoxyribonuclease
Elastinase
Fibrinolase
Gelatinase +(52%); +d (41%); (6%)
Hyaluronidase
Lecithinase +(81%)
Lipase: Tween 20 +
Lipase: Tween 40 +
Lipase: Tween 60 +
Lipase: Tween 80 +(86%)
Lipase: Tween 85 +(74%)
Lysine decarboxylase +(88%)
Ornithine decarboxylase +
Pectinase
Phenylalanine deaminase
serovar IV, avirulent; and serovar V, avirulent (Stevenson and Airdrie, 1984;
Daly et al., 1986). Later work showed that many infections were associated with
type II, which, in some instances, is as virulent as type I. Further complication
arose following the identification of the Australian salmon blood spot bacterium
(Llewellyn, 1980) as Y. ruckeri (de Grandis et al., 1988), which has now been
included in serovar III, although there is cross reaction with serovar I. Current
nomenclature, based on whole-cell serology, is summarized in Table 12.3
(adapted from Stevenson et al., 1993). Six whole-cell serovars (IVI) have been
proposed, although DNA homology analysis has discounted serovar IV.
However, these major whole-cell serotypes may be further subdivided,
according to the methodology applied. Lipopolysaccharide profiles obtained by
electrophoresis and immunoblot have provided a further basis for classification,
and six O-serogroups, based on LPS O-antigen, have been identified (Table
12.4) (Stevenson et al., 1993). Cross-reaction between whole-cell serovars I and
II appears to result from similar LPS profiles, although serological differences
are evident when absorbed sera are used (Stevenson et al., 1993). An additional
scheme for serological classification has been proposed by Romalde et al.
(1993), based on studies using absorbed sera and antigenic determinants (LPS
Phosphatase
Ribonuclease
Tributyrinase
Urease
Other reactions
Citrate utilization (Simmons) +(3%); +d (94%); (3%)
Growth in KCN +(24%)
Haemolysis + Alpha
Hydrogen sulphide production On triple sugar iron
Indole production
Motility +(82%)
Nitrate reduction +(85%); +d (12%); (3%)
Nitrite reduction
Table 12.2. Continued
Substrate/test Reaction Notes
Table 12.3. Whole-cell serotypes of Yersinia ruckeri (after Stevenson et al.,
1993).
Serovar Designation Notes
I Hagerman Cross-reacts with III and V
II Oregon Includes at least two further groups as
determined using absorbed sera
III Australian Includes I (salmonid blood spot)
IV Now excluded by DNA homology
V Colorado
VI Ontario
462
and membrane proteins). In this study, four serogroups were suggested (Table
12.5), incorporating the six predominant serovars proposed by Stevenson et al.
(1993). Regardless of serotype, DNA restriction fingerprinting showed that
Y. ruckeri is highly homogeneous (Romalde et al., 1993). However, plasmid
profiling showed that only isolates of the proposed serogroup O1 contained a 50
MDa plasmid (Romalde et al., 1993). Further complication of serological
nomenclature arises with the Norwegian classification of Y. ruckeri into serogroups
I, II and III. Many Norwegian isolates appear to be related to serovar I based on
LPS profiles, but differ serologically (Ibrahim et al., 1993). Furthermore, cross-
protection studies suggest antigenic similarities between Norwegian serogroup III
and Hagerman serovar I, while Norwegian serogroups II and III may differ
(Erdal, 1989). An interesting and thorough attempt at defining clonal groups for
Y. ruckeri, similar to those proposed for Escherichia coli was proposed by
Davies (1991a), based on serology, biotyping and outer membrane protein
(OMP) profiling. Six clonal groups were proposed, based on five OMP types and
two biotypes, using isolates of Y. ruckeri from diverse geographical origins
(Davies, 1991a). It is difficult in a short review to thoroughly evaluate the
numerous methods that have been reported for the intraspecies classification of
Y. ruckeri. Each method has its merits, but in all cases further research has
identified exceptions and anomalies; consequently, this remains an area
requiring further study.
Table 12.4. Lipopolysaccharide O-antigen serotypes of Yersinia ruckeri
(after Stevenson et al., 1993).
O-antigen serogroup Whole-cell serovars
O:1 I, III
O:2 IIa
O:3 IIb
O:4 IIc
O:5 V
O:6 VI
Table 12.5. New scheme of serotyping proposed by Romalde et al. (1993).
