Journal of Bacteriology & Parasitology

Small Animal Brucellosis: Associated Risk Factors, Seroprevalence and Characterization of Brucella Isolates in Two Districts of South Omo Zone Ethiopia

Received: 04-Mar-2021 Published: 25-Mar-2021, DOI: 10.35248/2155-9597.21.s9.001

Introduction

Brucellosis is a multi-species infectious and contagious bacterial disease causing economic losses for livestock production industry in many developing countries worldwide [1,2]. It is the most common zoonosis worldwide with over 500,000 cases every year, often considered as a neglected hazard for the public health [3,4]. Despite efforts made to establish brucellosis control program in different countries, it still represents an endemic disease in several regions worldwide, including the Central Asia, Middle East, Mediterranean region, and parts of Africa, Latin America [5]. Brucellosis is responsible for reproductive losses in livestock animals, that are commonly caused by Brucella melitensis or Brucella ovis in small ruminant, Brucella abortus in cattle, Brucella suis in pigs, and Brucella canis in dogs. Small ruminant brucellosis is the most frequent bacterial zoonosis in low-income countries including Ethiopia where the disease is a major cause of direct economic losses and an impediment to trade and exportation [6]. Brucella melitensis is the main species infecting small ruminants. Despite the economic losses incurred and the wide spread distribution of small ruminant brucellosis in Ethiopia, especially in South Omo Zone, less attention has been paid to the spread of the disease in pastoral areas. The accuracy of the diagnostic tests of brucellosis is an essential component in the success of test, eradication and control strategies. The isolation and identification of Brucella species in small ruminants are essential in these areas where livestock and pastoralists have close contact in their daily life. This could help authorities and decision makers to plan disease control and appropriate prevention strategies [7]. This represents an important challenge as brucellosis is endemic in Ethiopia and commonly causes retained fetal membrane, abortion, as well as infection of the accessory sex gland and orchitis in males [8]. It is a widely distributed neglected zoonotic disease, with poor awareness among the community, and cause serious economic losses in small ruminants’ production industry [9-11]. The incidence of brucellosis is generally considered higher in pastoral settings of Africa. However, because of the difficulty to access pastoral communities, the occurrence and the control of brucellosis is poorly understood both in humans and their animals in the pastoral settings of the sub-Saharan Africa where the burden of the disease could be high [12]. In Ethiopia, small ruminants are the main source of livelihood for small holders under extensive pastoral production system [12]. However, Ethiopia fails to optimally utilize this resource and brucellosis significantly affects livestock productivity. In Ethiopia, several studies showed individual seroprevalence ranging from 0.1%– 15.2% in different parts of the country that most of them are largely confined to serological surveys. Although, isolation of Brucella species is the gold standard for the identification and confirmation of animal brucellosis, there is little research done to isolate and identify causative agents in Ethiopia. A recent study showed poor community’s knowledge about brucellosis and high risk for Brucella infection among pastoralist communities of South Omo Zone [13]. Therefore, the isolation and identification of the prevailing Brucella species and the assessment of potential risk factors are necessary. Accordingly, the aim of this study was to use a combination of serological, biochemical and morphological characterization methods to evaluate the prevalence of Brucella infections among small ruminants as well as to identify the associated risk factors in two districts (Nyangatom and Dassenech) of South Omo Zone.

Materials and Methods

Study area

The study was conducted in two districts (Nyangatom and Dasenech) of South Omo Zone, in the Ethiopian Southern Nations, Nationalities and Peoples' Region (SNNPR) and it is located at 750 km south of Addis Ababa (Figure 1). This Zone has eight districts and two districts were selected for the purpose of this study. These areas were selected because of several factors like: the absence of enough data on the status of brucellosis, large population of small ruminants in the areas and source of the export animals, poor livestock management practices (no constructed houses for small ruminants), seasonal mixing of flocks of different origins. The two districts (Nyangatom and Dassenech) mainly rely on small ruminant production for their livelihood. Nyangatom and Dassenech have an animal resource which is estimated at 415, 292 cattle, 55,100 goats, 48,260 sheep, 11,218 donkeys and 5,474 chicken.

Figure 1: Map showing the two study Districts of South Omo Zone, Ethiopia.

