Lessons learned from dealing with the challenge of canine brucellosis in practice.

With more and more dogs being imported to the UK, and no requirement for preimport screening for Brucella canis, veterinary teams are now encountering canine brucellosis on an increasingly regular basis. At BVA Live Mark Moreton and Elizabeth McLennan-Green will reflect on their experiences of developing guidance to help practices manage the risks associated with this zoonotic pathogen.

Characterisation of Brucella species and biovars in South Africa between 2008 and 2018 using laboratory diagnostic data.

BACKGROUND: Brucellosis is an infectious zoonotic bacterial disease of humans and other animals. In the Republic of South Africa (RSA), animal brucellosis is widespread and the current available data on the prevalence of this disease rely solely on serological testing. The primary limitation of brucellosis serology is the lack of discriminatory powers to differentiate between Brucella species and biovars as well as the cross-reactivity observed with other Gram-negative bacteria. AIM: The aim of this study was to conduct a retrospective laboratory-based survey on Brucella species and biovars isolated from various animal species in SA between 2008 and 2018. MATERIAL AND METHODS: The isolation of Brucella species and biovar typing was performed using conventional microbiological techniques. RESULTS AND DISCUSSION: A total of 963 strains of Brucella species were included in this study with a frequency of detection for B. abortus (n = 883; 91.6%) followed by B. melitensis (n = 42; 4.4%), B. ovis (n = 29; 3.0%) and B. canis (n = 9; 0.9%). Of the 883 strains of B. abortus, 90.1% were typed as B. abortus biovar-1 while 5.7% as B. abortus biovar-2, and 3.3% and 0.5% were B. abortus S19 and B. abortus RB51 vaccine strains, respectively. Among the 42 B. melitensis strains, 71.4% were reported as B. melitensis biovar-1 and 26.2% as B. melitensis biovar-3 while 2.4% was B. melitensis biovar-2. CONCLUSION: A retrospective study, such as this one, provides useful information that can be critical in formulating policies and strategies for the control and eradication of brucellosis in animal populations in RSA.

Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs.

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term "orchitis" is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

Seroprevalence of Brucella canis in dogs rescued from South Dakota Indian reservations, 2015-2019.

Canine brucellosis, caused by Brucella canis, is an infectious disease with implications for canine as well as human health. The identification of infected dogs originating from and around two South Dakota Indian reservations prompted an examination of the seroprevalence of B. canis in stray or owner-surrendered dogs from these communities. Using results from in-clinic screening tests of 3898 dogs over more than 4 years, we determined an overall apparent B. canis seroprevalence of 6.8% (adjusted estimated true prevalence of 29.4%), with rates declining over time. The apparent rate was similar to other surveys of stray dog populations in the US. Older dogs were significantly more likely to be B. canis-positive than younger dogs, as were reproductively intact dogs versus altered dogs (although this difference was not statistically significant). There were geographic differences in seropositive rates as well, with higher rates found in dogs originating from one reservation compared to other locations. Current diagnostic tests lack sensitivity to effectively identify all B. canis-infected dogs, but results from this study are valuable for investigating differences among risk factors for infection. Because of the potential for B. canis to infect other dogs and people, stray dog populations should be screened for B. canis before those animals are placed in adoptive homes.

Investigation of the molecular epizootiological characteristics and tracking of the geographical origins of Brucella canis strains in China.

