Prevalence and risk factor ' s analysis of bovine brucellosis in peri-urban areas under intensive system of production in Gujarat , India

The economic wellbeing of dairy farmers depends upon healthy, productive and sound reproductive livestock. Among the various prevalent diseases which considerably affect production and reproduction performance of dairy animals, bovine brucellosis is perhaps the most economically important reproductive disease of the rapidly growing Indian dairy industry. In India, brucellosis was first recognized in 1942 and is now endemic throughout the country. The disease has been reported in cattle, buffaloes, sheep, goats, pigs, dogs and humans. Brucellosis in India is a very common but often neglected disease [1]. The most significant feature of bovine brucellosis epidemiology is the shedding of large numbers of organisms during 10 days after abortion or calving of infected cows and the consequent contamination of the environment [2]. The prevalence of infection in animal reservoirs provides a key to its occurrence in humans also. Therefore, the correct and prompt diagnosis is important in controlling and eradicating the disease in animals. The diagnosis of the disease can be challenging and is frequently delayed or missed because the clinical picture may mimic other infectious and non-infectious conditions [3]. Recently, ELISA has taken over as an important serological tool in the diagnosis of brucellosis because of its economy, sensitivity, specificity, rapidity, reproducibility, and easy interpretation through colorimetric end product [4]. Further, the advent of milk based I-ELISA (Milk-ELISA) brings revolution in screening of large population. Milk-ELISA performed on bulk milk samples are now routinely and effectively used for screening and monitoring dairy cattle for brucellosis [5]. Overall in country as a whole and Veterinary World, EISSN: 2231-0916 Available at www.veterinaryworld.org/Vol.7/July-2014/13.pdf