New serotype Subgroup Former serovar
O1 a I
b III
O2 a II
b II
c II
O3 n/a V
O4 n/a VI
n/a, not applicable.
DIAGNOSTIC METHODS
Provisional diagnosis of yersiniosis in fish is often made on the basis of
experience at a particular site, combined with the clinical signs seen in the fish.
However, as indicated above, these are typical of many other Gram-negative,
septicaemial infections; even the frequently cited reddening of the mouth may be
absent and, in any case, is seen in other types of infection. Additionally, small
fish may die virtually asymptomatically.
Confirmation of yersiniosis is most easily made by culture from tissues,
particularly the spleen, heart and kidney. Widely available media, such as
tryptone soya agar (TSA), sometimes supplemented with 5% blood, are the most
effective. The use of a preculture procedure, where the tissue suspected to be
infected is incubated in liquid media (tryptone soya broth (TSB) or TSB + blood)
at 18C for up to 48 h may increase the number of positives obtained from a
population (Daly and Stevenson, 1985). If required, confirmation can be
obtained by non-lethal sampling (Noga et al., 1988). The regular cyclical
shedding of Y. ruckeri which has been reported to occur from the intestinal tract
of carrier fish, however, may hinder isolation (Busch and Lingg, 1975).
Attempts have been made to produce diagnostic media to aid culture
(Waltman and Shotts, 1984; Rodgers, 1991b), but, as with most diagnostic
selective media, growth of the target organism may be reduced in addition to the
desirable reduction in growth of other species. Additionally, the requirement of
this medium that Tween 80 should be hydrolysed is not met by many isolates
(Austin and Austin, 1993), complicating the concept of its use. More recently,
differentiation based on virulence characteristics has been used successfully
(Furones et al., 1993), but, in general, standard TSA is effective and preferred for
isolation (Hastings and Bruno, 1985; Rodgers and Hudson, 1985).
Identification by the determination of attributes (see Table 12.2) uses
traditional methods, and laboratory minikits, such as API 20E, are also used
successfully (Santos et al., 1993). However, additional tests may be required
here if an exact definition of the species is required, since closely related species,
such as H. alvei, would not be differentiated by this method.
The use of immunodiagnostic methods has matured to the point where these
may also be described as traditional. Choice may be made between monoclonal
and polyclonal reagents in the protocols of these methods, although, for
commercial purposes, monospecific polyclonals are more robust and problem-
free. Antigen detection, using specific antibodies in enzyme-linked
immunosorbent assay (ELISA) formats, has been used in both research and
commercially in plate and rapid, on-stick formats. Antibody detection by latex-
agglutination tests has been used to determine subclinical infections, both
experimentally (Johnson et al., 1974; Hansen and Lingg, 1976) and
commercially (Romalde et al., 1995).
The most recent method of detection and identification, based on the
development of specific molecular probes and polymerase chain reaction (PCR),
is capable of detecting extremely low levels of Y. ruckeri (Argenton et al., 1996).
However, this laboratory method is not used routinely and its sensitivity
generally restricts its use to special, screening situations. Problems arise in
464
validation of this technique and in judging the relevance of the positives
obtained to farm husbandry situations (Hiney and Smith, 1998).
CONTROL AND TREATMENT
Transmission of the disease
Early work and descriptions of the disease were made by those working in
salmonid aquaculture, leading to the concept of yersiniosis as primarily a disease
of salmonids. Confirmed clinical outbreaks were seen in both trout (rainbow,
steelhead, cutthroat, brown and brook) and in salmon (coho, sockeye and
Atlantic) (Busch, 1982). Early epizootics in these species have been described in
detail by Ross et al. (1966), Rucker (1966) and Busch (1973). More recently, it
has been recognized that the host range of Y. ruckeri is more diverse, and a list of
the species from which it has now been isolated is given in Table 12.1. Whether
it is capable of a saprophytic existence as a part of the environmental flora
outside a host is a matter of debate; Rucker (1966) suggested that this may be the
case, but later authors, such as Klontz and Huddlestone (1976), argued against
this. Their findings reflected those seen for many other Gram-negative
coliforms, including E. coli and Aeromonas salmonicida, where the survival in
clean water in a laboratory experiment may be a matter of hours, but may be
prolonged for 23 months in sediments or where there is organic matter present
(Romalde et al., 1994). This allows transmission from fish to fish under natural
conditions without the need to propose that the organism has lived
saprophytically outside an animal host.
