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

DATA AVAILABILITY STATEMENT : All relevant data are within the paper and its Supporting Information files.SUPPORTING INFORMATION : DATA S1. Raw data in excel format and original gel images.Bovine brucellosis is endemic in Rwanda, although, there is a 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 seroprevalence was 20.7% (62/300) with RBT, 2.9% (8/300) with i-ELISA, and 2.9% (8/300) using both tests in series. 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 and this culture prevalence 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 a coordinated national bovine brucellosis vaccination and initiate a test-and-slaughter program that is not presently applicable in Rwanda.The Belgian Directorate-General for Development Cooperation, through its Framework Agreement with the Institute of Tropical Medicine.http://www.plosone.orgam2023Veterinary Tropical Disease

Mortality rates and survival analysis of owned, free-roaming dogs in a resource-limited community, Bushbuckridge, South Africa

Canine rabies can be successfully controlled in dogs through mass vaccination. In populations of free-roaming dogs in resource-limited settings, the maintenance of herd immunity through vaccination is challenged by the high population turnover. Understanding and describing mortality in these populations may therefore assist in the control of rabies. The objective of this study was to determine the rates and causes of mortality in owned, free-roaming dogs in Hluvukani village, Bushbuckridge, South Africa, from May 2014 through July 2015. From the Health and Demographic Surveillance System in Dogs in Hluvukani village, we followed a nested cohort of dogs one year and older over a 12-month period and puppies born to the cohort for 120 days, from May 2014. Deaths were recorded and investigated through verbal autopsy and post-mortem examination. Survival rates from enrolment (adults) or from birth (puppies) were compared using Cox proportional hazard regression models. Of the cohort of 367 adult dogs (203 males and 164 females), 27 died during the follow-up period. The mortality rate was 78 per 1,000 dog-years in the cohort. Adult females had a shorter survival time from enrolment (mean = 341.7 days) compared to adult males (mean = 356 days; p = 0.05). No difference in survival was detected between age groups. Enrolled litters were 62 and 329 enrolled puppies, 135 died before 120 days of age. Mortality in puppies was high with 2,390 deaths per 1,000 dog-years recorded, and a mean survival time of 60 days. No difference in survival was observed between males and females (p = 0.3). In adults and puppies, causes of death were identified as natural (43%), non-natural (53%) and euthanasia (4%). Mortality was low in adult dogs, but very high in the puppies. Despite high population turnover through births and deaths, the vaccination coverage was still sufficient to prevent rabies outbreaks in the village.Poster presented at the University of Pretoria, Faculty of Veterinary Science Faculty Day, August 25, 2016, Pretoria, South Africa.ab201

Characterization of Brucella spp. and other abortigenic pathogens from aborted tissues of cattle and goats in Rwanda

BACKGROUND : Abortions cause tremendous economic losses in food-producing animals and may lead to food insecurity. OBJECTIVES : This study aimed to characterize Brucella spp. and other abortigenic pathogens from aborted tissues of cattle. METHODS : For cattle, aborted tissues (n = 19) were cultured, and Brucella spp. were detected using the genus-specific 16S-23S ribosomal DNA interspacer region (ITS) assay and speciated using Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR assays. Brucella negative samples were screened using the eight abortigenic pathogens PCR panel. Samples from an abortion outbreak that occurred within a goat tribe were included in this investigation. Sera of females (n = 8) and males (n = 2) were analyzed using the Rose Bengal Test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA), while vaginal swabs (n = 3) and aborted tissues (n = 1) were cultured and characterized. RESULTS : The ITS-PCR detected Brucella DNA in cultures from two aborted tissues of cattle (10.5%, [2/19]), which were identified as B. melitensis (n = 1), and B. abortus (n = 1) using AMOS and Bruce-ladder PCR assays. Campylobacter fetus (n = 7) and Leptospira spp. (n = 4) including co-infections (n = 2) of C. fetus and Leptospira spp. were identified from the Brucella negative samples of cattle. Goats (100.0%, 10/10) were brucellosis seropositive on RBT and i-ELISA. Mixed infections caused by B. melitensis and B. abortus were isolated from the vaginal swabs (n = 3) and aborted tissues (n = 1). DISCUSSION AND CONCLUSIONS : This is the first identification of abortion-associated pathogens in aborted cattle indicating the enormous financial losses and a threat to public health. It is therefore essential to include these identified pathogens in the surveillance scheme of veterinary and human services.The Institute of Tropical Medicine, Belgium, and the Department of Veterinary Tropical Disease, South Africa.https://wileyonlinelibrary.com/journal/vms3hj2022Accountin

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

BACKGROUND : Bovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health. MATERIALS AND METHODS : A cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence. FINDINGS : Of the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle. CONCLUSIONS : This study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.The Belgian Directorate-General for Development Cooperation, through its Framework Agreement with the Institute of Tropical Medicine.https://journals.plos.org/plosntdsdm2022Veterinary Tropical Disease

Molecular characterization of Brucella spp. from seropositive herds of cattle farmed at the wildlife-livestock-human interface in Rwanda

DATA AVAILABILITY STATEMENT : The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.Seroprevalence studies showed that brucellosis is prevalent in cattle in Rwanda with no recent study on the characterization of Brucella spp. Therefore, this study aimed to characterize Brucella spp. in seropositive herds of cattle farmed at the wildlife–livestock–human interface. Whole blood samples (n = 118), milk (n = 41), and vaginal swabs (n = 51) were collected from 64 seropositive herds. All samples (n = 210) were inoculated onto modified Centro de Investigacion y Tecnologia Agroalimentaria (CITA) selective medium. Cultures were analyzed to detect Brucella spp. using 16S−23S ribosomal DNA interspacer region (ITS) PCR, the Brucella cultures were speciated using AMOS and Bruce-ladder PCR assays. Brucella spp. were detected in 16.7% (35/210) of the samples established from the samples using ITS-PCR. The AMOS PCR assay identified mixed Brucella abortus and B. melitensis (n = 6), B. abortus (n = 7), and B. melitensis (n = 1) from cultures from blood samples; mixed B. abortus and B. melitensis (n = 1) and B. abortus (n = 4) from cultures from milk samples; mixed B. abortus and B. melitensis (n = 6), B. abortus (n = 8), and B. melitensis (n = 1) from cultures from vaginal swabs. Bruce-ladder PCR assay confirmed B. abortus and B. melitensis cultures. The isolation of Brucella spp. was significantly associated with districts, with the Nyagatare district having more isolates than other districts (p=0.01). This study identified single ormixed B. abortus and B. melitensis infections in cattle samples in Rwanda, which emphasizes the need to improve brucellosis control at the wildlife–livestock– human interface and raise the awareness of cattle keepers, abattoir workers, laboratory personnel, and consumers of cattle products.The Belgian Directorate- General for Development Cooperation, through its Framework Agreement with the Institute of Tropical Medicine.https://www.frontiersin.org/journals/veterinary-scienceam2023Veterinary Tropical Disease

Mortality rates and survival analysis of a cohort of owned adult dogs and puppies in Hluvukani Bushbuckridge South Africa

Rabies is an acute, fatal, progressive, incurable viral encephalitis affecting all warm-blooded animals, including humans. Dogs are the primary reservoir of rabies virus (RABV) in Africa for both humans and animals. Although rabies can be successfully controlled through vaccination, high rates of dog population turnover through births and deaths make the maintenance of herd immunity through vaccination challenging in populations of free-roaming dogs in low-resource settings. Understanding these demographic processes may help find solutions to create stable, vaccinated populations. The primary aim of this study was to determine the rates and causes of mortality in owned, mostly free-roaming dogs in Hluvukani village of the Mnisi community, in Bushbuckridge Municipality, Ehlanzeni District, Mpumalanga Province. The study enrolled a cohort of adult dogs (one year and older) in May and June, 2014, and followed them for 12 months. Litters of puppies were also enrolled at birth and followed for 120 days each. Outcomes were recorded during frequent follow-up visits, and causes of mortality were determined through owner interview ( verbal autopsy?) and post-mortem examination. A cohort of 367 adult dogs was enrolled in the study (203 males and164 females). Of these, 27 (7.4%) died during the follow-up period, and seven (1.9%) were lost to follow-up. We observed a mortality rate of 77.6 per 1,000 dog-years in the cohort (50.8 per 1,000 dog-years in males and 112.7 per 1,000 dog-years in females). There was very high mortality in female dogs, especially from the age of 5 years and above (305.8 per 1,000 dog-years). Female dogs had a shorter survival time (mean = 341.7 days) compared to the male dogs (mean = 355.8 days); this difference was significant with the log-rank test (p = 0.04) and by Cox regression (p = 0.05). Adult dogs of age 5 years and above had shorter survival time (mean = 338.5) than dogs of ages 3-4 years, which in turn had higher survival times (mean = 358.4) than dogs of ages between 1-2 years old (mean = 348.5). The chi-square test gave a p-value of p = 0.06 and the Cox proportional hazard a p-value of p=0.3. Thus, there is a marginally significant difference in survival between the age categories of adult dogs. Twenty-seven verbal autopsy? results were collated including 15 natural deaths, 7 deliberate deaths, 3 accidental deaths and 2 euthanasia?s. Of the 15 deaths classified as natural?, two were considered to be due to snake envenomation. The remaining 13 were considered to be due to infectious and/or parasitic causes. Of the seven deaths classified as deliberate?, four were considered to have been caused by poisoning and three by trauma. The two accidental, non-anthropogenic deaths were due to animal bite wounds, and the single accidental, anthropogenic death was caused by a motor vehicle accident. Two dogs were euthanized because they were unwanted by the owner. Of the 164 females enrolled in the study, 57(34.8%) had at least one litter of puppies during the study period. The total number of puppies enrolled at birth for the study was 329, comprising 152 (46.8%) males, 148 (44.4%) females, and 29(8.8%) puppies of unknown sex. Of the 329 puppies enrolled, 135 (36.6%) died during the follow-up period, and 126 were lost to follow-up. The mortality rate for puppies was 2,389.3 deaths per 1,000 dog-years. Sex-specific mortality rates were 1,811.5 deaths per 1,000 dog-years for male puppies and 2,172.2 deaths per 1,000 dog-years for female puppies. The survival rates were not significantly different between male and female puppies (p=0.3). One hundred and thirty five verbal autopsy results were collated and analyzed from all dead puppies. Of the 54 puppy deaths classified as natural?, 53 were considered to be due to infectious and/or parasitic causes. Of the 23 deaths classified as deliberate?, six were considered to have been caused by poisoning and 17 by trauma. The 47 accidental, non-anthropogenic deaths were considered to be due to the following causes: eaten by mother (n = 18), starvation (n = 8), laid on by mother (n = 7), animal bite (n = 5), drowning (n = 4), suffocation (n = 3) and crushed by a brick (n = 2). The single accidental, anthropogenic death was caused by a motor vehicle accident. Five puppies were euthanized because they were unwanted. In conclusion, free-roaming dogs in this study were owned, and provided for by their owners without reliance on environmental resources. Mortality rate was low in the adult dog cohort. Birth rate in the female cohort was high and the mortality rate was also high in the puppies before they reached 120 days of life. Natural causes were the highest causes of death in puppies, but accidental and deliberate causes were also frequent. This study recommends future validation studies of methods of verbal autopsy in determining causes of death of dogs in underserved, resource-constrained communities like the Hluvukani village in the Mnisi community area of Mpumalanga Province, South Africa.Dissertation (MSc)--University of Pretoria, 2015.tm2016Veterinary Tropical DiseasesMS

Raw data in excel format and original gel images.

(XLSX)</p

Agarose gel electrophoresis for Bruce–ladder PCR products amplified from cultures of tissues from slaughtered cattle.

Lanes M: Gene Ruler 100 bp (ThermoFischer Scientific, Johannesburg, South Africa); lanes 1–5: B. abortus; lanes 6–8: B. melitensis; lane 9: positive control, B. suis ZW45, lane 10: positive control, B. melitensis rev 1, lane 11: B. abortus (REF 544), lane 12: positive control, B. abortus S 19, lane 13: negative control with sterile water.</p

Agarose gel electrophoresis for AMOS PCR products amplified from cultures of tissues from slaughtered cattle.

Lanes M: Gene Ruler 100 pb plus (ThermoFischer Scientific, Johannesburg, South Africa), lanes 1–4: Brucella abortus (496 bp), lanes 5–7: B. melitensis (731 bp), Lanes 9–10: mixed B. melitensis and B. abortus, lane 11: negative control containing sterile water, lane 12: positive control, B. abortus RF544, lane 13: positive control, B. melitensis rev 1.</p

Serological, bacterial culture, and PCR results of samples collected from slaughtered cattle in Rwanda.

Serological, bacterial culture, and PCR results of samples collected from slaughtered cattle in Rwanda.</p