On-line version ISSN 2224-9435
J. S. Afr. Vet. Assoc. vol.85 n.1 Cape Town Jan. 2014
Simbarashe ChinyokaI; Solomon DhliwayoI; Lisa MarabiniII; Keith DutlowII; Gift MatopeIII; Davies M. PfukenyiI
IDepartment of Clinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe, Zimbabwe
IIAWARE Trust Zimbabwe, Harare, Zimbabwe
IIIDepartment of Paraclinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe, Zimbabwe
A cross-sectional study was conducted in order to detect antibodies for Brucella canis (B. canis) in dogs from urban Harare and five selected rural communities in Zimbabwe. Sera from randomly selected dogs were tested for antibodies to B. canis using an enzyme-linked immunosorbent assay. Overall, 17.6% of sera samples tested (57/324, 95% CI: 13.5-21.7) were positive for B. canis antibodies. For rural dogs, seroprevalence varied from 11.7% - 37.9%. Rural dogs recorded a higher seroprevalence (20.7%, 95% CI: 15.0-26.4) compared with Harare urban dogs (12.7%, 95% CI: 6.9-18.5) but the difference was not significant (p = 0.07). Female dogs from both sectors had a higher seroprevalence compared with males, but the differences were not significant (p > 0.05). Five and two of the positive rural dogs had titres of 1:800 and 1:1600, respectively, whilst none of the positive urban dogs had a titre above 1:400. This study showed that brucellosis was present and could be considered a risk to dogs from the studied areas. Further studies are recommended in order to give insight into the epidemiology of brucellosis in dogs and its possible zoonotic consequences in Zimbabwe. Screening for other Brucella spp. (Brucella abortus, Brucella melitensis and Brucella suis) other than B. canis is also recommended.
Canine brucellosis, caused by Brucella canis (B. canis) was first discovered from episodes of abortion and reproductive failure in beagles in the USA in 1966 (Carmichael 1966). Brucella canis infection is a significant cause of reproductive failure in dogs worldwide (Wanke 2004). In pregnant bitches, the infection localises in the reproductive tract where it causes placentitis with subsequent abortions and stillbirths (Lopes, Nicolino & Haddad 2010). However, early embryonic deaths and resorption can occur a few weeks after mating and may be mistaken for failure to conceive (Lopes et al. 2010). Epididymitis, orchitis, testicular atrophy, poor sperm quality and infertility and loss of libido have been reported in male dogs (Carmichael & Kenney 1968; Hollett 2006). Despite being infected, many dogs in most cases remain asymptomatic and appear to be healthy (Behzadi & Mogheiseh 2011), but severe lymphadenitis involving the retropharyngeal and inguinal lymph nodes may be found (Wanke 2004). In humans, although infrequent, B. canis causes undulant fever (Ramacciotti 1980) or non-specific signs of recurrent fever, headache and weakness (Wallach et al. 2004). However, transmission to humans is reported to be rare, with only 30 cases documented worldwide since the isolation of B. canis in the late 1960s (Hollett 2006).
Infection due to B. canis is endemic in the southern states of the USA and South America but sporadic in Europe and Asia (Corrente et al. 2010). Except in Nigeria (Adesiyun, Abdullahi & Adeyanju 1986; Cadmus et al. 2011) and South Africa (Gous et al. 2005), there is dearth of information on canine brucellosis in Africa. The presumptive diagnosis of canine brucellosis is based on clinical signs and requires further confirmation through culture and isolation of the causative bacteria (Bae & Lee 2009). While culture and isolation is regarded as the gold-standard test for laboratory diagnosis of brucellosis, its sensitivity is low because the brucellae are fastidious micro-organisms that can easily be overgrown by contaminating bacteria. Thus, serological examinations are often used to detect evidence of exposure to B. canis since they are relatively easy to perform and may provide a practical advantage of estimating prevalence in populations (Bae & Lee 2009). Except for two B. canis isolations in two Harare dogs (Gomo 2013), the status of canine brucellosis in Zimbabwe is unknown. Hence, the objective of this study was to detect antibodies to B. canis in urban and rural dogs and to compare the prevalence in the two sectors. The baseline information obtained would provide a basis for future studies, management strategies and policies in controlling and preventing canine brucellosis in animals and humans.
Materials and methods
Study location and collection of serum samples
The study location and collection of serum samples has been described earlier by Dhliwayo et al. (2012) (Figure 1). Briefly, serum samples collected by the Animal and Wildlife Area Research and Rehabilitation Trust (AWARE) from five rural communities (Kariba, Machuchuta, Malipati, Marumani, and Ndhlovu, Victoria Falls) during dog spay or castration campaigns were used for this study. The serum samples were collected just prior to ovariohysterectomy and orchidectomy from apparently healthy rural-owned and stray dogs with unknown medical histories. Serum samples from the Harare urban area were taken from dogs presented to private veterinary practices for routine elective surgery. All rural dogs were considered to be free-roaming, which allows contact with other dogs in the same village. In contrast, based on owners' information, urban dogs were reared in confinement in individual homes.