Sampling methods

The target animals for this study were small ruminants of the Nyangatom and Dassenech districts in South Omo Zone. The sample size for serological study was estimated in accordance with previous reports on the seroprevalence of Brucella infection in aborted small ruminants. The sample size was calculated using the below formula with defined precision of 5%, at 95% confidence interval as follows [14].

n= [1.962*p (1-p)]/ d2

(n=required sample size, P=reported prevalence, d=desired absolute precision). A total of 124 small ruminants (sixty-two small ruminants from each district) were considered for this study, while parallel to this, a number of milk and vaginal swabs were sampled from sheep and goats for bacteriological culture. From each of the two districts, three sub districts were included in this study. These sub districts were selected using simple random sampling technique.

Blood, milk and vaginal swab samples collection

Blood samples (86 goats and 38 sheep), milk (8 specimens) and vaginal swab (24 specimens) were collected from selected districts in South Omo zone. The sera were gently decanted into sterile tubes and all samples transported in cold chain to Addis Ababa University, Aklilu Lemma Institute of Pathobiology, Immunology Laboratory, Addis Ababa, Ethiopia and stored at -20°C until further studies. Swab samples were collected with sterile applicator stick in Ames with Charcol Transport Medium (HiMedia, Mumbai, India). Similarly, milk sample were collected aseptically after washing, drying and disinfecting the whole udder and teats. Ten to 20ml mid-stream milk samples were collected from each teat into sterile 50 ml screw capped falcon tubes.

Questionnaire survey

A structured questionnaire survey was made to assess the degree of association between potential risk factors and of Brucella seropositivity. A pre-tested questionnaire on the risk factors was handled to 124 interviewees for 124 small ruminants (86 goats and 38 sheep) and the potential risk factors for brucellosis considered in this study included animal species, age, body condition, abortion history, frequency of abortion and parity number. The awareness and the way of prevention and control of the diseases were interviewed and the knowledge regarding the ways of the disease transmission was also evaluated among animal owners.

Laboratory diagnosis

Serological tests: All serum samples were screened using modified Rose Bengal Plate Test (mRBPT) and Complement Fixation Test (CFT) according to the described procedures [15]. For mRBPT, the agglutinations were recorded as 0, +, ++ and ++ +, according to the degree of agglutination [16]. Brucella positive and negative control sera were also tested along with the test sera to guide in the reading of the results. Then, positive sera were further tested using CFT for confirmation using standard B. abortus antigen S99 according to the proposed procedure of the World Organization for Animal Health.

Bacteriological test for milk and vaginal swab samples: Bacteriological tests were carried out under Biosafety level three (BSL3) with high personal protections at the Brucellosis laboratory. All individual milk and vaginal samples from serologically positive by mRBPT were subjected to bacterial culture. Primary isolation of Brucella spp. was done by inoculating the milk and vaginal samples on a Brucella selective supplement (HiMedia, Mumbai, India) with selective antibiotics supplement (FD005) (HiMedia, Mumbai, India) and inactivated 5% horse serum in Brucella agar (Himedia, Mumbai, India) and incubated for 10 days with or without CO₂ in 37°C with 5 and 10% CO₂ anerobically. Milk samples were centrifuged at 6000 rpm for 15 minutes and the cream and deposit were spread on Brucella selective agar base with supplement (Himedia Mumbai, India). After two weeks of incubation, the bacterial cultures were discarded if no growth was visible [17]. Brucella-suspected colonies were characterized by their typical round and honey drop-like appearance according to Alton [18]. Typical colonies of Brucella spp. were subject to further analysis to determine full identification. Also, suspected Brucella colonies were stained by Stamp’s modification of the Ziehl–Neelsen’s and Gram staining method for subsequent microscopic identification of the organisms. Brucella spp. were identified based on Gram negative, very tiny appearance and coccobacilli shape that were arranged mostly in single but some in pairs and also in clusters according to Alton [19].

Biochemical tests: Further biochemical characterization of the organism was done using (Oxidase test, catalase test, urea hydrolysis, nitrate reduction test, hydrogen sulphide (H2S) production and hemolyisis on blood agar), growth in the presence of thionin and basic fuchsin dyes incorporated at 20 to 40 μg/ml concentrations, Carbon dioxide requirement according to Alton [20].

Ethical consideration: Ethical clearance was obtained from Institutional Review Board of Addis Ababa University, Aklilu Lemma Institute of pathobiology (ALIPB IRB/019/2011/2019). The protocol for field studies and collection of samples from animals was approved by Nyangatom and Dassenech District’s agricultural and veterinary authorities of South Omo Zone and verbal consent from Livestock owners was approved by ethics.

