Seroprevalence and Molecular Detection of Bovine Brucellosis

Background: Brucellosis is one of the major zoonotic problems that exist worldwide. Brucellosis is clinically characterized by metritis, mastitis, repeat breeding, abortion in the last trimester of pregnancy, retention of placenta and reduced milk production in the female whereas epididymitis, orchitis and sterility in male. In humans can be highly variable, ranging from nonspecific, flu-like symptoms to undulant fever, arthritis, orchitis and epididymitis. Methods: A total of 567 bovine serum samples was taken from four districts of Brij region of UP. All the samples were processed to detection of prevalence of brucellosis by RBPT, STAT ELISA and confirmation of genes bcsp31, 16SrRNA, omp2 and IS711 by PCR. Result: The prevalence of brucellosis was found to be 07.93% (31/391), 08.69% (34/391) and 10.74% (42/391) shows positive by RBPT, STAT and I- ELISA respectively. In buffalo Out of 176 tested serum sample the seroprevalence was found to be 09.66% (17/176), 10.79% (19/176) and 12.5% (22/176) positive by RBPT, STAT and I- ELISA respectively. Out of 567 samples 18 were positive for Brucella genus specific gene. The higher prevalence of the disease in this region increases the risk of zoonotic transmission and it implies a serious threat to the human population as well as the huge impact on economy due to loss of productivity as well as loss of livestock population.

Epidemiological investigation for brucellosis in dogs of Thrissur

India is endemic to bovine brucellosis, and there is a high potential for transmission of disease from ruminants to dogs. A total of 18 bitches belonging to five different breeds at different stage of abortion (30 days to 65 days of gestation) were selected for this study. Majority of them were showing abortion (88.89 per cent) at 45 to 65 days of the gestation. Microscopic examination of Stamp stained smear obtained from the aborted foetal stomach contents revealed red coccobacillary organisms suggestive of Brucella spp.in three cases. RBPT on paired sera samples on day of presentation and three weeks after abortion showed agglutination within four minutes in five out of 18 female dogs. DNA extracted from the aborted tissues of a RBPT positive Labrador dog yielded amplicons of 193 base pair specific for Brucella spp. on PCR. The results obtained from this study stress the need for screening dogs for canine brucellosis in the current brucellosis surveillance and control programmes.

Evaluación del índice de frecuencia, sensibilidad y especificidad entre iELISA y PAL en el diagnóstico de brucelosis bovina en tanque de leche / Evaluation of the frequency, sensitivity and specificity index between iELISA and PAL in the diagnosis of bovine brucellosis in a milk tank

El objetivo del presente proyecto fue diagnosticar brucelosis bovina en tanque de leche con la Prueba Anillo de Leche (PAL) y ELISA indirecto (iELISA), así el índice de frecuencias para determinar una comparativa entre ambas pruebas para establecer costo beneficio, tiempo de análisis, precisión en los resultados. Se seleccionó una población de 276 bovinos hembra adultos lecheros raza Holstein-Friesian de 38 a 48 meses de edad. La brucelosis se diagnosticó en muestras de leche con la prueba PAL y iELISA, bajo los protocolos establecidos. Los resultados obtenidos con la técnica PAL se tuvieron 77 muestras negativas, 15 positivas y 4 control de la placa 1 con un total de 96 muestras; se obtuvieron 86 negativos, 6 positivos y 4 control de la placa 2 con un total de 96 muestras; y 83 negativos, 9 positivos y 4 control de la placa 3 con un total de 96 muestras. Se tuvo mayor porcentaje de detección 82.8% a Brucella abortus en las muestras analizadas por medio de iELISA que con las muestras analizadas con PAL 46.92%. Se concluye que el índice de frecuencias para determinar una comparativa entre ambas pruebas para determinar costo beneficio, entre PAL y iELISA, tiempo de análisis la PAL es más específica 48.93% que la prueba iELISA 19.83% y adicionalmente la prueba iELISA es más sensible 76.92% que PAL 14.85% para diagnosticar brucelosis bovina en tanque de leche. Se deduce que la prueba iELISA presento más frecuencias positivas, por lo que es una prueba más sensible que la PAL. Sin embargo, PAL es más rápida de realizar y es menos costosa que iELISA.

