Sero-Prevalence and Associated Risk Factors of Bovine Brucellosis in Selected Dairy Farms in Bishoftu Town, Oromia, Ethiopia

International Journal of Microbiological Research 9 (): 45-53, 018 ISSN 079-093 IDOSI Publications, 018 DOI: 10.589/idosi.ijmr.018.45.53 Sero-Prevalence and Associated Risk Factors of Bovine Brucellosis in Selected Dairy Farms in Bishoftu Town, Oromia, Eiopia 1 1 1 3 Hika Waktole, Ermiyas Geneti, Wahid M. Ahmed, Gezahegne Mammo and Fufa Abunna 1 Department of Microbiology, Immunology and Veterinary Public Heal College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box: 34, Bishoftu, Oromia, Eiopia Department of Animal Reproduction & A.I, Veterinary Research Division, National Research Centre, Giza, Egypt 3 Department of Clinical studies, College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box: 34, Bishoftu Oromia, Eiopia Abstract: A cross-sectional study was carried out from December 015 to April 016 to determine seroprevalence and associated risk factors of bovine brucellosis in dairy farms in Bishoftu town. A total of 400 blood samples were collected from cross breed dairy cattle and e Rose Bengal plate test (RBPT) was used as a screening test. Those serum samples reacting positively to RBPT were subjected to e complement fixation test (CFT) for confirmation. Accordingly, RBPT detected 15 of 400(3.75%) of e samples as brucellosis positive. The positive sera when furer retested using CFT, 1 out of e 15 RBPT positive sera were confirmed to be positive. The prevalence of brucellosis based on CFT in e study area was 3% and all positive sera were from female cattle. A Chi-square computed statistical analysis indicated at abortion history ( =10.67; P<0.001), abortion period ( =9.4; P<0.000), retained fetal membrane ( =14.5;P< 0.00) and parity ( =5.69; P<0.05) were e major risk factors for Brucella infection in e study area. In addition, result of e questionnaire survey revealed at percentage of 16.14% history of abortion and 17.7% history of retained fetal membranes. A total of 66 cattle attendants and farm owners were interviewed and 30.3% were found to have no knowledge of brucellosis, only 18.18% were wear protective gloves during handling of aborted material and 34.85% responded at ey consume raw milk. Therefore, in order to control spread of brucellosis implementation of better management practices like isolation of aborted animals, provision of separate parturition pen, proper disposal of aborted fetuses and fetal membranes should be practiced. Key words: Bishoftu Bovine brucellosis Dairy cattle Risk factors Sero-prevalence INTRODUCTION Brucellosis is e most widely spread zoonoses disease in e world [3]. It is an infectious bacterial Eiopia has huge livestock population, yet, ey disease caused by genus Brucella which is Gramwere affected by different diseases which greatly affect negative; intracellular coccobacillary comprised of e economy and public heal wiin e country. species based upon biochemical features and eir Among ese diseases brucellosis is one of e major correlation wi preferred host species [4]. Currently diseases affecting e dairy industry responsible for low ten species are recognized including e better known productivity [1]. It is an economically important disease six classical species comprised of B. abortus (cattle, of livestock causing reproductive wastage rough biovar 1-6 and 9), B. melitensis (goats, sheep, biovar 1-3), infertility, delayed heat, loss of calves, reduced meat and B. suis (pigs, reindeer and hares, biovar 1-5), B. ovis milk production, culling, dea from secondary infection (sheep), B. canis (dogs) and B. neotomae (desert wood from abortion and economic losses from international rats). More recently, new members to e genus include trade bans []. B. ceti and B. pinnipedialis (dolphins/porpoises and Corresponding Auor: Hika Waktole, Department of Microbiology, Immunology and Veterinary Public Heal College of Veterinary Medicine and Agriculture, Addis Ababa University, P.O. Box: 34, Bishoftu, Oromia, Eiopia. 45

seals respectively), B. microti (voles) and B. inopinata 45kms Sou East of Addis Ababa. The area is located at (reservoir undetermined). Of ese species, B. melitensis 9 N latitude and 40 E longitudes at an altitude of 1850 has e greatest risk for human infection followed by meters above sea level in central high land of Eiopia. B. suis and B. abortus, however several of e oer It has an annual rainfall of 866 mm of which 84% is in e species have been shown to be virulent for human [5]. long rainy season (June to September). The dry season Among e animal brucellosis, bovine brucellosis is extends from October to February. The mean annual e most important disease in many countries around e maximum and minimum temperatures are 6 C and 14 C