Data quality assurance

All data used for this study were primary data, and were collected by the principal investigator. Statistical analysis (multivariate logistic regression) was done using Stata version 14. Prevalence was computed by dividing the number of test positives by the total number examined multiplied by 100. The Chi-square (χ2) and logistic regression tests was employed to identify possible association between risk factors and reproductive characteristics with seropositive to Brucella infection. The degree of association was considered significant when a P-value of less than 0.05 is obtained or when the 95% confidence intervals of the odds ratio in the multivariable logistic regression analysis, which did not include 1 were considered as significant.

Results

Seroprevalence of brucellosis

The seroprevalence of brucellosis was 24% (95% CI: 0.17-0.32) using mRBPT while it was 21% (95% CI: 0.14-0.28) by CFT tests. Thus, the overall seroprevalence of Brucella infection in aborted small ruminants in Nyangatom and Dassenech districts of South Omo Zone was 21% by the combined mRBPT and CFT tests.

Association of risk factors with Brucella seropositivity

Analysis for association between environmental factor and Brucella infection on the basis of the combined mRBPT and CFT was done using Pearon’s chi-square and Fisher’s exact tests.

There was a significant association between majority of the kebeles (pastoral associations) and seroreactivity to Brucella infection (P<0.05). Among kekeles, high percentages of seropositivity was observed in charrii (32%), while lobot (0.2%) and nikiya (0.2%) were affected at lesser extent.

Age, body conditions, frequency of abortion and parity status were significantly associated to Brucella seropositivity, but no association was shown with animal species and gender (P<0.05).

The difference in seropositivity in chi square and logistic regression among the variable indicates, there were confounding factors (age, body condition, and frequency of abortion) in this study, as they were only significant with chi square, but not when compared with Multivariate logistic regression analysis (confidence interval of their Odds ratio included 1).

According to the multivariable logistic regression model fitted many of the putative risk factors considered: district (animal location), species, age, body condition, frequency of abortion and parity status were found to be significantly associated with Brucella seropositivity. Small ruminants in age group <3 years (OR=0.06, 95% CI: 0.07-0.44) were likely to be at higher risk for Brucella infection than animals in >3-5 and >5 years’ age groups. The multivariate analysis also revealed that increased parity of sheep and goats was more likely to be associated with an increasing risk of getting Brucella infection when evaluated collectively with other factors. Thus, animal with multiple parturition were at higher risk of encountering Brucella infection (OR=0.50, 95% CI: 0.399-0.63) than monoparous animals.

Isolation of Brucella: From a total of 124 seropositive clinical samples, 8 milk and 22 vaginal swabs were subjected to Brucella selective culture and the further characterization of Brucella isolates by biochemical tests. The result showed that 16.7% (5/30) of samples were positive for the Brucella spp. selective culture. All isolates, were from vaginal swabs of 4 goats (13.33%) and 1 sheep (3.33%), while no isolates were found from milk samples, as it may be spoiled, during transportation from long distance.

The isolates were initially recognized on the basis of colony morphology which was characteristic of Brucella growth with very small, smooth, glistening, pin-point and round like colonies with honey like appearance. Microscopic examination was performed and Gram stained cultures revealed small Gramnegative coccobacilli arranged singly and in pairs and on Modified Ziehl-Neelsen (MZN) stain, the Brucella organisms were stained red on a blue background. On different biochemical reactions, the present Brucella species were found to be positive for catalase, oxidase, urea hydrolysis, nitrate reduction tests and all the colonies were grown without 5%-10% CO₂. The isolates were further differentiated biochemically using parameters such as CO₂ requirement, H₂S production, and growth on thionin and basic fuchsin dyes incorporated into tryptic soy agar at different concentrations were tested.

Accordingly, biochemical test, CO₂ requirement and sensitivity to dyes of culture isolates recovered from seropositive goats and sheep vaginal swabs sample revealed that animals were infected by B. melitensis.