The prime source of infection is generally considered to be the shedding of
large numbers of bacteria from carrier or infected fish in the faeces. Such fish do
not normally shed quantities capable of causing infection, unless stressed
(Hunter et al., 1980). The level of carrier fish in a population depends on the
criteria and methods used. Busch and Lingg (1975) demonstrated that, 45 days
postinfection, 25% of a population of rainbow trout carried Y. ruckeri
asymptomatically in the lower intestine. The preculture/culture method used in
this case is sensitive and the data may reflect a situation in fish following the first
serious epizootic in a population. However, where a detailed investigation is
made, using culture, immunological and molecular methods together, in a
population grown under normal farm conditions for several months, a much
higher proportion of individuals is found to be positive and it is not improbable
that all are actually carrying the pathogen. Once infected, therefore, a fish
population can maintain the pathogen indefinitely.
Yersinia ruckeri has also been isolated from brood stock, which are
frequently, if not always, infected, but transmission through eggs where
disinfection has been properly carried out has not been reported (Dulin et al.,
1976).
Conditions predisposing populations to clinical infection relate primarily to
stress. Healthy laboratory populations can withstand exposure to high numbers
of cells without succumbing to clinical disease (Ross et al., 1966). Infection may
occur where fish are obese through poor feeding regimes, but poor water quality
is the prime cause. Common causes are high ammonia, due to poor water flow or
too high densities, low oxygen, due to poor flow and high temperatures, or the
presence of a high level of suspended organic and siliceous matter (Bullock and
Snieszko, 1975). When these conditions are marginal, handling stress may
trigger infections where the fish would have remained healthy if left untouched.
The expectation of trouble, therefore, is in summer conditions, where
temperatures rise and water flows are reduced. The peak is considered to be 15
18C, and monitoring oxygen and temperatures daily is an effective warning
system where yersiniosis is endemic. Infections have not been reported below
10C.
Prevention and control
Prevention of yersiniosis is achieved by avoiding the introduction of infected
stock and ensuring that all imported eggs have been properly disinfected.
Screening of fry obtained from areas where yersiniosis is endemic is not
sufficiently reliable.
In areas where yersiniosis is endemic, prevention is achieved by avoiding
those husbandry conditions described above which promote infection, although
in many areas, where adequate summer water flow cannot be guaranteed and
high temperatures occur, some degree of infection may be inevitable. The
problem can be contained by the reduction of handling, the lowering of
densities, treatment of water (Liltved et al., 1995; Liltved and Lundvald, 1996)
and the use of vaccination (Bruno and Munro, 1989; Rodgers, 1991a). Each of
these factors lowers the threshold at which a clinical outbreak may occur. If a
serious outbreak does occur, antibiotics may need to be administered.
Yersiniosis was one of the first diseases for which an effective commercial
fish vaccine was developed (Busch, 1978; Cossarini-Dunier, 1986) and
vaccination is widely employed as an effective aid in its control. Reviews have
been given by Ellis (1988) and more recently, by Stevenson (1997). Most
commercial vaccines are bacterins prepared from virulent isolates, some using
whole bacterial cells and others exposing internal bacterial cell components by
lysis at pH 9.8. Most commercial vaccines use the whole-cell serotype, serovar I
(Hagerman strain), which is the cause of the majority of disease outbreaks. Some
outbreaks have been attributed to serovar II (Cipriano et al., 1986), but vaccines
formulated against only serovar I appear to be cross-protective in North
American aquaculture and contain infection from either type. The situation in
Norway and South America, where other variants have been reported, may be
different. In Norway, although commercial bacterins based on serovar I
(Hagerman) gave adequate protection against Norwegian serogroup III,
protection was significantly lower against Norwegian serogroups I and II (Erdal,
1989). Addition of Norwegian serogroup II bacterin to the commercial
preparation enhanced protection against serovar I and Norwegian serogroup II
(Erdal, 1989). It has been suggested that some serovar I variants become more
common where antibiotic-resistant strains occur through the prolonged routine
466
use of antibiotics (H. Madrid, Chile, 1996; personal communication).
Commercial vaccines for yersiniosis are administered by injection or
immersion. In the latter case, the major source of uptake is the gill, where
antigen-trapping cells have been detected (Torroba et al., 1993). The
effectiveness of ERM vaccines varies under field conditions. In situations where
yersiniosis is serious, due to the fish being highly stressed, vaccines rarely
prevent disease completely, since the immune system is also affected by these
conditions (Betoulle et al., 1995). Even so, a marked difference is seen between
control and unvaccinated stocks (Tebbit et al., 1981). Where fish are grown
under less severe conditions, the incidence of disease in vaccinated populations
is frequently at a level sufficiently low for no medication to be required
throughout the growth cycle, and vaccination is therefore very cost-effective
(Horne and Robertson, 1987).