Testing for Brucella canis antibodies
The detection of IgG antibodies to B. canis in the collected dog sera (n = 324) was carried out using the Brucella canis ImmunoComb® Antibody Test Kit (also called a 'dot assay' or a modified enzyme-linked immunosorbent assay) (Biogal-Galed Laboratories, Israel) as previously described by Muhairwa et al. (2012). Except that for this test, a purified B. canis antigen is attached to the Comb, the procedure is similar to the test for canine Leptospira (Biogal-Galed Laboratories, Israel) described earlier by Dhliwayo et al. (2012). The results were read with a calibrated colour Comb Scale (graded S0 to S6), which was provided with the test kit. A scale of S3, which is equivalent to a positive immune response at a titre of 1:200 by an indirect fluorescent antibody (IFA) test, was considered as the 'cut-off' level of IgG antibodies (http://www.biogal.co.il). Hence, in this study, serum samples giving a Comb Scale score of > S3 (> 1:200 titre) were considered to be positive for B. canis antibodies.
The overall B. canis seroprevalence was calculated from the total number of samples tested and expressed as a percentage. Seropositivity was examined in relation to location (urban vs rural) and sex (female vs male). The X2-test was used to measure differences in proportions between categories and p-values of < 0.05 were considered significant. Association between seropositivity and location or sex was evaluated by calculating X2, the relative risk (RR) and the 95% confidence interval (CI) using Win Episcope software (version 2.0).
The distribution of sampled dogs and their B. canis seroprevalence according to different categories are shown in Table 1. A total of 324 dog serum samples were collected and the overall seroprevalence was 17.6% (95% CI: 13.5% - 21.7%). Overall, rural dogs recorded a higher seroprevalence (20.7%) compared with urban dogs from Harare (12.7%) but the difference was not significant (p = 0.07). No significant (RR = 1.2, 0.9 < RR < 1.4, X2 = 2.9, p = 0.09) association was recorded between B. canis seropositivity and location. For rural dogs, seroprevalence varied from 11.7% - 37.9%, with dogs from Machuchuta recording a significantly (p < 0.01) higher seroprevalence compared to those from Victoria Falls. Female dogs from both sectors had a higher seroprevalence compared with male dogs, but the differences were not significant (p > 0.05). Overall, the association between B. canis seropositivity and sex was not significant (RR = 1.1, 0.9 < RR < 1.5, X2 = 0.7, p = 0.2).
The majority (64.9%) of the seropositive dogs had a titre of 1:200, whilst 22.8% of the positive dogs recorded a titre of 1:400 (Table 2). Five and two of the seropositive rural dogs had titres of 1:800 and 1:1600 respectively, whilst none of the seropositive urban dogs had a titre above 1:400 (Table 2).
Bacteriological isolation and identification offers a definitive diagnosis of B. canis infection in dogs. However, bacteriological isolation is time consuming, difficult to perform and poses a health risk to personnel (Keid et al. 2007; Kim et al. 2006). For epidemiological studies to establish baseline data for canine brucellosis, serological tests of B. canis infection in dogs can be applied successfully without bacteriological methods (Flores-Castro et al. 1977). Rapid slide agglutination tests, 2-mercaptoethanol tube agglutination tests, indirect fluorescent antibody tests, agar gel immunodiffusion and enzyme-linked immunosorbent assays are some of the serological tests for B. canis that are routinely used (Wanke 2004). Serological cross-reactions between B. canis and other Gram-negatives are commonly detected using some tests, particularly the agglutination tests (Kim et al. 2006). In addition, titre variations between individual animals can occur when different tests are applied (Kim et al. 2006). Hence, to circumvent these problems, ELISA techniques including the 'dot-assay' that use purified species-specific antigens and/or monoclonal antibodies have been developed (Radojicic et al. 2001). Radojicic et al. (2001) found the dot-assay technique to be reliable and highly specific for the rapid detection of B. canis antibodies in dogs. Thus, given the high specificity of the 'dot-assay', the results observed in this study are likely to reflect a true exposure of the dogs to B. canis infection.
The present study provides the first serological evidence of B. canis infection in dogs in Harare urban areas and five selected rural communities of Zimbabwe. The survey shows that approximately 18% of the dogs studied had antibodies to B. canis by the ImmunoComb® Dot-ELISA test (Biogal, Israel). The ImmunoComb® Canine Brucella Antibody Test kit has a high sensitivity (98%) and a high specificity (93%) (http://www.biogal.co.il), thus reducing the possibility of false positive and false negative reactions. Despite the lack of test validation, antibodies to B. canis have been detected using a similar testing technique (Muhairwa et al. 2012; Radojicic et al. 2001). Therefore, since there is no vaccine for B. canis (Hollett 2006), the positive results obtained in the present study indicate exposure to B. canis. The very high titres (1:1600) observed in two of the rural dogs most likely point towards acute brucellosis at or around the time of sampling.