Brucellosis is a global pandemic infectious zoonosis. Brucella canis is a rare source of human brucellosis in China, and its public health significance remains under debate. Moreover, data pertaining to the epizootiological characteristics and geographical origin of B. canis on a nationwide scale are limited, and the risk to public safety posed by B. canis infections is unknown. The MLVA (multilocus variable-number tandem repeat analysis) assay can be helpful to analyse epidemiological correlations among Brucella isolates and to track their geographic origins. To accomplish this task, MLVA-16 was used to analyse the epidemiological links of 63 isolates obtained from dogs and humans. Sixty-three B. canis strains were sorted into three large clusters (A, B and C) and 50 different genotypes (GT1-50), and 43 unique genotypes were represented by single isolates, suggesting that these strains had no obvious epidemiological links and that canine brucellosis is predominantly sporadic in China. The other seven shared genotypes (among a total of 20 isolates) were each represented by two to eight isolates, indicating that strains from each shared genotype were epidemiologically correlated. Five of the shared genotypes were from 16 strains obtained from Beijing, indicating that canine brucellosis in Beijing originates from multipoint outbreaks with multiple sources of infection. Based on comprehensive case analysis of clinical B. canis infection, we preliminarily suggest that human B. canis infections are associated with Mycoplasma pneumoniae infection that results in decreased patient immunity. B. canis may have limited epidemiological significance for the healthy population, but it remains a significant threat to the canine breeding industry and to humans who come into close contact with dogs. Based on MLVA-11 data, B. canis strains were clustered into 16 genotypes and divided into five evolutionary branches; these data confirm that this population covers an extensive geographic area and exhibits characteristics of the origin and evolution of co-existing introduced and locally native lineages. We believe this study will contribute to strengthening efforts to prevent and control canine brucellosis and to improve public understanding of the health risks posed by B. canis.

Diagnosis of canine brucellosis: comparison of various serologic tests and PCR.

Canine brucellosis is an infectious and contagious disease associated with reproductive losses in breeding kennels. As a zoonotic disease, it poses a risk to human health, especially for veterinarians and breeders who handle materials potentially contaminated with Brucella canis. However, canine brucellosis is a neglected and underestimated disease given the difficulties in establishing a definitive diagnosis. We evaluated the frequency of detection of B. canis in 5 breeding kennels by using various serologic methods and PCR. Circulation of B. canis in these kennels was confirmed by bacterial isolation. The frequency of positive serologic results varied from 6.3% by AGID to 16.5% by dot-ELISA. There was no positive serology for smooth Brucella. PCR testing was positive in 13.9% of samples. The only detection tests with reasonable agreement were PCR and 2ME-MAT. The diagnosis of canine brucellosis remains challenging. The use of a single laboratory method, or even the use of different laboratory methods, may not be sufficient to reach a definitive diagnosis.

Risk factors associated with the occurrence of Brucella canis seropositivity in dogs within selected provinces of South Africa.

The growing population of free-roaming dogs in informal communities in South Africa may increasingly place humans at risk of possible zoonotic infections including, but not limited to, Brucella canis. Worldwide, the prevalence of B. canis infection has increased during the last two centuries, resulting in increased reports of dog and human infections. This study investigated the risk factors associated with B. canis infection in dogs in three predefined areas: Gauteng, the Eastern Cape and Western Cape provinces, of South Africa. Dogs aged 7 months and older presented to welfare organisations and breeders in the study areas were selected for sampling. A comprehensive questionnaire on dog ownership, general health and vaccination status was completed prior to sampling. One blood sample of 8 mL was collected aseptically per dog. Then, equal amounts (4 mL) were transferred to the different vacutainer tubes. The 2-mercaptoethanol-tube agglutination tests were used after validation. Fifty-two dogs out of the combined sample of 1191 dogs from the three study areas tested positive for B. canis, representing an overall occurrence of 4.4%. A binomial logistic regression model was fitted to identify risk factors associated with B. canis in dogs within the study areas. Dog age (0.371; p 0.05) and external parasite infestation (0.311; p 0.05) were significantly associated with the B. canis infection. Ownership and sterilisation need to be further investigated as possible risk factors because both had odds ratios of 1684 and 1107, respectively, in the univariate model.

Canine Brucellosis: Old Foe and Reemerging Scourge.

The genus Brucella is a primary cause of reproductive diseases. Widely known as a problem in livestock, Brucella is gaining notoriety as a cause of canine reproductive disease and as a scourge to dog breeders. Only within the last few decades has the risk of severe brucellosis in dogs, and the people who own and work with them, been more fully appreciated. This review summarizes the epidemiology, clinical signs, and advances in diagnosis and management of Brucella canis. Canine brucellosis prevention, owner education, and possible therapies for the future are also discussed.

Detection of Brucella spp. in dogs at Pantanal wetlands.