Figure 12.1 shows data for a farm cycle of 16 months from a Danish trout
farm where yersiniosis is endemic. Control fish were medicated after 5 months
to reduce the level of losses; vaccinated fish received no medication.
Yersinia ruckeri is sensitive to the range of antibiotics routinely used in
aquaculture (Inglis and Richards, 1991; Martinsen et al., 1992). However,
failure to remove the husbandry conditions which influence the severity of the
disease results in regular recurrences, requiring further treatments. Two or three
treatments are commonly needed throughout a summer season and, during this
time, resistance may easily develop (Ceschia et al., 1987). The compounds most
regularly used for control of yersiniosis are sulphamerazine, oxytetracycline,
tribrissen and the bacteriostatic agent, oxolinic acid (Inglis et al., 1995).
Fig. 12.1. Performance of Yersinia ruckeri vaccine in rainbow trout, Oncorhynchus mykiss.
PATHOGENESIS
Virulence factors
It is somewhat ironic that, in spite of numerous effective vaccination regimes,
little is known of the virulence determinants associated with Y. ruckeri. Perhaps
this may in part be explained by the complexity of the different serogroups and
the fact that relatively few studies have compared virulent and avirulent isolates
within single serogroups, concentrating predominantly on comparisons between
serogroups. The lack of research in this area may also reflect the early successes
with vaccination against this disease, reported as early as 1965 (Ross and
Klontz, 1965). Nevertheless, the data available suggest that the virulence of
Y. ruckeri, as with most pathogens, is multifactorial.
Plasmids
In the human pathogenic Yersinia species, Y. pestis, Y. enterocolitica and
Y. pseudotuberculosis, virulence is associated with a 4050 MDa plasmid
(Skurnik, 1985). Plasmid profiling of Y. ruckeri has identified a large plasmid of
7088 kb carried in many isolates of serovar I (de Grandis and Stevenson, 1982;
Toranzo et al., 1983, Stave et al., 1987). However, this plasmid was not
homologous with virulence plasmids from other yersiniae (Stave et al., 1987),
although strains of serovar I carrying the plasmid decreased the chemi-
luminescent response of striped bass macrophages (Stave et al., 1987).
Heat-sensitive factor
Furones et al. (1990) compared virulent and avirulent isolates of Y. ruckeri from
serovar I. They reported a strong correlation between the production of a heat-
sensitive factor (HSF), probably lipid in nature, and virulence. Indeed when
trout were infected by bath or intraperitoneal injection, only HSF
+
isolates
resulted in mortality. However, mortalities during bath challenge were variable
and the study concluded that other factors must also be involved.
Resistance to non-specific immune mechanisms
Strains of Y. pestis carrying the 4050 MDa plasmid produce antiphagocytic
factors, which reduce the oxidative microbicidal activity of mouse macrophages
(Charnetzky and Shuford, 1985), as determined by chemiluminescent (CL)
response. Stave et al. (1987) reported that serovar I isolates of Y. ruckeri
harbouring a 70 MDa plasmid also depleted the CL response of striped bass,
Morone saxatilis, macrophages, although the factors expressed by the plasmid
were not determined. Furthermore, one of the serovar II isolates, which did not
carry the plasmid, also showed a decreased CL response in striped bass
468
macrophages. The decreased CL response may reflect the method used.
Chemiluminescent assays detect reactive oxygen species (ROS); it may be that
the reduced CL response results from quenching of ROS by bacterial enzymes,
such as superoxide dismutase (SOD) and catalase. Indeed, all serovar I isolates
analysed contain at least one superoxide dismutase enzyme, depending on the
growth conditions (A.C. Barnes and P. Elsemere, unpublished results). These
enzymes may in themselves be important virulence factors, increasing resistance
to phagocytic killing. It remains to be determined whether they are plasmid- or
chromosomally encoded.
In addition to resistance to macrophage bactericidal activity, virulent
serovar I isolates of Y. ruckeri are reported to be resistant to killing by
non-immune rainbow trout serum, while avirulent serovar I isolates are sensitive
(Davies, 1991b). However, not all serum-resistant isolates are virulent,
reaffirming the multifactorial nature of virulence.
