British Journal of Dairy Sciences 2(3): 35-39, 211 ISSN: 244-244 Maxwell Scientific Organization, 211 Submitted: November 21, 211 Accepted: December 2, 211 Published: December 2, 211 Sero-prevalence of Brucellosis in Bovines at Farms under Different Management Conditions 1 Rakhshinda Munir (Late), 1 Umer Farooq, 1 Zahida Fatima, 1 Muhammad Afzal, 2 Zubair Anwar and 1 Muhammad Jahangir 1 Animal Health Program, Animal Science Institute, National Agricultural Research Centre, Park Road, Islamabad, 455, Pakistan 2 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan Abstract: Brucellosis is endemic in Pakistan. Keeping in view the importance of the disease and non availability of scientific information, a study was designed to carry out the sero-prevalence of brucellosis using an indirect and Rose Bengal Precipitation Test (). Serum samples were collected from three types of farm categories having different husbandry practices. (1) Government livestock farms located in Punjab province (2) Privately owned farms located in out skirts of Lahore and (3) Dairy cattle colony (Gawala colony) located in Lahore. I- detected sero-conversion in 15.2% buffaloes and 9% cattle, whereas, by using it was 8 and 6.5% in buffaloes and cattle, respectively. In cattle, more abortions were recorded at private farms (17.86%) followed by gawala colonies (11.61%) and government livestock farms (8.92%). Also, I- detected sero-conversion against brucellosis in 13.2% female and 1.3% male animals, while by using it was 7.9% in female and 1.6% in male animals, respectively. Key words: Brucellosis,, gawala,, sero-prevalence of brucellosis INTRODUCTION Bovine brucellosis is considered as the most wide spread zoonotic disease in the world (WHO, 1998). The importance of this highly contagious disease is due to its economic impact on livestock industry resulting in the form of abortions, loss of calves, retention of placenta, prolonged calving interval, reduced milk yield and infertility (McDermott and Arimi, 22). The disease is also a serious public health hazard as it is communicable to human beings through milk or as occupational hazard to farmers, butchers and veterinarians. Two main species associated with brucellosis in food animals i.e. Brucella abortus and Brucella melitensis have been reported from Pakistan (Akhtar et al., 1974; Mohiyudin, 1979) and the bacterium is capable of surviving and multiplying inside mononuclear phagocytic system (Munir et al., 28; Jarvis et al., 22). Prevalence of brucellosis in farm animals depends on a number of factors, the most important being the production system and chances of spread during parturition and breeding. Livestock production system in Pakistan is changing and more livestock farmers are now maintaining larger herds, thus increasing the chances of spread of brucellosis in Pakistan (Afzal, 1997). Studies on brucellosis have mainly been restricted to prevalence rates in Pakistan. These studies clearly indicate that the increasing prevalence rate of brucellosis in the country. While earlier studies indicated prevalence rates between.33 to.65 % (Sheikh et al., 1967; Mohiyudin, 1979), more recent studies indicate prevalence 21.5 to 26.1% (Munir et al., 28; Sarwar, 2; Akhtar et al., 199; Lodhi et al., 1995; Ramzan, 1996). Among animal handlers, prevalence of brucellosis ranging from.95 to 8.58 % has been reported by various workers (Masoumi et al., 1992; Afridi et al., 1993; Qazilbash, 1996). Brucellosis is endemic in Pakistan. However data regarding the magnitude of the disease in cattle and buffaloes is scanty and inconsistent. Keeping in view, the importance of the disease and non availability of scientific information, a study was designed to carry out the seroprevalence of brucellosis using an indirect and Rose Bengal