Progressive Area Elimination of Bovine Brucellosis, 2013–2018, in Gauteng Province, South Africa: Evaluation Using Laboratory Test Reports

Bovine brucellosis is a zoonotic disease of global public health and economic importance. South Africa has had a national bovine brucellosis eradication scheme since 1979; however, no published report on elimination progress from any province exists. We analysed laboratory test results of all cattle herds participating in the Gauteng Provincial Veterinary Services’ eradication scheme between 2013 and 2018. Herd reactor status and within-herd seroprevalence, modelled using mixed-effects logistic and negative binomial regression models, respectively, showed no significant change over the period. However, provincial State Vet Areas, Randfontein (OR = 1.6; 95% CI: 1.2–2.1; p < 0.001) and Germiston (OR = 1.9; 95% CI: 1.5–2.5, p = 0.008) had higher odds of reactor herds than the Pretoria Area and within-herd prevalence count ratios for these areas were 1.5-fold greater than the Pretoria State Vet Area (p < 0.001). Reactor herds were associated with increased herd size (p < 0.001) and larger herd sizes were associated with lower within-herd prevalence (p < 0.001). Despite no evidence of significant progress toward bovine brucellosis elimination in Gauteng province, variability in bovine brucellosis prevalence between State Vet Areas exists. A public health and farmer-supported strategy of ongoing district-based surveillance and cattle vaccination targeting small- to medium-sized herds combined with compulsory test and slaughter of reactors in larger herds is recommended for the province.

Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda

Bovine brucellosis is endemic in Rwanda, although, there is paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n=300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigación y Tecnología Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The RBT seroprevalence was 20.7% (62/300), and 2.9% (8/300) with i-ELISA and 2.9% (8/300) using both tests in parallel. Brucella specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3) and B. melitensis (n=5) while Bruce-ladder PCR also identified B. abortus (n=5) and B. melitensis (n=6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures which is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen the national bovine brucellosis control program through vaccination as well as test- and-slaughter.

Immunization with Brucella abortus S19Δper Conferred Protection in Water Buffaloes against Virulent Challenge with B. abortus Strain S544

Vaccination of cattle and buffaloes with Brucella abortus strain 19 has been the mainstay for control of bovine brucellosis. However, vaccination with S19 suffers major drawbacks in terms of its safety and interference with serodiagnosis of clinical infection. Brucella abortus S19∆per, a perosamine synthetase wbkB gene deletion mutant, overcomes the drawbacks of the S19 vaccine strain. The present study aimed to evaluate the potential of Brucella abortus S19Δper vaccine candidate in the natural host, buffaloes. Safety of S19∆per, for animals use, was assessed in guinea pigs. Protective efficacy of vaccine was assessed in buffaloes by immunizing with normal dose (4 × 1010 colony forming units (CFU)/animal) and reduced dose (2 × 109 CFU/animal) of S19Δper and challenged with virulent strain of B. abortus S544 on 300 days post immunization. Bacterial persistency of S19∆per was assessed in buffalo calves after 42 days of inoculation. Different serological, biochemical and pathological studies were performed to evaluate the S19∆per vaccine. The S19Δper immunized animals showed significantly low levels of anti-lipopolysaccharides (LPS) antibodies. All the immunized animals were protected against challenge infection with B. abortus S544. Sera from the majority of S19Δper immunized buffalo calves showed moderate to weak agglutination to RBPT antigen and thereby, could apparently be differentiated from S19 vaccinated and clinically-infected animals. The S19Δper was more sensitive to buffalo serum complement mediated lysis than its parent strain, S19. Animals culled at 6-weeks-post vaccination showed no gross lesions in organs and there was comparatively lower burden of infection in the lymph nodes of S19Δper immunized animals. With attributes of higher safety, strong protective efficacy and potential of differentiating infected from vaccinated animals (DIVA), S19Δper would be a prospective alternate to conventional S19 vaccines for control of bovine brucellosis as proven in buffaloes.

Brucella abortus S19 GFP-tagged vaccine allows the serological identification of vaccinated cattle

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.