Seroprevalence of small ruminant brucellosis in Werer Agricultural Research Center, Afar Region, North East Ethiopia

Academia Journal of Microbiology Research 3(2): 031-035, December 2015 DOI: 10.15413/ajmr.2015.0107 ISSN 2315-7771 2015 Academia Publishing Research Paper Seroprevalence of small ruminant brucellosis in Werer Agricultural Research Center, Afar Region, North East Ethiopia Accepted 19 th October, 2015 ABSTRACT Bezabih MK 1 * and BultoWC 2 1 College of Veterinary Medicine, Haramaya University, Dire Dawa, Ethiopia. 2 Werer Agricultural Research Center, Afar, Ethiopia. *Corresponding author. E-mail: migkafe@gmail.com; Tel: +251910083862; Fax: +251-0255530201. A cross sectional study was conducted from September 2013 to June 2014 to determine the sero-prevalence of small ruminant brucellosis in Werer Agricultural Research Center. A total of 400 serum samples; 200 from sheep and 200 from goats were collected from semi intensive management system with no history of vaccination with simple random sampling method and the samples were initially screened by Rose-Bengal-Plate Test (RBPT) and positive reactors were further tested by complement fixation test (CFT) for confirmation. Out of the total samples tested 14 (3.5%) of detected as seropositive by Rose Bengal Plate Test out of which only 9(2.25%) were positive by CFT, considered as the overall prevalence. The seroprevalence of brucellosis was found higher in goats (3.0%) than sheep (1.5%), in males (2.9%) than females (2.2%), in adults (2.7%) than young (0%). All of the risk factors have no significant difference with the prevalence of brucellosis (P > 0.05). The existence of the disease in the center indicates that the center will be the source of spread for the future. Accordingly, elimination of positive seroreactors, regular monitoring and strict biosecurity has been recommended to control and eradicate the disease from the center. Key words: Brucellosis, CFT, RBPT, seroprevalence, small ruminants. INTRODUCTION Livestock play a significant role, directly or indirectly, in achieving food self-sufficiency in the country. Of the total household cash income from crop and livestock, livestock account for 37 to 87% in different parts of the country (Ayele et al., 2003). The small ruminant population of Ethiopia is about 18.1 million sheep and 14.8 million goats (CSA, 2009). Owing to their high fertility, short generation interval and adaptation even in harsh environments, sheep and goats are considered as an investment and insurance to provide income for the purchase of food during seasons of crop failure. Furthermore, wool and manure are also important by-products of small ruminant productions (MoARD, 2005). There is also a growing export market for sheep and goats meat in the Middle Eastern Gulf states and some African countries. However, the effective livestock productivity remains very low due to lack of sufficient nutrition, poor husbandry practice, the presence of infectious diseases and inadequate disease control activity (DACA, 2006). Among many factors that limit economic return from small ruminants, production diseases stand in the front line. One of such disease which hampers the productivity of small ruminants is brucellosis (Ademosoum, 1994). Brucellosis is a disease caused by infection with bacteria of the genus Brucella and there is a general host preference within the genus. B. melitensis and B. abortus are responsible for late abortions and birth of weak and sickly newborns in cattle sheep and goats. B. melitensis is the main cause of disease in adult male and female sheep and goats (Robinson, 2003). Clinically, the disease is characterized by one or more of the following signs: abortion, retained placenta (Neta et al.,