Precipitation Test (). Sera were collected from three types of farm categories having different husbandry practices. C Government livestock farms located in Punjab province, where the animals are mainly kept for breeding improvement and conservation. These farms are also involved in selling milk and surplus stock to farmers and hence have a great influence on the livestock population of the Province. C Privately owned farms located in out skirts of Lahore. This is peri-urban market oriented dairying from where milk supply can mostly be made directly to consumers. Corresponding Author: Zubair Anwar, Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan 35
Br. J. Dairy Sci., 2(3): 35-39, 211 Dairy cattle colony (Gawala colony) located in Lahore. This is unique aggregation of cattle and buffaloes maintained on commercial basis for selling milk. Dry animals are continuously replaced with the animals coming from the entire country. The study will help us to chalk out control strategies for the rational control of disease. 35 3 25 2 15 1 1533 Cattle Buffalo 829 2362 1735 1294 329 MATERIALS AND METHODS 5 56 221 277 146 244 39 A total of 329 serum samples from adult cattle and buffaloes were collected between the year 27 to 29, from three types of farm categories; government farms (Livestock Research Station, National Agricultural Research Centre, Park Road, Islamabad; Government Livestock farms located at Jahingirabad, District Khanewal; Khizerabad, District Sargodha; Rakh Ghulaman, District Mianwali; Chak Katora, District Vehari) and semen production units (Barani Livestock Production and Research Institute, Kherimurat, District Attock and Semen Production Unit Qadirabad, District Sahiwal) located in Punjab province; peri-urban private farms around Lahore; and Gawala colony Lahore. Of these samples 2362 sera were collected from eleven government farms, 39 from ten private owned farms and 277 from Gawala colony. Among these sera 376 were from cattle and buffalo from two semen production units. All necessary information regarding number, type of animals kept at farm, vaccination schedule and history of abortion were recorded on a specially designed Performa. Sera were tested by an indirect and Rose Bengal Precipitation Test (). Rose Bengal precipitation test was performed following the method Alton et al. (1988). Briefly, 3 :L of serum was mixed with 3 :L antigen (N-vitro Diagnostikum, Germany) on glass slide. It was rocked at 22ºC for 4 min. The reaction was interpreted as when agglutination was observed and declared negative when the mixture was homogenous after 4 min. For further labortary analysis Indirect Enzyme Linked Immunosorbent Assay () was performed using a commercial indirect kit produced by Chekit, Bommeli Laboratories, Switzerland. Polystyrene flat bottomed 96 wells micro titration were pre-coated with an activated antigen. The test samples and appropriate sera controls (strong, weak, strong negative and weak negative) were diluted 1:2 in phosphate buffered saline Tween-2 (PBST) and added in the wells. These plates were incubated at 25ºC for 9 min in a humidified chamber and were washed three times. Then anti-ruminant IgG conjugate 1:2 dilution was added to each well. The plates were again incubated at 25ºC in a moist chamber for 3 min. After three washings chromogen ABTS (2, 2 azinobis 3-ethyle benzthiazolin) was added. All the washings were carried out using PBST. 4 3 25 2 15 1 5 Government Private Gawala Fig. 1: Blood collected from cattle and buffalo farms under different managemental conditions 115 34 48 197 Government Private Gawala 18 157 11 243 Buffalo Cattle Buffalo Cattle 25 59 354 62 24 18 14 91 14 112 7 153 Fig. 2: Comparison of and for brucellosis in buffalo and cattle at farms under different managemental