In Zimbabwe, there is limited information on canine brucellosis. However, two confirmed Brucella isolates were obtained by the Central Veterinary Laboratory from two dogs in Harare (Gomo 2013). One of the isolates was found to be the same genotype as the B. canis reference strain (REF RM6/66, Le Fleche et al. 2006), whilst the other could not be accurately assigned to a species, so was grouped with B. canis and Brucellus suis (B. suis) bv 3, 4 subcluster (Gomo 2013). The serological results of the present study indicate the presence of brucellosis in both urban and rural dogs. Given the isolation of B. canis in two Harare dogs, brucellosis could be considered to present a risk to dogs from the studied areas and further investigations are warranted. Furthermore, the zoonotic risk of exposure of pet owners, dog handlers and veterinarians to B. canis cannot be overemphasised.
Although not conclusive, the data suggest that rural dogs have a higher brucellosis seroprevalence than their urban counterparts. In addition, a higher seropositivity was recorded in females and this agrees with earlier studies (Cadmus et al. 2011; Xiang et al. 2013). This has been attributed to the fact that if a single male dog is infected and mates with several females, it can transmit the infection through infected semen (Cadmus et al. 2011). Although the transmission of B. canis has been shown to occur through ingestion of contaminated material (Wanke 2004), sexual transmission is believed to be important since the organism is secreted in significant numbers in the semen of infected male dogs (Shin & Carmichael 1999). Previous studies demonstrated a higher prevalence of infection in stray compared with non-stray dogs (Chikweto et al. 2013; Fredrickson & Barton 1974; Lovejoy et al. 1976). Boebel et al. (1979), Brown et al. (1976), Thierrmann (1980) and Wooley et al. (1977) also reported outbreaks of brucellosis in stray dogs. In addition to natural mating, other sources of B. canis include the foetus, placenta, foetal fluids, urine, and vaginal discharges after an abortion or stillbirth. All rural dogs are considered to be free-roaming, thus they have contact with other dogs in the same village and this could probably place them at a greater risk of exposure to brucellosis. However, there is need for further studies to better understand the epidemiology and risk factors of canine brucellosis in urban, kennel and rural settings in the country.
Although B. canis is the main cause of canine brucellosis (Wanke 2004), Bruella abortus (B. abortus), Brucella melitensis (B. melitensis) and B. suis infections have also been reported in dogs (Baek et al. 2003; Barr et al. 1986; Cadmus et al. 2011; Forbes 1990; Hinic et al. 2010). Infection with B. abortus in dogs is associated with ingestion of aborted foetal tissue from infected livestock (Baek et al. 2003; Forbes 1990). The suspected role of dogs in spreading of B. abortus and B. melitensis to neighbouring herds, flocks and humans was reported (Baek et al. 2003). In Zimbabwe, B. abortus, B. melitensis and B. suis have been reported in livestock (Gomo 2013; Gomo et al. 2012; Matope et al. 2009; Mohan et al. 1996). During the present study, only B. canis was screened and further studies should screen for B. abortus, B. melitensis and B. suis to determine if dogs in the country are also exposed to these organisms.
The findings of this study should be viewed in the light of its limitations. Due to lack of clinical details of the studied dogs, limited conclusions can be drawn. In addition, no bacteriological confirmation of seropositive cases was done. Despite these limitations, the study showed the presence of B. canis seropositivity in both urban and rural dogs. The only reported B. canis isolation in Zimbabwe to date is that from two dogs in Harare. Further studies are recommended to give insight into the epidemiology of brucellosis in dogs and its possible zoonotic consequences in Zimbabwe. Such studies should include samples for serology as well as bacteriology from different categories of dogs originating from different ecological regions of the country. Screening for other Brucella species (B. abortus, B. melitensis and B. suis) other than B. canis is also recommended.
The authors are grateful to the Council for Assisting Refugee Academics (CARA) and the AWARE Trust of Zimbabwe for funding this project. Part of the research was supported by funds from the University of Zimbabwe Research Board Grant Number RB/102/2011.
The authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article.
D.M.P. (University of Zimbabwe) was the project leader and responsible for the study design, data analysis and reviewing the manuscript. G.M. (University of Zimbabwe) was the project co-leader and was responsible for the study design and manuscript writing. S.C. (University of Zimbabwe), S.D. (University of Zimbabwe), L.M. (AWARE Trust) and K.D. (AWARE Trust) were responsible for sample collection and testing, literature review and drafting the manuscript.
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PO Box MP 167, Mt Pleasant
Received: 18 July 2013
Accepted: 11 Oct. 2013
Published: 07 Apr. 2014