Canine brucellosis is an infectious disease that produces reproductive disease in both males and females. Although Brucella canis is more common, the infection by Brucella abortus is more frequent in dogs sharing habitats with livestock and wild animals. We decided to investigate the role of dogs in the maintenance of Brucella spp. in the Pantanal wetland. Serum and whole blood samples were collected from 167 dogs. To detect antibodies against B. abortus and B. canis, buffered acidified plate antigen (BAPA) and agar gel immunodiffusion (AGID) tests were performed. To detect Brucella spp., B. abortus and B. canis DNA, PCR was performed using the bcsp31, BruAb2_0168, and BR00953 genes, respectively. To confirm the PCR results, three bcsp31 PCR products were sequenced and compared with sequences deposited in GenBank. The seropositivity rates of 7.8% and 9% were observed for the AGID and BAPA tests, respectively. Positivity rates of 45.5% and 10.8% were observed when testing bcsp31 and BruAb2_0168, respectively, while there was no positivity for BR00953. The sequenced products had 110 base pairs that aligned with 100% identity to B. abortus, B. canis, and B. suis. Considering our results, dogs may be acting as maintenance hosts of Brucella spp. in the Pantanal region.

Brucella canis infection in a young dog with epididymitis and orchitis.

INTRODUCTION: The following case report describes the clinical and diagnostic procedure for suspected brucellosis infection in a dog. A 21 month old intact male Border Collie was presented with an enlarged right testicle and epididymis. The dog was imported to Switzerland from Germany at the age of three months, but was never abroad since then. Clinical and laboratory diagnostic investigation included bacteriology and histology. An initial serological evaluation by means of rapid slide agglutination test (RSAT) was negative. Repeated examination of the same serum by a chromatographic immunoassay (ICT) revealed a positive result. Brucella canis infection was confirmed by culture. The present case is intended to underline the importance of the suspected diagnosis of 'brucellosis' in the presence of reproductive tract problems in dogs. In addition, Brucella canis has zoonotic potential and it is imperative to comply with strict hygiene management.

INTRODUCTION: Le rapport de cas suivant décrit la procédure clinique et diagnostique en cas de suspicion d'infection par la brucellose chez un chien. Un Border Collie mâle intact de 21 mois a été présenté avec un grossissement du testicule et de l'épididyme droits. Le chien avait été importé d'Allemagne en Suisse à l'âge de trois mois, mais n'avait si non jamais été à l'étranger depuis lors. Des examens diagnostiques cliniques et de laboratoire, notamment bactériologie et histologie ont été effectués. Une première évaluation sérologique au moyen du test d'agglutination rapide sur lame (RSAT) était négative. Un examen ultérieur du même sérum par une immunoanalyse chromatographique (ICT) a révélé un résultat positif. L'infection à Brucella canis a été confirmée par culture. Le présent cas souligne l'importance du diagnostic présumé de «brucellose¼ en présence de problèmes de l>appareil reproducteur chez le chien. De plus, Brucella canis a un potentiel zoonotique et il est impératif d'appliquer des mesures d'hygiène strictes.

Seroprevalence of brucellosis in Mississippi shelter dogs.