conditions 38 4 354 35 3 25 2 15 243 153 216 1 59 5 25 52 38 Government Private Gawala Fig. 3: Comparison of and RBPR for brucellosis at farms under different managemental conditions The plates were kept for 15 minutes in dark for color development. The reaction was stopped by adding 4% SDS solution and optical density of plates were recorded using 45 and 492 nm differential filters (Biancifiori et al., 1997; OIE, 2; Wright et al., 1993). In the end qualitative data of and was analyzed by Chi-square test (Steel and Torie, 1984) for statistical analysis RESULTS Blood samples collected from cattle and buffalo at farms under different managemental conditions is summarized in Fig. 1. Further comparison of and for Brucellosis in Buffalo and cattle at farms under 25 216 36
Br. J. Dairy Sci., 2(3): 35-39, 211 Table 1: Comparison and P2 distribution of and for brucellosis in buffalo and cattle at farms under different managemental conditions Brucellosis () ------------------------------------------------- -------------------------------- Animal Farms Negative Positive Not sure None Positive Buffalo Government 698 115 16 829 767 62 829 (.14,84.2) (1.,13.9) (.11,1.9) (1.24,1) (.3,92.5) (.32,7.5) (.34,1) Gawala 171 48 2 221 23 18 221 (.84,77.4) (6.12,21.7) (3.93,.9) (7.91,1) (.,91.9) (.,18.1) (.,1) Private 25 34 5 244 22 24 224 (.3,84) (.27,13.9) (.1,2) (.39,1) (.9,9.2) (.98,9.8) (1.6,1) 174 197 23 1294 119 14 1294 (1.1,83) (7.36,15.2) (1.15,1.8) (-,1) (.11,92) (1.3,8) (-,1) P 2 9.55* 1.42 Probability (.4) (.49) Cattle Government 1379 128 26 1533 144 91 1531 (.1,9) (.81,8.3) (.1,1.7) (.91,1) (.4,94.1) (.64, 5.9) (.68,1) Gawala 43 11 2 56 49 7 56 (.97,76.8) (6.96,19.6) (1.21,3.6) (9.14,1) (.22,87.5) (3.16,12.5) (3.37,1) Private 127 18 1 146 132 14 146 (.8,87) (1.75,12.3) (.85,.7) (2.67,1) (.15,9.4) (2.21,9.6) (2.36,1) 1549 157 29 1735 1621 112 1733 (1.14,89.3) (9.51,9) (2.6,1.7) (-,1) (.41,93.5) (6.,6.5) (-,1) P 2 12.72** 6.41* Probability %.1.4 Table 2: Comparison and P2 distribution of and for brucellosis at farms under different managemental conditions Brucellosis () ----------------------------------------------------- ------------------------------------- Farms Negative Positive Not sure None Positive Government 277 243 42 2362 227 153 236 (.49,87.9) (3.96,1.3) (.5,1.8) (4.49,1) (.11,93.5) (1.41,6.5) (1.51,1 ) Gawala 214 59 4 277 252 25 277 (2.79,77.3) (21.9,21.3) (.12,1.4) (24.81,1) (.11,91) (1.39,9) (1.49,1) Private 332 52 6 39 352 38 39 (.1,85.1) (.9,13.3) (.7,1.5) (1.7,1) (.29,9.3) (3.72,9.7) (4.,1) 2623 354 52 329 2811 216 327 (3.37,86.6) (26.76,11.7) (.24,1.7) (-,1) (.5,92.9) (6.51,7.1) (-,1) P 2 3.38** 7.1* Probability % 1.3 Table 3: Comparison and P2 distribution of and for brucellosis in buffalo and cattle Brucellosis () ------------------------------------------------------ --------------------------------------- Animal Negative Positive Not sure None Positive Buffalo 174 197 23 1294 119 14 1294 (1.93,83) (13.85,15.2) (.3,1.8) (15.81,1) (.1,92) (1.35,8) (1.45,1) Cow 1549 157 29 1735 1621 112 1733 (1.44,89.3) (1.33,9) (.2,1.7) (11.79,1) (.8,93.5) (1.1,6.5) (1.8,1) 2623 354 52 329 2811 216 327 (3.37,86.6) (24.18,11.69) (.4,1.72) (-,1) (.18,92.9) (2.35,7.1) (-,1) P 2 27.6** 2.53 Probability % 1.11 Table 4: Comparison and P2 distribution of and for brucellosis in females and males Brucellosis () ------------------------------------------------------ --------------------------------------- Sex Negative Positive Not sure None Positive Female 2254 349 5 2653 2441 21 2651 (.82,85) (4.89,13.2) (.44,1.9) (6.14,1) (.17,92.1) (2.18,7.9) (2.35,1) Male 369 5 2 376 37 6 376 (5.78,98.1) (34.51,1.3) (3.7,.5) (43.37,1) (1.18,98.4) (15.41,1.6) (16.58,1) 2623 354 52 329 2811 216 327 (6.6,86.6) (39.4,11.7) (3.51,1.7) (-,1) (1.35,92.9) (17.59,7.1) (-,1) P 2 49.51** 18.94** Probability % 1 1 37
