Introduction

Ethiopia has a large number of small ruminants, estimated at 31.30 million sheep and 32.74 million goats (1). They are managed in all agroecological zones, mainly in arid and semiarid environments. Most pastoral communities, including the Borana community, rear a large goat population in addition to camels and cattle because of their adaptive nature in harsh environments and use for immediate cash income, apart from a source of food (2-4).

However, despite its significant importance of small ruminants’, reproductive diseases have prevented their productivity. Reproductive problems are usually manifested by; infertility, abortion, stillbirth and weak offspring (5). Among others, brucellosis has been recognized as one of the neglected tropical zoonotic diseases responsible for reproductive problems due to the causative agents affinity to reproductive organs.  The causative agent is fastidious bacteria under the genus Brucella (6,7). Different species of the genus Brucella are responsible for the disease among those, B. melitensis and B. ovis are the most common cause of brucellosis in sheep and goats (8,9), while B. melitensis is the primary cause of goat brucellosis (10).   

Brucellosis is an overlooked disease by animal owners and policymakers, which affects the economy and human health in various ranges. Among health problems reported, retarded growth of the newborn, decreased kidding rate, culling due to infertility, decreased milk yield, abortions, stillbirths, weak neonate, absenteeism at working hours (in humans) and cost of treatment (11-13). Generally, the risk of infection is influenced by the species of Brucella involved such as B. melitensis more serious public health hazard than B. abortus and it is responsible for 70% of all infections (6,14). 

In Ethiopia, the disease was reported in 1970 (15). There was no vaccination practice in Ethiopia before and after the first report. Any positive case reported is designated as caused by natural infection. Following the first report, various studies have demonstrated seropositivity associated with abortion, retained placenta or stillbirth in small ruminants (16,17). Observation showed that 58.68 % of goats and 17.73 % of sheep flocks had abortions in a 12-month survey, with a prevalence of 16.1 % and 12.6 % in doe and ewes at the individual level (18). Furthermore, the overall seroprevalence of brucellosis in small ruminants was higher in goats than in sheep (19).  The overall seroprevalence ranges from 0.24% in Chiro West Haraghe zone (20) to the highest seroprevalence of 17.36% reported in Elwaya, Borena zone (21).  In the Borena pastoral community, few districts were studied, despite several districts and large livestock populations in the area (22).  Most reports are related to serological surveys and isolation of the organism is minimal due to facility shortage (23).  

Wachile district is among the neglected districts in the Borana zone due to road inaccessibility. Although there are frequent abortion cases reported in small ruminants of the district, no survey was conducted. In line with this information gap, this study was designed to address it. Therefore, the objective of the current study was to assess the seroprevalence of brucellosis in small ruminants, associate it with assumed risk factors, and assess the perception of the community.

Materials and methods

 Description of the study areas

The study was carried out in the Wachile district of the Borena zone, Oromia region. Wachile district covers an area of approximately 13,000 km² and is divided into 13 kebele (the smallest government administrative units). It is located at a distance of 695 km south of Addis Ababa and geographical coordinates of 04.55⁰ N latitude and 39.06⁰ E longitude with an altitude of 1095.0 m.a.s.l. The agroecology of the district is characterized by lowland. The mean annual rainfall of the study area ranges from 250 to 700 mm, and the mean annual temperature varies from 19 to over 42ºc. The estimated livestock population of Wachile district is 101,436 Camels, 130,772 Goats, 90,618 Sheep, 1,383,616 Cattle, 3,350 Mules and 5,411 Donkeys (24, 25). 

Study population

The study population is small ruminants kept under the extensive management system in study districts. Small ruminants found in thirteen kebeles were considered in the study population.  Among these, five kebeles were used as the target population, and the determined sample size was selected proportionally based on the population of small ruminants that existed in selected kebeles.   Sheep and goats regardless of sex and age above six months were included in the selection. Kebeles used for sampling were Wachile, Harajarte, Webi, Walensu and Kakallo. All these kebeles were managed under the extensive pastoral management system. There was no Brucella vaccination history.   