Academia Journal of Microbiology Research; Bezabih and Bulto. 032 2010), orchitis, epididymitis and rarely arthritis. Abortions in the mid third of gestation that are associated with retained placenta and fetal membranes as well as reduced milk production may be indicative (Godfroid et al., 2004). Diagnosis depends on the isolation of Brucella from abortion material, udder secretions or from tissues removed at post-mortem. Presumptive diagnosis of Brucella infection can be made by assessing specific cellmediated or serological responses to Brucella antigens (Radostits et al., 2000). The spread of an infection from country to country or within the same country generally follows the transfer of infected animals. Brucellosis is also transmitted from farm to farm through wild animals and dogs responsible for carrying around aborted fetuses. Mixing herds at pasture and keeping the animals in shelters during the night, particularly if in such areas parturition takes place, represent major factors for transmission of the infection. Cattle can also be infected from sheep and goat. Dogs and rodents in contact with infected animals may acquire infection. In the transmission cycle, insects and ticks may also be involved. The phenomenon of latency, so common in cattle, has been confirmed also in B. melitensis infected sheep and goat (FAO 2010). Risk factors for brucellosis can be categorized into those determinants necessary for the transmission and maintenance of the disease within herds (herd immunity type of housing stocking density use of maternity pen) and those factors that are required for the transmission of the disease between herds (lack of biosecurity intermingling with other herds sources of water). Factors related to the host the agent the environment and management practices determine the extent of exposure spread and maintenance of brucellosis in a geographical area (Godfroid, 2002). Brucellosis is a zoonotic disease that leads to considerable morbidity resulting in significant loss of working days across the globe and thus perpetuates poverty. Humans develop brucellosis when they come in contact with contaminated animals or animal products. The main species causing zoonotic disease in descending order of severity are B. melitensis B. abortus and B. canis but B. ovis is not zoonotic agent. In humans, the disease is characterized by a multitude of somatic complaints, such as fever, sweating, anorexia, malaise, weight loss, depression, headache and joint pains and is easily confused with malaria and influenza (Seifert, 1996). Small ruminant brucellosis is widely distributed in Ethiopia as whole and in pastoral areas like that of Afar Somali and Borena but few studies have been conducted in the country. The disease was detected in the majority (90%) of the afar regional state districts (Ashenafi et al., 2007) from which Werer Research Center bought majority of animals for research purposes. Therefore, the objectives of this study were to estimate the seroprevalence of small ruminant brucellosis at Werer Agricultural Research Center and to assess the risk factors associated with occurrence of ruminant brucellosis at Werer Agricultural Research Center and to assess the risk factors associated with occurrence of the disease. MATERIALS AND METHODS Description of the study area A cross-sectional study was conducted in Werer Agricultural Research Center from September 2013 to June 2014 which is located at 9 16'N 40 E, and 275 km east of Addis Ababa in the middle Awash valley at an altitude of 740 m.a.s.l. Temperature ranges between a mean maximum of 37 C in June and a mean minimum of 12.8 C in December. The rain fall ranges between 300 and 660 mm per year with annual mean of 550 mm (IAR, 1988). Study population The study animals were exotic, crossbreds and indigenous breeds of sheep and goats kept under semi-intensive management system. The sampling were included all sheep and goats above 6 months of age with no history of previous vaccination against brucellosis. Sample size determination and sampling method Sample size was determined by using standard procedures as described by Thrusfield (1995) with 16% expected prevalence of small ruminant brucellosis in Afar region (Yibeltal 2005), 95% confidence level and 5% desired absolute precision (d 2 ). n= 1.96 2 Pexp (1-Pexp) d 2 Hence, the sample size required (n) as per the above formula is 207 heads of sheep and goats together. However, to increase precision a total of 400 animals (200 sheep and 200 goats) were sampled by simple random sampling method. Data collection Relevant data of the study animals were recorded along with blood specimen s collection. The individual animal details such as the identification number, sex, age, breed and species. Furthermore, was recorded from production record book of the farm. Blood sample collection and serological tests About 8 ml of blood was collected from the jugular vein of