world due to its economic importance [, 6 & 7]. Bovine respectively, wi mean relatively humidity of 61.3% [8]. brucellosis is an infectious and contagious disease known Study population: The target population was dairy cattle for its impact on reproductive performance of cattle and is at were managed under e intensive production predominantly a disease of sexually mature animals [8, 9]. system, which consists of breeding females, replacement The disease is primarily caused by Brucella abortus and heifers and available bulls. Breed of cattle in dairy farms occasionally by Brucella melitensis where cattle are kept were crosses of local Zebu wi Holstein Frisian breed. togeer wi infected sheep or goats and A total of 400 animals of above six mons of age were characteristically associated wi abortion at first selected of which 54 were breeding females, 134 were gestation and is mainly caused by biovar (mainly biotype- heifers and 1 were serving bulls. None of e animals 1) of B. abortus [10, 5]. Chronic infection of e mammary tested were vaccinated against brucellosis. glands due to Brucella suis has also been reported [11]. Clinically bovine brucellosis is characterized by impaired Study Design fertility specifically wi abortion, metritis, orchitis and Sampling Meodology: A cross sectional study design epididymitis [1]. was employed in is study. Farms were selected Since e first report of brucellosis in e 1970s in purposively based on e willingness of e owners and Eiopia [13, 14] e disease has been noted as one of animals wiin e farm were selected using simple random e important livestock diseases in e country [15-18]. sampling meod. The sample size of e dairy cattle was A large number of studies on bovine have been reporting calculated on e basis of 5.6% prevalence of bovine individual brucellosis seroprevalence ranging from 1.1 to brucellosis in and around Addis Ababa [15]. Therefore, to.6% in intensive management systems [19-] and 0.05 determine e sample size of dairy cattle in is area, 11.% -15.% in extensive management system [3-7]. was used as Pexp and 95% confidence interval and 5% Cross breeding indigenous cattle wi high yielding required precision [9]. exotic cattle is e main policy established by e Eiopian government to bridge e gap between supply ZxPexp x(1- Pexp) n = and demand for dairy products. Owners of dairy cattle and d institutions promoting e dairy industry require current, where n = e required sample size, Z =Confidence level reliable scientific data on such important diseases as (regular value=1.96), P = expected prevalence (5.6%) and brucellosis and moreover in Eiopia, despite several d=desired absolute precision (0.05). researches have been undertaken in e area of bovine brucellosis in different parts of e country e disease is still a major problem demanding much research and investigation. This manuscript was put forward wi e objectives of determining e seroprevalence of bovine brucellosis and its associated risk factors in dairy farms in Bishoftu town and to assess knowledge, attitude and practices of e farm owners and attendants regarding to brucellosis. MATERIALS AND METHODS Study Area: The study was conducted in Bishoftu town from December 015 to April 016. Bishoftu is located (1.96) x 0.056 x (1-0.056) n= = 81 (0.05) However, a total of 400 dairy cattle were sampled in order to increase e precision and reliability of e data collected and because of e availability of adequate resources. Collection of Blood Samples: Approximately 10 ml of blood sample was obtained from e jugular vein of each animal using plain vacutainer tubes and sterile needles. After identification, each animal was labeled on e corresponding vacutainer tube; e tubes were set tilted overnight at room temperature to allow clotting. 46

Next morning, sera were decanted into cryovials and each Corporation) and analyzed using STATA version 11.0 cryovial containing e serum was labeled. Then Rose for Windows (Stata Corp. College Station, Texas, USA). Bengal Plate test was conducted. Finally, serum samples The seroprevalence was calculated as e number of were kept at -0 C at Addis Ababa University, College of seropositive samples divided by e total number of Veterinary Medicine and Agriculture, microbiology samples tested. To identify association of seropositivity laboratory until e positive sera were submitted for wi e potential risk factors (age, herd size, parturition complement fixation test to e National Veterinary pen, abortion history, abortion period, retained fetal Institute (NVI), Bishoftu, Eiopia. membrane and parity) were computed by Pearson s Chi-square test. Serological Tests Rose Bengal Plate Test (RBPT): All sera samples RESULTS collected were initially screened by RBPT using RBPT antigen (Veterinary Laboratories Agency, New