Introduction
The economic wellbeing of dairy farmers depends upon healthy, productive and sound reproductive livestock.Among the various prevalent diseases which considerably affect production and reproduction performance of dairy animals, bovine brucellosis is perhaps the most economically important reproductive disease of the rapidly growing Indian dairy industry.In India, brucellosis was first recognized in 1942 and is now endemic throughout the country.The disease has been reported in cattle, buffaloes, sheep, goats, pigs, dogs and humans.Brucellosis in India is a very common but often neglected disease [1].The most significant feature of bovine brucellosis epidemiology is the shedding of large numbers of organisms during 10 days after abortion or calving of infected cows and the consequent contamination of the environment [2].
The prevalence of infection in animal reservoirs provides a key to its occurrence in humans also.Therefore, the correct and prompt diagnosis is important in controlling and eradicating the disease in animals.The diagnosis of the disease can be challenging and is frequently delayed or missed because the clinical picture may mimic other infectious and non-infectious conditions [3].Recently, ELISA has taken over as an important serological tool in the diagnosis of brucellosis because of its economy, sensitivity, specificity, rapidity, reproducibility, and easy interpretation through colorimetric end product [4].Further, the advent of milk based I-ELISA (Milk-ELISA) brings revolution in screening of large population.Milk-ELISA performed on bulk milk samples are now routinely and effectively used for screening and monitoring dairy cattle for brucellosis [5].Overall in country as a whole and Gujarat in particular, the village milk production system is being transforming into a milk production industry which is more concentrated in and around the city areas (peri-urban areas).Ahir, Bharvad, Rabari communities as well as other farmer communities are focusing on this profitable business on a large scale and taking dual benefits of already established milk co-operatives infrastructure and direct market of city areas.A parallel milk marketing system is growing rapidly in and around each city due to readily available raw milk market.Earlier research work carried out on bovine brucellosis has mostly focused on organized government farms or farmers under milk co-operatives.These studies did not cover peri-urban milk producers maintaining good quality animals under intensive system of production and which were reluctant to provide necessary support and information.
The present study was carried out after the necessary permission of institutional ethical committee.
Six selected cities Ahmedabad, Anand, Surat, Navsari, Valsad and Vapi covering middle and south Gujarat were included in the present study.Five peri-urban areas of each city were randomly selected for the present work.From each peri-urban area, five farmers following intensive system production with herd size 10 milking animals were included in the study.The species (cattle and Buffalo) and breed-wise bulk and individual milk samples at approximately 10 per cent of total milking animals, were collected for Milk-ELISA.A total of 199 bulk and 582 individual milk samples were collected aseptically in 5 ml sterile screw capped plastic sample collection vials and kept in insulated ice-box with prefreezed ice-packs during transportation up to laboratory.The actual numbers of samples collected were differed from above mentioned selection criteria due to mixed farming (cattle and buffaloes or different breeds of cattle or buffaloes) practice on dairy farms.The available facilities of Department of Veterinary Medicine and Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University (AAU), Anand, Livestock Research Station, NavsariAgricultural University (NAU), Navsari and Regional Animal Disease Investigation Offices (ADIO) of Department of Animal Husbandry (Ahmedabad and Navsari) were used for present study.After removal of creamy part by centrifugation, milk samples were transferred to another vials and stored at -20ºC, till further use.
indirect ELISA test kits were procured from VMRD, Inc., U.S.A and the tests were performed as per the protocol outlined in the user manual at LRS, NAU, Navsari or ADIO, Ahmedabad.
The epidemiological information and necessary history regarding various risk factors were collected in surveillance performed during the present study.Atotal of 45 risk factors were taken into consideration.They were grouped into four major categories i.e. 1) Risk factors on general characteristics of farms, 2) Risk factors on introduction of infection to farms, 3) Risk factors on management systems of farms and 4) Risk factors on exposure of disease.
Data pertaining to prevalence based on milk testing and risk factors were analyzed on IBM SPSS statistical software version 20.0 using chi square test (probability at 5% and confidence interval at 95% level) as per method described by Snedecor and Cochran [6].
In the present study, an overall 67 (33.70%) out of 199 bulk milk samples were found positive for a antibodies on milk-ELISA.The present result is in accordance to the previous reports on herd prevalence of bovine brucellosis based on milk-ELISA [7][8][9][10].Similarly, an earlier report of Asfaw [11] also found 100% herd prevalence in peri-urban, 30% in intra-urban and 12.5% in interurban areas with overall herd prevalence of 33.30%.In a single report from Gujarat, Varasada [12] reported only 3.53% herd prevalences on tank bulk milk testing by milk-ELISA.During the present study, the overall animal prevalence based on milk-ELISA was 11.90%.Present finding is in accordance with previous reports based on milk tests which reported 10-15% animal prevalence [13][14][15].Whereas, Patel [16] and Aulakh . [17] recorded still higher animal prevalences on milk tests with corresponding figures of 28.30 and 18.26%, respectively.Whereas, few reports from India and neighboring country reported animal prevalence below 10% , the animal prevalence (11.90%) observed in present study was found higher than a wide area based bovine brucellosis prevalence (6-7%) by ELISA conducted by Renukaradhya .
[1].Further, the reports from India on sero-prevalence of bovine brucellosis also showed more or less similar trend of animal prevalence of bovine brucellosis [22][23][24].The species and peri-urban areas-wise herd and animal prevalence are presented in Table-1.The prevalence was found non-significantly higher in cattle than buffaloes.Further, it was varied non-significantly between the peri-urban areas.The higher prevalence in present study may be attributed to highly intensive production system and frequent replacement of animals without prior testing.
The risk factors showed significant associations with occurrence of bovine brucellosis were discussed whereas the information of distribution of those risk factors had statistically nonsignificant association with occurrence of brucellosis are given in respective table.
The present investigation had been planned to study bovine brucellosis in dairy herds of peri-urban areas using milk-ELISA.-2.The highest prevalence (87.50%) was observed in a group of herds with herd size between 51-75 animals whereas it was the lowest (21.70%) for a group of herds with herd size below 25 animals.Increase in prevalence with increased herd size was observed with highly significant asso-ciation with occurrence of bovine brucellosis (p= 0.000).Such finding is in accordance with the earlier report of Tun [10] who reported significantly higher risk when the herd size is greater than 50 animals as it was 28.6% for herd size above 50 and only 3.1% for herd size less than 50 animals.Likewise, scientists had also reported significant association of herd size with prevalence of brucellosis [11,19,[25][26][27][28][29][30][31][32].On contrary, non-significant association between herd et al. size and prevalence of brucellosis was also reported by Kebede .