Academia Journal of Microbiology Research; Bezabih and Bulto. 033 Table 1. Seroprevalence of small ruminant brucellosis between species in WARC. Risk factors No of sera tested RBPT Positive CFT Positive (%) (95% CI) χ 2 (P value) Species Ovine 200 6(3%) 3 (1.5%) (0.31-4.3) Caprine 200 8 (4.0%) 6 (3.0%) (1.11-642) 1.023 (0.312) Male 34 2 (5.9%) 1 (2.9%) (0.07-15.3) Female 366 12 (3.3%) 8 (2.2%) (0.95-4.26) Young 65 2 (3.1%) 0 (0%) (0-5.52) Adult 335 12(3.6%) 9 (2.7%) (1.24-5.04) None 36 0 0 (0%) (0-9.74) Once 94 2 (2.1%) 1 (1.1%) (0.03-5.79) Multiple 236 10 (4.2%) 7 (3 %) (1.2-6.02) 2.082(0.776) 1.786(1.81) 2.03 (0.362) Total 400 14 (3.5) 9(2.25%) (1.03-4.23) Each sheep and goats using plain vacutaioner tubes and needles. The blood was allowed to clot for 1-2 h at room temperature, stored horizontally overnight at 4 C, and then the serum was separated from the clot by centrifugation at 2000-3000 rpm for 10-15 min. Then the separated serum was labeled and kept under refrigeration (-20 C) until tested. Rose Bengal plate test (RBPT): The Rose Bengal Plate Test (RBPT) was used as a screening test for the presence of Brucella agglutinins. The test was conducted at Laboratory of National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta. The interpretations of the results were done according to the degree of agglutination. Agglutinations were recorded as 0, +, ++ and +++ which represent the absence of agglutination, barely visible agglutinations, fine agglutination and coarse clumping respectively. Those samples with no agglutination (0) were recorded as negative while others were recorded as positive (OIE, 2008). Complement fixation test (CFT): All sera which tested positive by the RBPT were retested using CFT for further confirmation. The CFT was done at National Animal health Diagnostic and Investigation Center, Sebeta. Standard B. abortus antigen for CFT is used to detect the presence of anti-brucella antibody in the sera. Sera with strong reaction, more than 75% fixation of complement (3+) at a dilution of 1: 50; at least 50% fixation of complement (2+) at a dilution of 1:10 and about 25% fixation of the complement (1+) at dilution of 1:20 were classified as positive (OIE, 2004). Data management and analysis All the collected data were stored in the Microsoft excel spread sheet program and analyzed by using SPSS version 20. Chi-square test was used to determine presence of association of different risk factors with seropositivity and, 95% confidence interval (CI) at 5% cut-off value were set for significance. RESULTS Among the total of 400 sera (200 goats and 200 sheep) tested, 14(3.5%) were positive for brucellosis by RBPT out of which 9(2.25) sera were found positive for Brucella antibodies by CFT. Therefore, the overall prevalence of brucellosis in small ruminants of Werer Agricultural research center was proved to be 2.25% (Table 1). Potential risk factors of small ruminant brucellosis In this study, the seroprevalence of Brucella antibodies was found to be 1.5% in sheep and 3.0% in goats, 2.2% in females and 2.9% in males, 2.7% in adults and 0% in young, and 3%, 1.1% and 0% in females which give birth more than once, only once and didn t give birth respectively but statistical analysis didn t revealed any significant difference

Academia Journal of Microbiology Research; Bezabih and Bulto. 034 Table 2. Seroprevalence of Caprine brucellosis according to risk factors in WARC. Risk factor No of Sera tested RBPT Positive CFT Positive χ 2 (P Value) Females 177 7(4%) 5(2.8%) Males 23 1(4.3%) 1(4.3%) 0.162 (0.687) Young 50 1 (2%) 0 (0%) Adult 150 7 (4.7%) 6 (4%) 2.062 (0.151) None 20 0 (0%) 0 (0%) Once 36 2 (5.6%) 1 (2.8%) 0.684 (0.71) Multiple 121 5 (4.1%) 4 (3.3%) Table 3. Seroprevalence of Ovine brucellosis according to risk factors in WARC. Risk factor No of Sera tested RBPT Positive CFT Positive χ 2 (P Value) Females 189 6 (3.2%) 3(1.6%) Males 11 0(0%) 0(0%) 1.77 (0.674) Young 15 1(6.7%) 0(0%) Adult 185 5(2.7%) 3(1.6%) None 16 0(0%) 0(0%) Once 58 1(1.7%) 0(0%) Multiple 115 5(4.3%) 3 (2.5%) 0.247 (0.619) 1.962 (0.375) among the risk factors (P > 0.05) (Table 1). Also statistical analyses of these risk factors were done separately for sheep and goat. Accordingly, the prevalence of brucellosis in female and male goats was 2.8 and 4.3% (Table 2) while 2(1.0%) and 0(0%) in sheep (Table 3) respectively. The prevalence of brucellosis in females which give