Haw, Among e 400 serum samples at were tested by Addlestone, Surrey, KT15 3NB, United Kingdom) e RBPT for screening of brucellosis, 15 (3.75%) were according to OIE [3]. Briefly, sera and antigen were taken positive. Out of 15 RBPT positive sera, 1 were positive from refrigerator and left at room temperature for half an for CFT and all of em were female cattle. The overall hour before e test to maintain to room temperature and seroprevalence of bovine brucellosis in e study area processed following e recommended procedures. was 3% (Table 1). Out of e tested cows of above years age (54), Complement Fixation Test (CFT): Sera at tested ere were 41(16.14%) and 45(17.7%) cows wi history positive to RBPT were furer tested using CFT for of abortion and retained fetal membrane respectively confirmation using standard B. abortus antigen S99 (Table ). (Veterinary Laboratories Agency, New Haw, Addlestone, A Chi-square analysis revealed abortion history, Surrey, T15 3NB, United Kingdom). Preparation of e abortion period, retained fetal membrane and parity were reagent was evaluated by titration and performed significantly associated (P< 0.05) wi seropositivity of according to protocols recommended by World bovine brucellosis an among oer factors considered Organization for Animal Heal [48] Sera wi strong during e study (Table 3). reaction, more an 75% fixation of complement (3+) at a From 66 farms studied, 64.58, 7.73 and 100% of e dilution of 1: 5 or at least wi 50% fixation of complement farm owners and attendants in small, medium and large (+) at a dilution of 1:10 and above were classified as herd sizes responded as ey were aware of brucellosis positive and lack of fixation/complete hemolysis was respectively. It was also found out at all farm owners of considered as negative. e study area were dependent on culling of e known Brucella infected animals while most of farm owners Questionnaire Survey: The questionnaire was designed dispose after bir to open dump in small and medium to obtain information from cattle attendants in e farms herd size farms. From a total of 66 cattle attendants and and data wi regard to e farms such as herd size, use of owners interviewed, only 18.18% wear protective gloves maternity pens, breeding meod, disposal of aborted during handling aborted material and 34.85% responded materials and replacement strategies. During each sample at ey consume raw milk (Table 4). collection e sex, age, parity, breed, history of abortion The study, based on e questionnaire survey, and retained fetal membranes were recorded. Furermore, revealed at all farms in e study area had no frequent knowledge on brucellosis, wheer ey use protective contact wi oer herds. Of e 66 farms assessed by gloves during handling of aborted materials or not and questionnaire survey, it was found at 87.5% of small consumption of raw milk was included in e farms, 63.64% of medium farms and 71.43% of large farms questionnaire survey. used AI for breeding purpose. The practices of provision of separate parturition pens were 100% in large farms, Data Analysis: Data generated from questionnaire survey 54.55% in medium farms and only 6.5% for small farms. and laboratory investigations were recorded and coded Moreover, all of e farms cull eir animals based on e and entered in to Microsoft Excel spreadsheet (Microsoft reproductive and non-reproductive problems (Table 5). 47

Table 1: Seroprevalence of overall bovine brucellosis in Bishoftu Dairy farms. Number of sera tested RBPT positive CFT positive Prevalence 400 15(3.75%) 1(3%) 3% Table : Percentage of examined animals wi of history of abortion and retained fetal membrane Total number of cows Abortion Retained fetal membrane 54 41(16.14%) 45(17.7%) Table 3: Association of risk factors wi Brucella seropositivity Variables Number of tested animals CFT negative CFT positive (P-value) Age 6 mons to years 9 9(100%) 0(0%) 3.69(0.055) > years 308 96(96.1%) 1(3.9%) Herd size Small 0 0(100%) 0(0%) 5.37(0.068) Medium 101 101(100%) 0(0%) Large 79 67(95.7%) 1(4.3%) Parturition pen No 54 54(0%) 0(0%) 1.94(0.164) Yes 346 334(96.5%) 1(3.47%) Abortion history No 13 07(97.18%) 6(.8%) 10.67(0.001**) Yes 41 35(85.37%) 6(14.63%) Abortion period No abortion 13 07(97.18%) 6(.8%) 9.4(0.000**) First trimester 1 1(100%) 0(0%) Second trimester 8 8(100%) 0(0%) Third trimester 1 15(71.43%) 6(8.57%) Retained fetal membrane No 09 04(97.61%) 5(.39%) 14.5(0.000**) Yes 45 38(84.44%) 7(15.56%) Parity Primiparous 79 79(100%) 0(0%) 5.69(0.017*) Pluriparous 175 163(93.14%) 1(6.86%) * Significant; ** Highly significant Table 4: Knowledge, attitudes and practices (KAP) of farm owners and attendants about Brucella infection in small, medium and large herd size in e study area Proportion of respondents(n) Herd size ------------------------------------------------------------------------------------------------------------------------------------------------- Variables Small(n=48) n (%) Medium(n=11) n(%) Large(n=7) n(%) Total n(%) Awareness about brucellosis No 17(35.4) 3(7.7) 0(0) 0(30.3) Yes 31(64.58) 8(7.73) 7(100) 46(69.7) Brucella infected animal Culling 48(100) 8(7.73) 0(0) 56(84.85) Test and slaughter 0(0) 0(0) 0(0) 0(0) Bo 0(0) 3(7.7) 7(100) 10(15.15) After bir disposal Burning 0(0) 0(0) (8.57) (3.03) Burying 18(37.5) 5(45.45) 5(71.43) 8(4.4) Open dump 30(6.5) 6(54.55) 0(0) 36(54.55) Wearing protective glove No 48(100) 10(90.91) 1(14.9) 59(89.39) Yes 0(0) 1(9.09) 6(85.71) 7(10.61) Raw milk consumption No 30(6.50) 7(63.64) 6(85.71) 43(65.15) Yes 18(37.50) 4(36.36) 1(14.9) 3(34.85) n=number, %=percent 48

Table 5: Summary of e proportion of variables in e ree herd (farm) size Herd size ------------------------------------------------------------------------------------------------------------------------------------------------- Variables Small(n=48) Frequency (%) Medium(n=11) Frequency (%) Large(n=7) Frequency (%) Total No. (%) Frequent contact wi oer herd No 48(100) 11(100) 7(100) 66(100) Yes 0(0) 0(0) 0(0) 0(0) Service type AI 4(87.5) 7(63.64) 5(71.43) 54(81.81) Bull 0(0) 3(7.7) 1(14.8) 4(6.06) Bo 6(1.5) 1(9.09) 1(14.9) 8(1.1) Parturition pen No 45(93.95) 5(45.45) 0(0) 50(75.76) Yes 3(6.5) 6(54.55) 7(100) 16(4.4) Cleaning of calving pen Flushing wi water 34(70.83) 6(54.55) 0(0) 40(60.61) Bo* 14(9.17) 5(45.45) 7(100) 6(39.39) Replacement stock Buy in (4.17) 0(0) 0(0) (3.03) Raise own stock 4(87.5) 8(7.73) 5(71.43) 55(83.33) Bo 4(8.33) 3(7.7) (8.57) 9(13.64) Culling criteria Reproductive problem 0(0) 0(0) 0(0) 0(0) NRP 0(0) 0(0) 0(0) 0(0) Bo 48(100) 11(100) 7(100) 66(100) Bo*=Flushing wi water and disinfection wi detergent, n=number, %=percent NRP=Non-reproductive problem DISCUSSION zone who reported only female positive reactors. The explanation for is finding could be at male animals are In e present study, e overall prevalence of bovine kept for a shorter time an females and us e chance brucellosis in Bishoftu dairy farms was 3%. This overall of exposure is lower for males [37]. It was also reported seroprevalence of 3% was comparable wi e findings at serological response of male animals to Brucella of oer auors in Eiopia; 3.% in Tigray region by infection is limited. It was specified at e testes of Berhe et al. [3], 3.1% in Jimma zone by Ibrahim et al. [18], infected male animals were usually observed to be non-.9% in central oromia by Jergefa et al. [30] and 3.5% in reactors or showed low antibody titers [38]. On e oer souern and eastern Eiopia by Megersa et al. [7]. hand, Asfaw et al. [15] reported a 0.11% seroprevalence Similarly, comparable seroprevalence was reported from among male animals while Hailemelekot et al. [0] reported some oer countries: 4.% in Eritrea [31], 3.3% in Central.11% seroprevalence in extensive management system. Africa [3]. However, higher seroprevalence rates an e Even ough age was not significantly associated present study were reported by Eshetu et al. [16], 10% in wi Brucella seropositivity (P> 0.05) a seroprevalence of Addis Ababa by [33], 14.96% in Norwestern parts of 3.9% was found among adult age group (> years of age) Eiopia; Dinka and Chala [4], 11.% in East Showa zone whereas no Brucella seorpositivity was observed in of Oromia regional state and Megersa et al. [34], 8% in young age group (6 mons to years) of dairy cattle in Pastoral region of Eiopia. The reasons for e low e study site. Several previous reports have indicated prevalence of bovine brucellosis in is study area might at higher seroprevalence of brucellosis in adult age be due to better hygienic practices, use of maternity pen group of cattle [7, 39 & 40] similar to e findings of is and/or separation of cows during parturition, cleaning and study. This could be explained by sexual maturity and disinfection activities, culling of infected animals, pregnancy due to e influence of sex hormones and depending on own herds for replacing stock and farm placenta eryritol on e paogenesis of brucellosis [41]. owners knowledge of brucellosis in ese intensive farms. The present study revealed at a history of The prevalence of e disease in male was nil previous abortion and retained fetal membrane were compared to female animals. This finding is in agreement significantly associated (P<0.05) wi brucellosis wi e work done by Abebe [35] in Tigray region, seropositivity. Using e questionnaire survey, 16.14% Tolosa [36] in Jimma Zone and Degefu et al. [6] in Jijjiga abortion and 17.7% retention of fetal membranes were 49