Br. J. Dairy Sci., 2(3): 35-39, 211 25 2 15 1 5 18 16 14 12 1 8 6 4 2 25 2 15 1 5 197 Government Private Gawala 121 1 17 148 Cattle abortion 14 1357 56 146 1559 Cattle tolal 157 31 35 45 111 Buffalo abortion 112 Fig. 4: Comparison of and RBPR for brucellosis in Buffalo and Cattle 629 221 244 149 Buffalo total Fig. 5: Comparison of abortions in buffalo and cattle 4 35 3 5 6 Male 349 Female 21 Fig. 6: Comparison of and RBPR for brucellosis in female and male animals different managemental conditions with their statistical analysis is presented in Table 1 and Fig. 2. Next comparison of and for Brucellosis at farms under different managemental conditions and their statistical analysis is presented in Table 2 and Fig. 3. Comparative analysis of and for Brucellosis in Buffaloes and cattle and their statistical analysis is shown in Table 3 and Fig. 4. And the comparison of abortion in cattle and buffaloes and their statistical analysis is shown in Table 4 and Fig. 5. In the comparison of and for Brucellosis in females and males is presented in Fig. 6. DISCUSSION AND CONCLUSION The multi fold herd size was present at government farms as compared to private and gawala colony. More than 5 % animals from each farms category were bled for detecting sero-conversion against brucellosis. The highest buffaloes (21.7%) were observed in Gawala colonies followed by government (13.9%) and private farms (13.9%), while in cattle it was also highest (19.6%) in Gawala colonies followed by private (12.3%) and government farms (8.3%). In case of the highest buffaloes (18.1%) were detected at Gawala colony followed by private farms (9.8%) and government farms (7.5%), while highest cattle (12.5%) was observed in Gawala colony followed private farms (9.6%) and government farms (5.9%). Interestingly, the highest (21.3%) animals were at gawala colonies followed by private (13.3%) government farms (1.3%), while highest animals (9.7%) were at private farms followed by gawala colony (9%) and government farms (6.5%). When I- and were compared for the detection of brucella infection, it was observed that detected a higher number of cases as compared to which detected higher number of negative cases. In a campaign for the control of a disease like brucellosis, it is desirable that the screening test should be reliable and detects almost all cases in a herd. In our studies I- detected more samples as compared with and therefore, it would be a better choice to launching a campaign for the control and eradication of brucellosis. Also acceptable method for screening should minimize false negatives results especially when used for the detection of infection with serious consequences. In the present study, it was observed that I- detected more sera as compared to this observation was recorded by Nielsen et al. (1996) and Dajer et al. (1998), who compared I- with different serological tests and reported its sensitivity up to 1%. Molnar et al. (1998) and Poester et al. (1998), have also observed the higher sensitivity of I-. It was observed that I- detected seroconversion in 15.2% buffaloes and 9% cattle, whereas, by using it was 8% and 6.5% in buffaloes and cattle, respectively. In cattle, more abortions were recorded at private farms (17.86%) followed by gawala colonies (11.61%) and government livestock farms (8.92%); this might be due to their husbandry practices and adopted hygienic measures. I- detected sero-conversion against brucellosis in 13.2% female and 1.3% male animals, whereas it was 7.9 and 1.6% by for female and male animals, respectively. Most of the male animals were kept by the government farms while only few male animals were kept at private and gawala colonies. 38
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