birth more than once, once and those which did not give birth were 3.3, 2.8 and 0% in goat (Table 2) and 3(2.5%), 0(0%) and 0(0%) in sheep (Table 3) respectively. Based on age, the prevalence of brucellosis was 4.0 and 1.6% in adult goats and sheep respectively but (0%) in both young goats and sheep (Tables 2 and 3). DISCUSSION This study demonstrated that the overall seroprevalence of small- ruminant brucellosis in Werer Agricultural research center to be 3.5% by the RBPT and 2.25% by CFT out of which 3.0 and 1.5% in goats and sheep respectively. This finding had an agreement with the finding of Teshale et al. (2006) around Afar and Somali regions, 2.6 and 1.1% in goats and sheep respectively. This prevalence is lower than prevalence recorded in previous studies 9.7% in Afar and Somali; 16% in Afar Region (Yibeltal 2005). The difference in the prevalence of brucellosis between the current and previous studies might be attributed to the differences in management systems. In Afar, Pastoral parts of Oromiya and Somali Region large numbers of different species of animals are raised on communal pastures under limited watering areas, where as the livestock management in the current study area is characterized by semi intensive management, animals are raised separately. From a total of 200 goats sera tested, 6(3%) were seropositive for CFT but from 200 sheep sera tested only 3(1.5%) seropositive sheep was detected. This finding is in agreement with the reports of Ashenafi et al. (2007) which indicated that goats (5.3%) are at higher risk of acquiring Brucella infection than sheep (3.2%). All sero reactors were adult animals which agree with the explanation that Brucellosis is essentially a disease of sexually matured animals. This may result from the fact that sex hormones and erythritol, which stimulate the growth and multiplication of Brucella, tend to increase in concentration with age and sexual maturity (Radostits et al., 2000). The present study showed that 2.2% CFT positive sera

Academia Journal of Microbiology Research; Bezabih and Bulto. 035 were found in female sheep and goats and 2.9% males. This could probably disagree with the reason justified by Jain et al. (2012), male animals are less susceptible to infection, due to the absence of erythritol. Moreover, the serological response of male animals to Brucella infection is limited and testes of infected male animals were usually observed to be non-reactors or showed low antibody titers. The controversy of this result is probably due to the low number of male animals sampled since low number of males found in the research center. CONCLUSION AND RECOMMENDATION This study indicated that brucellosis was known to occur among small ruminants in Werer Agricultural Research Center. Breed, age, sex and species, of animals were considered to influence the prevalence of brucellosis among small ruminants in the study area. Among the risk factors high prevalence has been observed in local breeds, adults, and in goats than crossbreeds, young, and sheep respectively. Especially high seroprevalence in local breed sheep and goats indicates the presence of the disease in the region where the local breed small ruminants were brought from. This will be a future risk for the area and other locations where the center distribute the improved breeds. Therefore (i) The seropositive animals should be culled from the farm. (ii) There should be regular diagnosis of brucellosis for the flock and also for newly purchased/introduced animals. (iii) There should be strict biosecurity ACKNOWLEDGMENTS The researchers are grateful to Werer Research Center for funding this study, and National Animal Health Diagnostic and Investigation Center (NAHDIC), Sebeta, for their kind cooperation and generous help during the laboratory work. paper 52. ILRI (Inteernational Livestock Research Institute), Nairobi, Kenya. pp. 35. CSA (2009). 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Livestock marketing in Ethiopia: A review of structure, performance and development initiatives, Socio-economic and policy Research working p Cite this article as: Bezabih MK, Bulto WC (2015). Seroprevalence of small ruminant brucellosis in Werer Agricultural Research Center, Afar Region, North East Ethiopia. Acad. J. Microbiol. Res. 3(2): 031-035. Submit your manuscript at http://www.academiapublishing.org/ajmr