Study design and sample size determination

A cross-sectional study was conducted from August 2023 to January 2024 in the Wachile district of Borena Zone. The total sample size was estimated by the formula given for a simple random sampling procedure (26), and calculated using a 95 % confidence interval level at 5% desired absolute precision. Expected prevalence used from nearby district data since there was no previous report of the prevalence of sheep and goat brucellosis in Wachile district. Therefore, the prevalence of 9.2% in goats and 6.1% in sheep from the Yabello district of the Borena zone (near the same agroecological zone) was reported (27). So, we used an expected prevalence of 9.2 % and 6.1% for goats and sheep, respectively; to get a minimum sample size. n= (1.96)2Pexp (1-Pexp)/d² formula is used.

Where n = required sample size, d = desired absolute precision and P = expected prevalence by substituting the value; the calculated value of 216 sample sizes was determined. However, the sample size increased to 324 small ruminants by adding around (50%) to increase precision. Out of these sample sizes, 188 goats and 136 sheep were sampled. 

Sampling Strategy  

A multistage sampling method was used to select sampling units from different flocks.  Wachile district was selected purposively among Borena zone districts based on the information on the study gap. A total of 5 kebeles were selected out of 13 kebele because of high small ruminant population compared to other kebeles. To select individual animals from selected kebele, the proportion of total goat and sheep population in each kebele was calculated and then, based on the size of goat and sheep, the determined sample size was allocated to five selected kebeles. To get a proportional sample size in each kebele the total sample size was divided by the total size of the population in five selected kebeles and calculated into percentages (26). Accordingly, for sheep the sample size was 132 and the total population were 43362 which was calculated as (136/43,362) gotten 0.00314 and for goats, the sample size was 188 and the total population in five kebele were 52,990, which was again calculated as (188/52,990) obtained 0.0036 and these two outputs used as multiplying factor in each kebele population. Then the allocated sample size in each kebele was further again reallocated to flocks within each selected kebele; if the total flock size was less than five animals, then all animals were sampled and if the flock size was above five then a maximum of five animals per flock has been sampled in each kebeles until the required sample size in that kebele attained. Each  animals were selected using a systematic sampling technique from each flock by putting animals in a fence or yard.

Sample collection techniques

Information was collected about individual animals at the time of blood collection.  Sex, species, body condition scores and flock size, pregnancy stage, abortion history, reproduction status, history of retained placenta, kind of kids at birth and introduction of new to the flock were recorded. The flocks were categorized as small (when less than 15 sheep/goats), medium (between 15 to 30 sheep/goats) and large (when greater than 30 sheep/goats) flock sizes. Parity categorization, body condition scoring and age classification are recorded based on the guidelines established by research institutions and scholars (28-34). Abortion was defined as the loss of a fetus before 140 days of pregnancy (28). The gestation stage of aborted fetuses was categorized as first trimester (less than 50 days pregnancy), second trimester (between 51-100 days) and third trimester (101-154 days) (32). The consent of the animal owner was obtained before blood sample collection. If an animal owner refused to accept the consent, then proceeded to the next owner.   Sample collection was managed early in the morning while small ruminants were in their collecting pen otherwise animals would be released for browsing and grazing. It would be difficult to collect samples once they were scattered in the bush. Five to eight millilitres of blood were taken from the jugular vein of each selected animal using a venoject needle and non-heparinized vacutainer tube. After collection, each test tube was coded to coincide with the record sheet. Then the sampled blood was placed in a slant position to allow clotting at room temperature and then serum was decanted into cryovial test tubes. Then Serum samples were kept at -20°C in the Wachile district veterinary clinic until transported using cold chain to the Yabello regional veterinary laboratory to conduct a serology test.

Serological test                               

Two types of serological tests were employed for the detection of Brucella antibody: Rose Bengal Plate Test (RBPT) (as a screening test) and i-ELISA (indirect Enzyme-Linked Immunosorbent Assays) as a confirmatory test (DEXX Montpellier and ID vet (IDScreen®) in Louis Pasteur institute France, respectively). All serum samples prepared were subjected to screening tests using Rose Bengal Plate Test (RBPT), after screening all positive samples were further confirmed by iELISA (indirect Enzyme-Linked Immunosorbent Assays) at Yabello regional veterinary laboratory, according to the standard given by (9). Following this protocol, it was done by adding 25μl of antigen and 75μl of serum onto a plate. The antigen and test serum were then completely mixed with the plastic applicator, shaken for 4 min, and the degree of agglutination was visually inspected and recorded as positive or negative for the presence or absence of agglutination (8, 34, 35). RBPT-positive blood sample should be confirmed by a definitive test (36) because it is highly sensitive which results in a false negative. Therefore, Sera tested positive by RBPT were subjected to ELISA (Enzyme-Linked Immunosorbent Assays) for confirmatory test popular and more specific test as a standard assay for the diagnosis of brucellosis, it measures IgG, IgA and IgM antibodies (37).