Discussion

Given the important role of small ruminants and reliable data on the infection prevalence, several risk factors were described for brucellosis. Among them, animal species, age, body condition, frequency of abortion, and parity were reported as the most important risk factors. This study showed that the overall seroprevalence of brucellosis in small ruminants with history of recent abortion reached 24% (n=30) and 21% (n=26) with mRBPT and CFT, respectively. This report was higher as compared to the seroprevalences of small ruminant brucellosis reported elsewhere in Ethiopia including 7.52% reported in Afar Region, 9.6% in Yabello pastoral Area and 9.11% in Dire Dawa. It was also higher than the report of Mugizi and his colleague, who reported 8% and 11% prevalence in sheep and goats with a history of abortion and retained placenta, respectively in Soroti towns of Uganda. Due to difference in animal husbandry, communal grazing of range lands and watering areas as well as the influence of climatic conditions. The prevalence reported in this study using CFT was higher than the prevalence of 4.2% in small ruminants reported in the same study area. Teshale also reported a seroprevalence of 1.7% in goat and 1.6% in sheep in Somali pastoral areas. Another study also reported seroprevalences of 1.3% in goat and 1.5% in sheep in central highlands of Ethiopia. However, it is important to note that the current study was carried out on animals that had history of recent abortion, which could increase the chance of the seropositivity for brucellosis, since Brucella is a major cause of abortion in small ruminants. The higher prevalence reported in our study could be also due to variation in sensitivity and specificity imparted by the various tests, agroecological location and the number of sampled animals, management and production systems. Most of the prevalence studies used standard Rose Bengal Plate Test (RBPT) for screening, but this study used mRBPT. This simple modification is achieved by increasing the amount of sera for the test dose from 25 to 75 μl, while maintaining the antigen volume at 25 μl. This may significantly increase the sensitivity of the test without affecting the specificity. All other risk factors considered in this study including district, age groups, body conditions, frequency of abortion and parity numbers were found to be associated with Brucella infection in small ruminants as observed by using multivariable logistic regression analysis. Brucellosis is a disease mainly affecting sexually matured animals. Our results showed that adult age (above three years) categories were more likely to be seropositive than young animals (less than one year of age). This study also showed that the frequency of abortions and parity was significantly associated with seropositivity for Brucella infection in the studied animals. This indicates that abortions or stillbirths and retained placenta are typical outcomes of brucellosis in the region. Regarding the distribution of brucellosis in small ruminants having history of recent abortion among the different pastoral Kebeles, the highest seroprevalence was recorded in Charrii followed by Kakuta, Lobot, Nikiya and Trongole and Lorekacho.

There is a significant difference (P<0.05) in the brucellosis prevalence among the four pastoral kebeles of the district. Age, frequency of abortion and parity status remained significant in multivariable logistic regression analysis. Seropositivity for Brucella infection increases approximately 6 times in animals with age >5 years old when compared to ≤ 3 and >3-5 years old. This is in accordance with several previous studies showing a higher seroprevalence of brucellosis in adult age groups of small ruminants. Sexually mature animals are more susceptible to Brucella infection than sexually immature animals which is due to the fact that sex hormones and erythritol, stimulating the growth and multiplication of Brucella spp., tend to increase in concentration with age and sexual maturity.

The multivariate analysis also revealed that increased parity of sheep and goats was associated with an increasing risk of Brucella infection when evaluated collectively with other factors. Thus, animals with multiple parturition were at higher risk of encountering Brucella infection (OR=0.50, 95% CI: 0.399-0.63) than monoparous animals. Interestingly, multivariable logistic regression revealed that the risk of seropositivity was approximately 5.5 times higher in pluriparous animals when compared to monoparous animals. Higher parity was also significantly associated with the disease which is in agreement with the findings of Ashagrie. Generally, animals abort once during the mid-third of gestation but re-invasion of the uterus occurs in subsequent pregnancies with shedding in fluids and fetal membranes. Isolation of Brucella species is the gold standard for identification and confirmation of animal brucellosis. Previous studies in various parts of Ethiopia indicated that the disease is widespread among small ruminant populations. However, most surveys of brucellosis in Ethiopia rely on serological tests only, and there is little evidence for bacteriological isolation of Brucella species. To the best of our knowledge, only one study reported the isolation of B. meltensis from aborted goats in Afar Region. In present study, all isolates were obtained from vaginal swabs, while no isolates were recovered from milk. Shedding of Brucella organisms through body secretion was an important source of infection in humans. Brucella isolates were harvested form vaginal swabs which appeared to be very important in disease epidemiology since farmers assist delivery without any personal protection which aggravates the disease circulation. This stresses the need to coordinated brucellosis prevention strategies for human and animals. This is particularly the case for retained placenta, as the pastoralists usually use their bare hands to pull the placenta out of the vulva. These habits and practices expose them to high risk of contracting brucellosis. Based on biochemical characterization, B. melitensis was recovered from 22.7% (5/22) of vaginal swabs. This is in accordance with results obtained by Tekle et al., in Afar Region (Ethiopia), where 6 out 28 (21.43%) vaginal swabs were infected by B. melitensis. In goats, about two thirds of acute infections which were acquired naturally during pregnancy lead to infection of the reproductive organs, like vagina, and excretion of the bacteria in vaginal secretions during subsequent lactation and there is also evidence that indicated reproductive tract environment favored the growth of this bacteria.