Antimicrobial activity
Yersinia ruckeri has been reported to produce water-soluble antimicrobial
activity capable of inhibiting Vibrio anguillarum, Aeromonas hydrophila and
Aeromonas salmonicida (Michel and Faivre, 1987). This is not without
precedent, as strains of Y. pestis produce a potent murein-hydrolysing
bacteriocin, pesticin (Ferber and Brubaker, 1979). However, the activity
produced by Y. ruckeri does not inhibit other strains of Y. ruckeri and, as such,
does not fulfil the criteria of a bacteriocin (Michel and Faivre, 1987), although it
is important to state that it is unclear which serotypes were used in this study.
Whether or not the production of antimicrobial activity confers a genuine
selective advantage on Y. ruckeri remains contentious, as mixed infections with
both A. salmonicida and V. anguillarum could be obtained experimentally in
rainbow trout (Michel and Faivre, 1987).
Survival under starvation conditions
Survival under conditions of starvation is important to pathogenesis in two
ways. Firstly, the organism is at an advantage if it can survive outside the host for
extended periods to facilitate transmission. In this respect, Y. ruckeri is capable
of surviving in unsupplemented fresh or brackish water (salinity 020 p.p.t.) for
at least 4 months, while survival is greatly reduced in water of salinity 35 p.p.t.
(Thorsen et al., 1992). Furthermore, Romalde et al. (1994) reported that
Y. ruckeri was capable of survival for over 100 days in river water, lake water,
estuarine water and sediments. While acridine orange direct counts remained
constant, viable counts decreased over the experimental period, with culturable
cells persisting for longer periods in sediments than in water (Romalde et al.,
1994). In all cases, dormant bacteria could be revived by addition of fresh TSB
to the microcosms and virulence was maintained throughout the dormant state.
Although non-culturable cells showed marked changes in shape, size and
metabolic rate, no changes were reported in membrane proteins or plasmid
profiles, while minor changes were detected in LPS (Romalde et al., 1994).
Long-term survival may be facilitated by replication of the genome before the
onset of starvation is complete, with starved cells being able to carry up to six
copies of the genome (Thorsen et al., 1992). The second important factor is the
ability to acquire iron during the early stages of infection. Availability of iron in
the host is largely restricted by its association with iron-binding proteins, such as
haemoglobin and transferrin. The ability of a pathogen to acquire iron is
therefore of utmost importance. It has been reported that Y. ruckeri has a
siderophore-mediated iron-uptake system, along with several outer membrane
proteins associated with iron-restricted growth conditions (Romalde et al.,
1991a).
Other factors
Other putative virulence-associated factors which have been studied in
Y. ruckeri include cell-surface hydrophobicity and haemagglutination. Santos et
al. (1990) reported no haemagglutinating activity against human or trout
erythrocytes for all serovar I and II strains tested. This study also reported a lack
of hydrophobicity and no adherence to epithelioma papillosum cyprini (EPC)
cells for the same strains. However, adherence would appear to be dependent
upon the cell line used in the study, with Y. ruckeri strains showing moderate
adhesion and invasiveness in the chinook salmon embryo cell line CHSE-214
(Romalde and Toranzo, 1993). In addition, Romalde and Toranzo (1993)
reported that extracellular products from Y. ruckeri were strongly toxic for fish,
and displayed haemolytic activities for trout, salmon, sheep and human
erythrocytes.
In conclusion, the pathogenicity of Y. ruckeri is poorly understood,
predominantly as a result of the scarcity of good data. This lack of data may
reflect the general view that yersiniosis represents a problem solved through
the early success of vaccination regimes. However, the limited data that are
available suggest that pathogenesis is complex and multifactorial, and highlights
the necessity for further study of this organism.
IMMUNITY
Although Y. ruckeri O-antigen has been used as a model antigen for the study of
the kinetics of immune responses in rainbow trout (Anderson et al., 1979a,b,c)
and carp (Lamers and Muiswinkel, 1984), little is known of the key antigenic
components of Y. ruckeri to which the fish responds during infection or
following vaccination.