 Questionnaire survey

Open and closed semi-structured and structured questionnaires were designed to evaluate community perception of small ruminant brucellosis in the study area. Information about each flock was recorded by interviewing owners or herders using the local language (Afaan Oromo). The consent from each interviewee was obtained before the beginning of the questionnaire. For this survey, respondents were selected purposely, from flock owners whose sheep and goats were sampled were selected and interviewed. For the questionnaire survey, heads of household or any individual from the family member whose age is greater than 18 years were considered for interview. Accordingly, a total of 73 pastoral households were interviewed, of these 50 male and 23 female respondents were involved.   

Data management and analysis

Data were recorded and coded in Microsoft Excel spreadsheets before being transferred to statistical software for analysis (Stata TM 14.0, Stata Corporation, and College Station, Texas, USA). The data generated were analyzed for seroprevalence using descriptive statistics and the association of seroprevalence with kebele, species, sex, flock size, age group, BCS and entry of new animals to the flock were analyzed with chi-square (χ2) test using Fisher exact test; Individual seroprevalence was calculated the number of individual positive animals divided by the total samples size and multiplied by 100. Whereas, flock level prevalence was also calculated by dividing the number of flocks having at least one positive animal by the total number of examined flocks and multiplied by 100.   For all analyses, a P <0.05 is taken as statistically significant.

Results

Seroprevalence of brucellosis in small ruminant

The study conducted in small ruminants at Wachile district revealed seventeen positive samples using Rose Bengal Plate Test, of these positive samples only four of them were confirmed positive using I-ELISA (4/324*100= 1.23%). Among these positives goats and sheep share different seroprevalence of 1.6% and 0.74%, respectively. The seroprevalence of brucellosis at the kebele level shows that only two kebele had positive results as shown in Table 1. The difference between species showed statistical significance (p<0.05), where goats had high seroprevalence compared to sheep.  

Sero-prevalence of brucellosis in different assumed biotic risk factors

Seroprevalence results using i-ELISA showed different prevalences as indicated in Table 4. The sex of small ruminants showed females were relatively higher than males. Out of 73 flocks, only four flocks showed at least one positive recorded (4/73) *100 = 5.5% overall flock seroprevalence obtained.   Seroprevalence at the flock level showed large flock size had the only positive flock with seroprevalence of 10.8% [9.7-11.9] compared to zero prevalence in small and medium flock sizes. The adult age group had relatively higher than the young age group with a seroprevalence of 1.4 and 95% CI of 1.24 – 1.56, whereas the seroprevalence of the young was 0.93 [0.75-1.11].  Small ruminants with poor body condition scores showed higher than medium and good body condition scores. Animals contact with a new group of animals either at the market or introduced in the flock showed all positive for I-ELISA (Table 2). 

Reproductive parameters and brucellosis seroprevalence in small ruminant

Reproductive parameters were measured to learn about any difference in seroprevalence of brucellosis. Among sampled animals’ brucellosis sero-prevalence difference were not significantly (p>0.05) different between monoparity and multiparity.  Whereas, only lactating animals had seropositive under the physiological status of animals, all others (pregnant, dry and young) were negative. Animals with abortion history and abortion at third-trimester pregnancy and with a history of the retained placenta had seropositive results as shown in Table 3.

Seroprevalence was a significant association (p<0.05) with abortion history, trimester period and retained placenta.  Weak lamb and kidding were not associated with seropositive results. Among females’ animals with the history of abortion is 20% (43/216) which is quite large, but still brucellosis seropositive is lower compared to the percentage history record. There are several pathogens responsible for second and third-trimester pregnancy abortion.  Among animals with a history of abortion, 46.5% (20/43) were aborted in the trimester gestation period.

Questionnaire results

Demographic characteristics of respondents

The study comprised a total of 73 participants, selected from 5 Pastoral associations located within Wachile, district of Borena zone. All of interviewed participants were engaged in rearing small ruminant, 68.5% of them were male and 41% of them were in the age group greater than 41 years old. While a substantial proportion of the interviewed participants 79.5% (n=58) were owners of the flock and 78% (n=57) were illiterate as shown in Table 4. 