Conclusion

This study revealed that among risk factors for brucellosis in small ruminants, the history of abortion and parity numbers were remarkably associated to Brucella infection in pastoral areas of Ethiopia. Animal owners have poor awareness on brucellosis and proper management of the animals could considerably help to control the spread of the disease in these regions. Among, key features that may aid to improve farmer safety and reduce the transmission of the disease, regular cleaning of the housings and the safe disposal of aborted materials appeared to be essential. In order to prevent brucellosis transmission within the flock or to the other healthy flocks, the regular screening of small ruminants for brucellosis along with the vaccination of animals after birth should be implemented in pastoral areas.

REFERENCES

1. Lawalata HJ, Sembiring L, Rahayu ES. Molecular identification of lactic acid bacteria producing antimicrobial agents from bakasang, an Indonesian traditional fermented fish product. Indonesian J Biotechnol. 2011;16(2);93-99.
2. da Costa RJ, Voloski FLS, Mondadori RG, Duval EH, Fiorentini AM. Preservation of meat products with bacteriocins produced by lactic acid bacteria isolated from meat. J Food Quality. 2019;68(7):1-12.
3. Abushelaibi A, Al-Mahadin S, El-Tarabily K, Shah NP, Ayyash M. Characterization of potential probiotic lactic acid bacteria isolated from camel milk. LWT-Food Science and Technology. 2017;79(1):316-325.
4. Lawalata VN, Irawadi TT, Syamsu K, Suparto IH. Process engineering of langsat fruit peel (Lansium domesticum var. langsat) extraction as antibacterial and antioxidant. IPB University Bogor Indonesia. 2012.
5. Davidson PM, Parish ME. Methods for testing the efficacy of food antimicrobials. Food Technology. 1989;43(1):148-155.
6. Holt JG, Krieg NR, Sneath PH, Stanley JT, Williams ST. Bergey`s manual of determinative bacteriology (9th edn), Williams and Wilkins, Philadelphia, USA, 1994.
7. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39(4):783-791.
8. Saif FAAA. Efficacy of lactic acid bacteria isolated from some fruits and vegetables. Egypt J Microbiol. 2016;51(1):13-28.
9. Malik V, Devi U, Yadav RSN, Mahanta J. 16s Rrna based phylogenetic analysis of Lactobacillus plantarum isolated from various fermented food products of assam. J Microbiol Biotech Food Sci. 2015;5(1):20-22.
10. Wassie M, Wassie T. Isolation and identification of lactic acid bacteria from raw cow milk. Int J Advanced Research in Biol Sci. 2016;3(8):44-49.
11. Daliéa DKD, Deschamps AM, Richard-Forget F. Lactic acid bacteria-potencial for control of mould growth and mycotoxin: review. Food Control. 2010;21(4):370-380.
12. Anggraini L, Marlida Y, Wizna W, Jamsari J, Mirzah M, Adzitey F, et al. Molecular identification and phylogenetic analysis of GABA-producing lactic acid bacteria isolated from indigenous dadih of West Sumatera, Indonesia. F1000Res. 2018;7(1):1663.
13. Behera SS, Ray RC, Zdolec N. Lactobacillus plantarum with functional properties: an approach to increase safety and shelf-life of fermented foods. Biomed Res Int. 2018;2018(1):9361614.
14. Pitcher DG, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol. 1989;8(1):151-156.
15. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-1549.
16. Henry DE, Halami PM, Prapulla SG. Lactobacillus plantarum Mcc2034, a novel isolate from traditional indian lactic fermented preparation: molecular identification and evaluation of its in vitro probiotic potential. J Microbiol Biotech Food Sci. 2015;4(4):328-331.
17. Hiraishi A, Kamagata Y, Nakamura N. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens. J Fermentation Bioeng. 1995;79(1):523-529.
18. White TJ, Bruns TD, Lee SB, Taylor JW. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) Amplification and direct sequencing of fungal RNA genes for phylogenetics (2nd edn), Academic, San Diego, USA, 1990; Pp:315-322.
19. Kalalou I, Faid M, Ahami AT. Extending shelf life of fresh minced camel meat at ambient temperature by Lactobacillus dlbrueckii subsp. delbrueckii. Electronic J Biotechnol. 2004;7(3):1-6.
20. Prawan K, Bhima B. Isolation and characterization of lactic acid bacteria for probiotic application from plant sources. Int J Advanced Research. 2019;5(4):869-874.