The kinetics of the antibody response to O-antigen has been thoroughly
documented in rainbow trout. Peak numbers of antibody-producing cells are
detected in the spleen 14 days after antigen administration by flush. The titres of
470
circulating antibody increase to a maximum after about 28 days, with the onset
of detection coinciding with maximum numbers of antibody producing cells
(Anderson et al., 1979c). Immunization of rainbow trout, using commercial
Y. ruckeri bacterin, also demonstrated a significant secondary response to
antigen injected intraperitoneally 146 days after first exposure (Cossarini-
Dunier, 1986a). This demonstration of an anamnestic immune response to
Y. ruckeri in rainbow trout is important. However, there is a lack of correlation
between antibody titre and protection; Cipriano and Ruppenthal (1987) found
that, although humoral agglutinins were detected in brook trout 24 h after
passive immunization by intraperitoneal injection with immune sera, these
antibodies conferred no resistance to disease. Furthermore, Lillehaug et al.
(1996) demonstrated that protection in fry could not be achieved by passive
transfer of antibodies maternally. Brood-stock females were vaccinated
repeatedly by injection, and an increase in antibody titre, determined by ELISA
and agglutination, was obtained against serovars I and II in the serum of
vaccinated fish. Low levels were also detected in eggs and fry 1 week
posthatching, although mortalities were not reduced in the offspring of
vaccinated mothers compared with unvaccinated controls. This suggests that
other immune mechanisms, such as a localized or cell-mediated response, must
be involved in protection against yersiniosis.
Cell-mediated response to Y. ruckeri has been less thoroughly studied. Jones
et al. (1993) reported that killed cells of serovar I and serovar II strains
stimulated proliferation of nave peripheral-blood leucocytes, and that the two
strains were similarly mitogenic. However, the study concluded that this was
irrespective of the immune status of the fish.
Antibodies produced by rainbow trout appear to be serotype-specific
(Cipriano and Ruppenthal, 1987). Furthermore, antibodies against serovar I do
not recognize LPS in immunoblot, while those raised against 0:2/serovar II do
bind LPS (Stevenson et al., 1993). Additionally, a significantly higher dose of O-
antigen from serovar I is required to elicit a response in rainbow trout than with
O-antigen from serovar II (Anderson and Dixon, 1980).
In spite of the serological differences between Y. ruckeri serovars, there are
numerous reports of cross protection. Indeed, Cipriano and Ruppenthal (1987)
reported that, although immunization by intraperitoneal injection with bacterins
against Y. ruckeri resulted in circulating antibody only to the specific serovar
used in the bacterin, brook trout immunized in such a manner were protected
during experimental challenge with both serovars I and II. Furthermore, a
bacterin prepared from an avirulent serovar II isolate performed equally well
compared with bacterins produced from virulent isolates (Cipriano and
Ruppenthal, 1987). It is unlikely that this is a result of non-specific cross-
protection, as Amend and Johnson (1984) reported that there was no antigenic
competition between Y. ruckeri, A. salmonicida, V. anguillarum and Reni-
bacterium salmoninarum when administered to rainbow trout. The antigens
responsible for cross-protection have not yet been clearly identified; however, a
recent review suggested that native flagellar antigens seem to be antigenically
common among motile serogroups (Stevenson, 1997).
The generally successful vaccination of salmonids against yersiniosis using
bacterins based on serovar I and the numerous studies reporting cross-protection
have perhaps led to a degree of complacency and a general consideration that
other serovars are unnecessary in commercial vaccines (McCarthy and Johnson,
1982). With regard to the latter, as reported above, experience in Norway has
suggested otherwise (Erdal, 1989).
FUTURE DIRECTION
Many questions concerning Y. ruckeri remain unanswered. Its taxonomic
position is unclear and requires further study, probably at the molecular level.
The serology is vastly complex, and consensus between different countries
regarding nomenclature should be achieved. Furthermore, the methodologies on
which serotyping is based need to be standardized, such that results from
different laboratories may be reliably compared.
Very little is known of the pathogenesis of Y. ruckeri, and no model for its
virulence has yet been proposed. Clarification of the role of plasmids, along with
studies of its interaction with host defences, may shed some light on this. Studies
involving comparisons of avirulent isolates or mutants with virulent
representatives of the same serotype would be most informative.
Perhaps most importantly, the complacency resulting from the generally
successful commercial vaccines must be overcome, as their mode of action and
the specific antigens involved in protection are unknown. Further work needs to
be undertaken to determine the mechanisms of protection and cross-protection
against this pathogen in salmonids and, in particular, the nature of the antigens
involved. Without this vital knowledge, aquaculturists may find themselves
helpless if vaccines begin to fail.
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