recorded. The prevalence of abortion in e study area is in agreement wi at of Geresu et al. [40] in which he reported a prevalence rate of 17.39% in Asella and Bishoftu towns. This could be explained by e fact at abortions and retained placenta are typical outcomes of brucellosis. In addition, in highly susceptible nonvaccinated pregnant cattle, abortion after e 5 mon of pregnancy is cardinal feature of e disease [4]. In contrary to is findings, a relatively lower prevalence was reported by Tesfaye et al. [] (4.5%) in Addis Ababa dairy farms and [43] (6.7%) in Nor Gondar, Eiopia. There was statistically significant association (P<0.05) between abortion period and sero- positivity of brucellosis in e present study. This could be explained by e presence of higher seropositivity in cows in e last trimester may be due to e preferential localization of Brucella in e uterus in which allantoic fluid factors such as eryritol could stimulate e grow of Brucella and elevate in e placenta and fetal fluid from about e 5 mon of gestation [44, 41]. This study also revealed at ere is association between parity and seropositivity of bovine brucellosis wi P-value < 0.05 and hence, parity was one of e potential risk factors in e study area. This is probably due to increased contact wi fetal materials and vaginal discharge from infected cows ere by increasing e chance of being infected by B. abortus. This association was in agreement wi e finding of oer investigators [45, 46]. The questionnaire survey of 66 cattle attendants and owners in e farms indicates at 30.3% have no knowledge of brucellosis, only 10.61% wear protective gloves and 34.85% consume raw milk. Presence of high association between brucellosis and abortion as well as retained fetal membranes (Table 4) is indicative of risk to cattle attendants and professionals working in e area wiout precautions and protective cloes. Most cases of brucellosis in human are occupational and occur in e farm attendants, veterinarians and butchers [41]. In addition, most of e respondents in is study wi e small herd size (54.55%) did not bury afterbir (aborted fetus, still bir and retained fetal membrane) raer left em on open dump. These factors combined wi e poor cleaning practice by e owners could pose a great risk of spread of e disease to unaffected animals [36]. Since 34.85% of cattle attendants have habit of milk consumption wiout boiling or pasteurization, e risk from e disease could be high. The possibility of infection occurring by drinking milk necessitates e pasteurization or boiling of milk [47, 48]. CONCLUSIONS Bovine brucellosis caused by B. abortus has a major impact on human heal, besides causing significant economical losses in dairy industry. In e present study, e seroprevalence recorded revealed at brucellosis is an established disease in dairy farms of Bishoftu town. Even ough age was not significantly associated wi brucellosis adult animals were highly infected an younger animals and all of e positive reactors were female animals. In addition, history of abortion, abortion period, retained fetal membrane and parity were significantly associated wi Brucella seropositivity. From questionnaire survey, poor hygienic practices like improper disposal of aborted fetuses and fetal membranes, were identified as potential risk factors which could create favorable condition for e entry and establishment of brucellosis in e dairy farms and consumption of raw milk and absence of habit of using a protective cloes and gloves also act as a risk factor for human brucellosis. In conclusion, e prevailing Brucella seropositivity in e dairy farms indicates e importance of brucellosis in dairy cattle industry of e area and potential public heal implication for human population in e study area. Therefore, awareness creation among farm owners and attendants about e nature and effect of e disease; relative to large herd size dairy farms, hygienic practices are poor in medium and small herd size dairy farms. So at implementation of better management practices like isolation of aborted animals, provision of separate parturition pen and proper disposal of aborted fetuses and fetal membranes practice; since brucellosis has zoonotic importance, habits of pasteurizing of milk before consumption and using of protective gloves during handling of aborted fetus and fetal membrane and continuous surveillance to detect e presence of infection in e dairy farms and quarantine policy designed and implementation were forwarded as recommendations. ACKNOWLEDGEMENTS This study was funded by e Office of Research and Technology Transfer of e Addis Ababa University. We acknowledge College of Veterinary Medicine and Agriculture for e provision of laboratory facilities, National veterinary Institute for CFT tests and Dairy farm owners for allowing us to carry out is study on its farm animals. 50

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