Canine brucellosis is an emerging disease and compatible with a One Health management approach. Previous research has found higher prevalence of Brucella canis in stray dog populations than in owned animals, and shelter dogs may represent a zoonotic risk to pet owners. Dogs may also contract other Brucella spp., including Brucella suis, which is carried by some feral swine in the United States and poses a public health risk. Diagnostic tests for Brucella spp. are imperfect. Misclassification of disease status can result in serious repercussions for canine and human health including the unnecessary euthanasia of false positive dogs or failure to identify and remove false negative dogs from susceptible populations. Correct interpretation of any diagnostic test requires knowledge of the pre-test probability of disease in the population, therefore the objective of this cross-sectional study was to estimate the seroprevalence of B. canis and B. suis in Mississippi shelter dogs to guide evidence-based diagnostic testing and inform policy recommendations. Banked serum samples from 571 dogs collected in 2016-2017 as a representative sample of the Mississippi shelter dog population were tested for B. canis using a rapid slide agglutination test (RSAT) and for B. suis using a buffered acidified plate agglutination test. No dogs were seropositive for B. suis antibodies. Twenty-eight dogs (4.9%) were seropositive for B. canis antibodies on the RSAT, with 13 dogs (2.3%) remaining positive when retested with the addition of 2-mercaptoethanol to increase specificity. Test prevalence by shelter ranged from 0 to 8.6%. True prevalence was estimated using stochastic modeling to account for test performance and clustering of dogs by shelter. Approximately 65% of modeled shelters did not have seropositive dogs. For shelters where B. canis was present, the mean modeled seroprevalence was 17.8%. This cross-sectional study reveals important information regarding the distribution of B. canis seroprevalence in Mississippi shelter dogs. Current diagnostic tests lack the sensitivity needed to correctly identify individual infected dogs, but population testing may provide a reasonable estimate of disease. Eradication or control measures may be most efficiently allocated to shelters where canine brucellosis has been identified to effectively minimize transmission among dogs and to humans.

Caracterización de la variabilidad genética de cepas de campo de Brucella canis aisladas en Antioquia / Characterization of the genetic variability of field strains of Brucella canis isolated in Antioquia

Brucella canis, un patógeno intracelular facultativo, es responsable de la brucelosis canina, una enfermedad zoonótica que afecta a los caninos y al hombre. En los primeros causa abortos y fallas reproductivas; en el ser humano genera síntomas inespecíficos. En el año 2005 se demostró la presencia de B. canis en Antioquia (Colombia). Las cepas halladas se identificaron como tipo 2. La secuenciación del genoma completo de una cepa de campo denominada Brucella canis str. Oliveri mostró indels específicos de especie; a partir de estos se buscó conocer características genómicas de las cepas de B. canis aisladas y establecer relaciones filogenéticas, así como el tiempo de divergencia de la cepa Oliveri. Se realizó PCR convencional y secuenciación de 30 cepas de campo, se identificaron 5 indels reconocidos en B. canis str. Oliveri, se empleó ADN de Brucella suis, Brucella melitensis y cepas vacunales de Brucella abortus como controles. Se determinó que las cepas de campo estudiadas comparten 4 de los 5 indels de la cepa Oliveri, lo que indica la presencia de más de una cepa de B. canis circulando en la región. El análisis filogenético se realizó con 24 cepas de Brucella mediante secuencias concatenadas de genes marcadores de especie. Se probó la hipótesis del reloj molecular y adicionalmente se realizó test de tasas relativas de Tajima. De esta manera se demostró que la cepa Oliveri, al igual que las otras cepas de B. canis analizadas, divergen de B. suis. Se rechazó la hipótesis del reloj molecular entre las especies de Brucella y se demostró una tasa de evolución y una distancia genética similar entre las cepas de B. canis.

Brucella canis is a facultative intracellular pathogen responsible for canine brucellosis, a zoonotic disease that affects canines, causing abortions and reproductive failure; and the production of non-specific symptoms in humans. In 2005 the presence of B. canis in Antioquia was demonstrated and the strains were identified as type 2. The sequencing of the genome of a field strain denoted Brucella canis str. Oliveri, showed species-specific indel events, which led us to investigate the genomic characteristics of the B. canis strain isolated and to establish the phylogenetic relationships and the divergence time of B. canis str. Oliveri. Conventional PCR sequencing was performed in 30 field strains identifying 5 indel events recognized in B. canis str. Oliveri. ADN from Brucella suis, Brucella melitensis and vaccine strains from Brucella abortus were used as control, and it was determined that all of the studied field strains shared 4 out of the 5 indels of the sequenced Oliveri strain, indicating the presence of more than one strain circulating in the region. Phylogenetic analysis was performed with 24 strains of Brucella using concatenated sequences of genetic markers for species differentiation. The molecular clock hypothesis and Tajima's relative rate test were tested, showing that the Oliveri strain, similarly to other canis species, diverged from B. suis. The molecular clock hypothesis between Brucella species was rejected and an evolution rate and a similar genetic distance between the B. canis were demonstrated.