Community awareness about the disease is limited. The result of this study shows that most of the respondents, 89 % (n=65) and 93% (n=68) did not hear about brucellosis and have no awareness about its zoonotic potential, respectively. A large proportion of participants 49.3 % (n= 36), normally assist delivery. However, all of them that means 100 % (n=73) were used their bare hands (without using protective glove) while helping their animals during parturition and only 19 % (n= 26) of them was properly wash their hands after contact with abortion materials.  Furthermore, 85 % (n=62) of the respondents said that they consume raw animal milk regularly. In addition to raw milk; 2 % (n=3) of the respondents were consumed raw meat.

Despite, around 49.3 % (n=36) of respondents had experienced abortion in their flocks; the majority of households in this study were unable to implement any control measures to prevent the spread of disease. None of the respondents indicated to neither bury nor burn aborted fetuses and fetal membrane; rather 17.8 % (n=13) and 31.5 % (n=23) of them have experience of throw it on the field and feeding them to dogs, respectively as shown in (Table 5 and 6).

Discussion

This study shows that the overall small ruminant seroprevalence of brucellosis in selected pastoral associations of  Wachile district was 1.23% with 95%CI [1.11-1.35], whereas seroprevalence in goats and sheep was 1.6% and 0.74%, respectively; seroprevalence difference between species was not statistically significant(p>0.05).  The current finding was lower than with seroprevalence of 3.2 % and at species level seroprevalence of 3.7 % and 1.4 % in goat and sheep, respectively; from the same agroecological zone of Borena pastoralist area (38).  In addition, other studies in the Borena zone also showed high seroprevalence, where the overall seroprevalence of 8.1% and at species level seroprevalence of 9.2% and 6.1% in goat and sheep, respectively (39); the overall seroprevalence of 4.8%, and species level seroprevalence of 5.8% in goats and 3.2% in sheep reported from Afar region (35). The current seroprevalence of brucellosis in small ruminants is relatively similar with overall seroprevalence of 1.6% as reported by (40, 41). A seroprevalence of 1.6% was reported from Konso (42). Seroprevalence of 1.23% from Korahey Zone, Somalia region (43) and with seroprevalence of 3.5% from Southern Tigray reported by (44). Moreover, seroprevalence findings of 1.7% in goats and 1.6% in sheep from Afar and Somali pastoral regions reported by (45) and seroprevalence of 1.9% in goats and 1.2% in sheep from the Somali region were almost the same seroprevalence with the current study findings (16). Asmare et al (2013) (46) also reported at individual animal level analysis, seroprevalence of 32.5 % (95 % CI 21.9, 43.0) was recorded in pastoral production system, followed by agro-pastoral, 13.0 % (95 % CI 7.0, 19.0) and sedentary production system, 3.6 % (95% CI 1.3, 6.0), complement fixation test was used for confirmatory diagnosis, which was higher than the current study findings (46). The observed difference in seroprevalence could be due to the variation in the use of confirmatory tests and even the skill of the individual to conduct the test, sensitivity and specificity of the various test, agro-ecological location and sample size and sampling strategy, management and production system could play a major role.

It was observed that goats were relatively had higher seroprevalence than sheep, though it was not statistically significant. This finding agrees with reports from Ethiopia and other African countries.  5.8% and 3.2% in goats and sheep, respectively reported from Afar region, North Eastern Ethiopia (35). This finding agrees also with the results of (47) who reported a seroprevalence of 19.6% and 9.4% in caprine and ovine respectively from animals slaughtered at abattoirs in Abuja, Nigeria, which indicated seroprevalence of brucellosis in caprine dominates compared to ovine. This may be associated with high susceptibility of goats than in sheep and higher excretion of the pathogens and transmission might be more than in sheep.

Seroprevalence association between sex, flock size, age group and body condition score showed a non-significant association (p>0.05). Prevalence is also very low compared to other studies, this might be associated with management or other unforeseen factors. However, the history of animals in contact with newly introduced ones showed a significant seroprevalence association (p<0.05), which might indicate that animal movement has a great contribution to the spread of disease. Other studies indicated that large flock size has high seroprevalence compared to small flock size. As reported by (44) who stated larger flock sizes were found to be significantly (p<0.05) associated with Brucella seropositivity in small ruminants. This finding indicates that herd size and animal density are directly contributing to seroprevalence of the disease (5). There is a tendency of increasing prevalence among adults kept in larger flock (46) and reproductive inefficiency has been associated with Brucella exposure (44). There was significant difference observed between age group and sex in other studies contrary to the current study. This study might be associated with low seroprevalence in the area. Sexual maturity in female animals plays a role in Brucella multiplication (5). The high seroprevalence of brucellosis in females might be due to high concentration of erythritol, which is scarcely produced in males’ reproductive organs. The extended period of stay of female animals on the farm exposes them to Brucella organisms and hence the chances of acquiring infection are more than the males (5). Statistically significant (p<0.05) seroprevalence of brucellosis was observed in animals with a history of abortions, those at their third-trimester gestation stage, and animals with a history of previous retained fetal membranes.  This finding is in agreement with the study reported by (48) in central Ethiopia and (49) in northern Ethiopia who reported 44.3%and 40.3%, respectively. 

Pregnant animals are more susceptible to infection by the organism than sexually immature animals. Susceptibility also increases with pregnancy, as stage of gestation increases (50).  This indicates that abortions or stillbirths and retained placenta are typical outcomes of brucellosis (5).  This might be associated with the tropism/preference of Brucella species to the key target cells called trophoblast. Growth of Brucella inside trophoblast is apparently enhanced synergistically in the presence of high concentrations of steroid hormones and erythritol during the final gestation of ruminants. The capacity to replicate readily and extensively in trophoblasts can compromise the integrity of the placenta and infection of fetus, resulting in abortion or birth of week offspring (8,51). 

In this study serological test (I-ELISA) is used for confirmatory diagnosis whereas most other studies, especially those with high seroprevalence used, complement fixation test (CFT) confirmatory diagnosis used. The difference in seroprevalence might also be associated with the types of sensitivity and specificity of diagnostic kits.  According to comparative study conducted among RBPT, CFT and I-ELISA diagnostic tests showed that the highest specificity and sensitivity was observed in I-ELISA (52). In these comparative studies sensitivity of I-ELISA was 100%, which was higher than  RBPT and CFT but similar specificity with CFT (100%). The skill of the laboratory technician and unexpected inflation of the result might be the reason for the difference. The low seroprevalence of the current study despite more sensitivity of I-ELISA in the area dictates brucellosis in small ruminants is very low. Problems associated with abortion and other reproductive problems might be associated with other overlooked etiological agents. Further investigation is important to rule out the major reproductive problems in the area. As it was reported in other districts of Borena zone reproductive problems, especially in abortion cases could be due to chlamydiosis, coxiellosis in addition to brucellosis (53). Community awareness towards brucellosis infection and its zoonotic importance in this study is very low. Their practice knowledge on managing aborted fetuses and fetal membranes is very risky, so they either throw aborted materials into the open field or give it to a nearby dog. Such conditions significantly contribute to the dissemination of agents in the area.

Conclusion

A seroprevalence study conducted at Wachile district showed low seroprevalence. However, the finding is associated with the introduction of new animals to the flock, and seroprevalence is also associated with reproductive problems like abortion at the last gestation period, and retained placenta. According to the information obtained from the owners every small ruminants at their first kidding experienced abortion. This may draw attention to further study. Community educational status is very low which accompanied by low awareness about disease zoonotic importance and poor management of aborted waste is very pronounced. In line with this, the following recommendation is forwarded:

1. The community needs to be trained to prevent the entry of new animals into their flock as this can contaminate the healthy flock
2. Communities also need to be trained about zoonosis and ways of protecting mechanisms for their own and their families
3. The community has to be trained as to how to dispose of wastes associated with dead animals and aborted materials.

Acknowledgements

The authors highly appreciate animal owners for their support during sample collection. Almost all animal owners who were requested for consent have responded positively. Yabello Regional Veterinary Laboratory is acknowledged for being collaborative in conducting the serological test.

Ethical approval

The study was approved by the research ethics review committee (RERC) of the College of Natural and Computational Science under the reference number CNCS-REC031/23 on date 01 November 2023. To obtain consent from the animal owner and keep the welfare of animals while animal handling and blood collection.

Conflict of interest

Both authors declare there is no conflict of interest.