Out of 199 herds, 155 (77.90%) were of indigenous animals whereas only 44 (22.10%) herds had crossbred animals.But, the prevalence of brucellosis was significantly (p=0.012)higher in herds of crossbred animals (52.30%) than indigenous animals (28.40%).The highest prevalence (57.50%) was observed in herds of Holstein-Frisian crossbreds followed by 34.70% for Mahesani buffaloes, 33.30% for other breed of buffalo, 25.00% for Jersey crossbred, 17.40% for Gir cattle, 11.10% for Jafarabadi buffaloes and the lowest (10.00%) in Bunni buffaloes.The difference in prevalences due to breed was also statistically significant (p=0.013).The results are in accordance to earlier reports indicated significantly higher prevalence in crossbred than indigenous animals [23,[36][37][38].In this regard, Swai .[39][40] reported significant association of exotic blood with prevalence of brucellosis.Tesfaye .[41] also observed higher prevalence in crossbreds than local breed but it was statistically non-significant.The findings of Kebede .[33] and Chand and Chhabra [35] reported seropositivity, independent to breed and species, respectively.Only a single report of Karimuribo .[ ).The overall effect of age groups of owner was significant (p=0.002).Further, the prevalence in herds owned by owners having inherited knowledge of dairying was significantly (p=0.034)higher (36.00%) than herds owned by owners who gained knowledge of dairying by themselves (16.70%).In this regard, Tebug .[43] reported that higher awareness in farmers about the existence of zoonotic infections and practices with above primary education and more than six years of dairy farming experience.A total of six different risk factors on introduction of infection to farms were studied for the first time in Gujarat.Of these, distribution of four risk factors and their association with herd prevalence of bovine brucellosis is given in Table-3.None of risk factors showed significant association with occurrence of bovine brucellosis.Risk factors such as quarantine practice and testing before introduction were not followed in the herds covered under the study and therefore, statistical significance could not be drawn out.The results observed in present study are in accordance to the findings of Tun [10] who reported statistically non-significant effects of the risk factor variables (vaccination, breeding, milking methods and production system) concerned with the introduction of the infection into the herd.Vaccination against disease is considered to be a protective factor as reported by Azevedo [44].This was also supported by observation of Muma .[45] who found negative correlation of vaccine history with prevalence of brucellosis.Further, unrestricted movement of animals [46] and purchase of animals for farm replacement or breeding [36,[47][48] and removal of sero-positive reactors after testing [49] are considered to be important risk factors for introduction of infection.
Similar to risk factors on introduction of infection to farm, 11 different risk factors were also studied under this group.The distribution of 10 risk factors and their association with prevalence of bovine brucellosis is given in Table-3.In relation to risk factor on feeding practice in herds covered, all herds followed manual feeding system and therefore, its association with occurrence of bovine brucellosis could not be draw out.None of the risk factors had statistical significant association with occurrence of brucellosis.keeping good hygiene at dairy farm [26] and zero grazing [39,50] are considered as a protective factor for brucellosis, unhygienic practices were identified as factors that will facilitate the spread of infections [51].
A total 15 different risk factors were covered under this group.Except reproductive disorders, remaining risk factors were covered for the first time under this study.The distribution of 12 different risk factors on exposure of disease and their association with prevalence of bovine brucellosis is presented in Table-4.In none of the herds, provision of calving box and isolation of diseased animals was in practice.Proper disposal of aborted material was followed in all herds.Therefore, statistical significance of these three risk factors could not be drawn out.Majority of herds were not having control over visitors (82.40%) and stray animals (96.50%).It is a known fact that restriction over visitors and stray animals is helpful in reducing spread of infection which is further supported by an observation of Tun [10] who found significant increased prevalences with poor bio-security measures like control of visitors and stray animals.
None of the herd was practicing isolation of diseased animal and providing calving box to down calver as they were maintaining high density of animals in lesser space due to high market price of each square feet of land area in peri-urban areas.It is a fact that the shedding of large numbers of organisms occurs during 10 days after abortion or calving of infected cows is the most significant feature of bovine brucellosis epidemiology and therefore, provision of calving box to down cowers is definitely helpful to reduce the chance of spread of infection if any [2].The awareness of brucellosis among dairymen was also reported as significant risk factor [10,36,50].In the present study, a risk factor such as veterinary help/aid in cases of abortion/still birth showed statistically non-significant association with prevalence of bovine brucellosis.However, scientists had reported the presence of adequate veterinary services as protective factors in prevalence of brucellosis [25].
As per Table-4, the risk factors such as history of abortion, retention of placenta, still birth and metritis/ endometritis had statistically significant effects on prevalence of brucellosis.The results are in accordance to the findings of scientists who had reported significant association with reproductive disorders like abortion, retention of placenta and repeat breeding [17,35,43,44,50,[52][53][54][55].Some scientists also found higher prevalence of brucellosis with reproductive disorders but their association with prevalence was non-significant [22, 26, 28-29, 33, 56-60].
It can be concluded that prevalence of bovine brucellosis in dairy herds maintained under intensive system of production in peri-urban areas was comparatively higher than overall prevalence of brucellosis.Risk factors like larger herd in close confinement without adequate sheds, type of animal, type of breed and knowledge/awareness of dairyman, unrestricted animal market, replacement without prior testing, reproductive disorders with absence of their testing are the important risk factors under the intensive production system of peri-urban areas.
MDP carried out the study, tabulated and analysed the data, drafted the manuscript.PRP helped to draft the manuscript and approved the final draft.MGP and ANK helped in sample collection and processing.KKT helped in data assembling and statistical analysis.ABF rendered necessary infrastructure facilities and grant for purchasing necessary kits and other materials required during the study period.All authors read and approved the final manuscript.

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Risk factors on general characteristics of farms:The distribution of 11 risk factors on general characteristics of farms and their association with herd prevalence of bovine brucellosis is given in Table

Table - 1
: Species-wise and peri-urban area-wise prevalence of bovine brucellosis under intensive system of production.

Table - 2
: Distribution of risk factors on general characteristics of farms and their association with herd prevalence of bovine brucellosis.

Table - 3
: Distribution of risk factors on introduction of infection to farms and management systems of farms and their association with herd prevalence of bovine brucellosis.

Risk factors on introduction of infection to farms: Risk factors on management systems of farms: Table-4:
Distribution of risk factors on exposure of disease and their association with herd prevalence of bovine brucellosis.