Investigation and characterization of Brucella canis infections in pet-quality dogs and associated human exposures during a 2007-2016 outbreak in Michigan.

OBJECTIVE To estimate Brucella canis seropositivity rates for purebred dogs being bred by noncommercial breeders, describe epidemiological findings in infected commercial dog-production facilities, and characterize B canis infection in pet dogs and the risk to human health. DESIGN Retrospective descriptive study. SAMPLE 2,799 canine specimens submitted to the Michigan State University Veterinary Diagnostic Laboratory for B canis testing and records of B canis reports provided to the Michigan Department of Agriculture and Rural Development from 2007 through 2016. PROCEDURES Results of B canis laboratory tests and epidemiological findings for reported cases of B canis were reviewed and summarized. Federal and state public health officials were interviewed regarding human B canis infection. State veterinarians were interviewed regarding canine brucellosis reporting and control procedures. RESULTS Estimated B canis seropositivity was 0.4% among purebred Michigan dogs owned by noncommercial breeders. Infection was confirmed in dogs from 17 commercial dog-production facilities, 3 shelters, and 1 rescue agency. Estimated infection prevalence in production facilities ranged from 2 of 22 (9%) to 5 of 6 (83%). Transfer of infected dogs involved 22 Michigan counties and 11 states. Seven of 20 privately owned infected dogs had diskospondylitis; I also had uveitis. Fifty-three veterinary hospital or diagnostic laboratory personnel had inadvertent exposure to the pathogen. Brucella canis was isolated from 1 commercial production facility owner. CONCLUSIONS AND CLINICAL RELEVANCE B canis was uncommon in purebred dogs being bred by noncommercial breeders but endemic in Michigan commercial facilities producing dogs destined to become household pets. Infected pet dogs caused human B canis exposure, and several pet dogs had debilitating disease not associated with the reproductive system.

Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates.

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Sequencing and phylogenetic characterization of Brucella canis isolates, Ohio, 2016.

Brucella canis is one of zoonotic pathogens causing infection in human. In this study, we isolated and sequenced 38 B. canis strains from 11 cases. Core genome multilocus sequence typing analysis classified all B. canis isolates into two genogroups, GI and GII. All 38 isolates cluster together forming a 2016 Ohio cluster, in which they form five subclusters reflecting their geographical differences. Unlike GI, the isolates of the GII are from diverse geographical locations including Asia, America, Africa, and Europe and form Asia and South America clusters. Overall, our findings could be useful to investigate and track B. canis of future outbreaks.

[Characterization of the genetic variability of field strains of Brucella canis isolated in Antioquia]. / Caracterización de la variabilidad genética de cepas de campo de Brucella canis aisladas en Antioquia.

Brucella canis is a facultative intracellular pathogen responsible for canine brucellosis, a zoonotic disease that affects canines, causing abortions and reproductive failure; and the production of non-specific symptoms in humans. In 2005 the presence of B. canis in Antioquia was demonstrated and the strains were identified as type 2. The sequencing of the genome of a field strain denoted Brucella canis str. Oliveri, showed species-specific indel events, which led us to investigate the genomic characteristics of the B. canis strain isolated and to establish the phylogenetic relationships and the divergence time of B. canis str. Oliveri. Conventional PCR sequencing was performed in 30 field strains identifying 5 indel events recognized in B. canis str. Oliveri. ADN from Brucella suis, Brucella melitensis and vaccine strains from Brucella abortus were used as control, and it was determined that all of the studied field strains shared 4 out of the 5 indels of the sequenced Oliveri strain, indicating the presence of more than one strain circulating in the region. Phylogenetic analysis was performed with 24 strains of Brucella using concatenated sequences of genetic markers for species differentiation. The molecular clock hypothesis and Tajima's relative rate test were tested, showing that the Oliveri strain, similarly to other canis species, diverged from B. suis. The molecular clock hypothesis between Brucella species was rejected and an evolution rate and a similar genetic distance between the B. canis were demonstrated.

Detection of Brucella canis infection in dogs by blood culture and bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis.