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Effect of Treatment with Tri-Sodium Citrate

Alone and in Combination with Levamisole HCl
on Total Milk Bacterial Count in Dairy Buffalo
Suffering from Sub-Clinical Mastitis
Arfan Yousaf1, Imran Sarfaraz1, Muhammad Arif Zafar1, Rao Zahid Abbas2, Alamdar Hussain3, Asad Manzoor1
1

Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan. 2Department of Parasitology,
University of Agriculture, Faisalabad, Pakistan. 3Department of Pathology, University of Agriculture, Faisalabad, Pakistan

ABSTRACT
Mastitis is one of the most important diseases of dairy cattle and buffaloes and continues to have major economic impact
on the dairy industry throughout the world. The present study was carried out to investigate the affect of treatment with
tri-sodium citrate alone and in combination with levamisole HCL on total milk bacterial count in sub-clinically mastitic
dairy buffaloes. Twenty four sub-clinically mastitic buffalos were divided into four equal groups i.e. A, B, C and D. Animals
in Group-A were treated with tri-sodium citrate orally at dose rate of 30 mg/kg b.wt. for 8 days consecutively while the
buffalos in Group-B received levamisole HCl orally at dose rate of 2.5 mg/kg b.wt. for 3 days consecutively. The animals
in Group-C received tri-sodium citrate (30 mg/kg b.wt for 8 days consecutively) as well as levamisole HCl (2.5 mg/kg
b.wt. for 3 days consecutively) orally .The mastitic buffalos in Group-D served as untreated control. Milk samples were
collected at day 0, 3 and 10 for total bacterial count. The average total bacterial counts in Group A, B and C showed a
decrease of 11.53%, 10.16% and 11.05%, respectively at day 3 post treatment as compared to that at day 0 post
treatment. Average reductions of 39.6%, 30.2% and 45.4% in total bacterial counts were observed in animals of Group
A, B and C, respectively at day 10 post-treatment as compared to the bacterial counts at day 0 in the respective groups.
An almost 100% increase in total bacterial count from initial count (day 0) was observed in group D at day 10 and day
3, respectively. A significant decrease in total bacterial count was observed in all the treated groups. The combination
treatment with tri-sodium citrate and levamisole HCl proved to be most successful in reducing the bacterial count
significantly at day 10 post treatment in sub-clinically mastitic buffalos. The results of present revealed that combined
oral administration of tri-sodium citrate and levamisole HCl are possible substitute of antibiotic therapy for sub-clinical
mastitis

Keywords: subclinical mastitis, treatment, tri-sodium citrate

INTRODUCTION
Livestock sector plays an important role in the economy of Pakistan as 8 million families in our country are related to
livestock with their 30-35% of income dependent on livestock production activities. The importance of livestock can be
realized from the fact that it is contributing 11% in the GDP which is more than the total aggregate of crop section share
i.e. 10.3 percent. Dairy industry of Pakistan mainly comprises of cows and buffaloes. The total population of cattle and
buffalo in Pakistan is 31.8 and 29 million heads respectively (Anonymous, 2007). The buffalo contributes about 75% of milk
consumed all over the country (Sarwar et al., 2002). Despite this huge dairy animal population, Pakistan is still facing acute
shortage of milk supply in major urban areas (Siddiqui, 1999). The main factors responsible for milk shortage include low
genetic potential of animals, feeding imbalanced low grade rations and dairy diseases e.g. mastitis (Dodd, 1983).
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Financial losses due to mastitis occur for both sub-clinical and clinical stages of the disease (Fetrow et al., 1991). In
USA, sub-clinical mastitis is responsible for about 60-75% of economic losses due to mastitis (Allore, 1993). During
1978, losses due to clinical mastitis were estimated to Rs. 240 million per annum in Punjab province of Pakistan
(Chaudhry & Khan, 1978). A study conducted in Pakistan reveals that mastitis affected Nili-Ravi buffaloes had on an
average a 57 days shorter lactation period and 438 liter less milk production (Cady et al., 1983).
Treatment of mastitis is still highly controversial. Poor management, resistance against drugs, severity of udder pathology and elimination of predisposing factors are major causes associated with bovine mastitis treatment failure. Although
various preventive measures and management practices can be effective, but there is still a great need for well designed
therapeutic measures to substantiate appropriate treatment regimens against mastitis. Keeping in view the limitations
of antibiotic therapy and antibiotherapy, alternative approaches for the treatment and control of bovine mastitis has
been planned.
MATERIALS AND METHODS
Twenty four buffaloes positive for sub-clinical mastitis were selected for the drug trails on the basis of Surf Field Mastitis
Test (SFMT) as described by Muhammad et al., (1995). The animals were divided into four groups A, B, C and D, containing six buffalos in each. Buffaloes in Group-A received trisodium citrate at dose rate of 30 mg/kg b.wt. orally for eight
consecutive days. Animals in Group-B were treated with levamisole HCl orally at dose rate of 2.5 mg/kg b.wt. for three
consecutive days while buffaloes in Group-C were administered 30 mg/kg b.wt trisodium citrate orally for eight consecutive days along with levamisole HCl (2.5 mg/kg b.wt. orally) for three consecutive days. Group-D was served as control.
Milk samples from treated and non-treated animals were collected at day 0, 3 and 10 post-treatment and used to
determine total bacterial count (Cruickshank et al., 1975).
Collection of samples:
Before collection of milk samples, each teat was scrubbed with a pledged of cotton moistened with 70% ethyle alcohol.
A separate pledged was used for each teat. After sanitizing the teat opening with cotton gauze swabs, SFMT was
performed on all samples for the detection of sub-clinical mastitis as described by the Muhammad et al, (1995). Composite milk samples from Surf Field Mastitis Test positive buffalos were collected in 5 ml sterilized syringes after discarding
first few streams of milk. After collection, milk samples were immediately transported under cold storage to the Mastitis
Research Laboratory, Department of Clinical Medicine & Surgery, University of Agriculture, Faisalabad and bacteriological
examination were done.
RESULTS AND DISSCUSION
Number of teat that became negative for mastitis as detected by SFMT at different days post treatment has been shown
in Table-1. At day 3 and post treatment highest number of teats (9 and 17) was detected negative in group A. An equal
number of teats (8 and 11) became mastitis negative both on day 3 and 6 post treatment in both Group B and Group
C. On day 10 post treatment highest number of mastitis negative teats was observed in Group A (17) followed by that in
Group B (13). No teat became mastitis negative at any day post treatment.
The Overall highest cure rate was observed in Group A that received tri-sodium citrate alone.
The ph of mastitic milk has been reported to be alkaline. This alkaline nature of the mastitic milk favors bacterial growth.
Decrease in citric acid level of mastitic milk is said to be responsible for this increase in milk pH (Oshima and Fuse, 1981).
The normal pH of milk is nearly 6.5 and is not suitable for bacterial growth while in mastitic milk this pH increases to 7.27.6 that favors bacterial growth. When we treat the sub-clinical mastitis by tri-sodium citrate it decreases the pH of milk
thus making milk unsuitable for bacteria growth (Dhillon et al., 1991; Dhillon et al., 1995). As the bacterial growth is
inhibited, the teats showing sub-clinical mastitis before treatment become negative. As far as far percent reduction in
bacterial count, the best results were observed in Group C that received tri-sodium citrate and levamisole in combination.
It is established that mastitis cause immunosupression. Levamisole in lower doses acts as immunotherapeutic agent for
bovine mastitis. It is reported to increase level of immunoglobulins and activity of macrophages and polymorpho nuclear
cells which play a major role in the immune system of mammary glands (Ishikawa and Shimizu, 1981). The higher reduction
in bacterial count in the group that received levamisole HCl in addition to tri-sodium citrate may due the combined
systemic and localized inhibition of bacterial growth caused by the two drugs, respectively. Similar results have been
reported by Kalorey et al. (1993). These workers reported cultural examination based reduction in infection rate from
83.33% before treatment to 33.33% and 8.33% on 6th and 21st day post-treatment, respectively in lactating cows.
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Table 1: Number Of teat

Found Negative for Mastitis as Detected by Surf
Field Mastitis Test in different groups of mastitic buffalos receiving tri-sodium
citrate alone or in Combination with Levamisole HCl

Group-A= Tri-sodium citrate orally at dose rate of 30 mg/kg b.wt. for ten consecutive days.
Group-B= Levamisole HCl orally at dose rate of 2.5 mg/kg b.wt. for three consecutive days.
Group-C= Tri-sodium citrate (30 mg/kg b.wt.) for ten consecutive days along with levamisole HCl (2.5 mg/kg b.wt.)
for three consecutive days

Figure 1. Percent Reduction

in Total bacterial Count at
Different Days Post- Treatment in Mastitic Buffalos
Treated with Trisodium Citrate Alone or In Combination
with Levamisole HCl

REFERENCES
1.Allore. 1993. A review of the incidence of the mastitis in buffaoes and cattle. Pakistan Veterinary Journal, 13: 1-7
2.Anonymous. 2007. Economic survey of Pakistan 2006-2007. Finance Division, Economic Advisors Wing, Government of Pakistan, Islamabad
3.Chaudhry, N. A.; Khan, B. B. 1978. Estimation of economic losses due to animal diseases in Punjab. Final Report of Research Project, University
of Agriculture, Faisalabad
4.Cady, R. A., S. K. Shah, E. C. Schermerhorn and R. E. McDowell, 1983. Factors affecting performance of Nili-Ravi buffaloes in Pakistan. Journal Dairy
Science, 66: 578-586
5.Cruickshank, R., Dugaid, J. P.; Marmion, B. P.; Swain, R. H. A. 1975. Medical Microbiology, 12th Ed. Vol. 11, pp: 305-310. Churchill livingstone,
Edinburg London and Newyork.
6.Dodd, F. H. 1983. Mastitis-Progress on control. Journal of Dairy Science, 66: 1773-1780
7.Dhillon, K.S., Sandhu, H.S.; Singh, T.J.; Singh, J.; Gill, B.S. 1991. Novel mastitis therapy in buffaloes. Ind. J. Anim. Sci., 61: 1262-1264
8.Dhillon, K.S., Singh, T.J.; Sodhi, S.S.; Sandhu, H.S.; Dwivedi, P.N.; Singh, J.; Gill, B.S. 1995. Milk bacteriology: Pre and post-trisodium citrate
mastitis treatment in buffaloes. Ind. J. Anim. Sci., 65: 9-11
9.Fetrow, J., Stewart, S.; Eickher, S.; Farnsworth R.; Bey, R. 2000. Mastitis: An economic consideration. In Proceedings of 39th NMC Annual
Meeting, Atlanta Georgia. pp 3-47
10.Ishikawa, H.; Shimizu, T. 1981. Protein composition of whey from sub-clinical mastitis and effect of treatment with levamisole. J. Dairy Sci.,
65:658.
11.Kalorey, D. R., Kolte, R. M.; Dhoot, V.M.; Dakshimkar, N.P.; Harne, S.D. 1993. Non-antibiotic treatment for subclinical mastitis in cows. Ind.
J. Vet. Med., 13: 24
12.Muhammad, G., Athar, M.; Shakoor, A.; Khan, M.Z.; Rehman, F. Tauseef, M. 1995. Surf field mastitis test. An inexpensive new tool for
evaluation of wholsomeness of fresh milk. Pak. J. Food Sci., 5:91-93.
13.Oshima, M.; Fuse, H. 1981. Citric acid concentration in subclinical mastitic milk. J. Dairy Res., 48: 387-92
14.Sarwar, M., Khan, M. A.; Mahr-un-Nisa; Zafar, I. 2002. Dairy Industry in Pakistan: A Scenario. International Journal of Agriculture & Biology, 4:
420-428
15.Siddiqui, M. M. 1999. Dairy industry in Pakistan. Veterinary News and Views, 2:4
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