Transmission of Brucella canis in a canine kennel following introduction of an infected dog.

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

Brucella suis biovar 1 infection in a dog with orchitis in Germany.

In 2021, a case of canine brucellosis diagnosed in a dog with orchitis was presented to a veterinary practice in Germany. Serological testing excluded Brucella (B.) canis as a causative agent, but molecular analysis revealed the presence of B. suis biovar 1. Since biovar 1 is not endemic in Europe and the dog had no history of travel to endemic areas, a comprehensive epidemiological investigation was conducted using whole genome sequence data to determine the source of infection. We describe the clinical progress of the animal and the potential infection of a veterinary clinic employee. The findings highlight the importance of considering less common Brucella species as possible causes of canine brucellosis. The data also emphasize that it is quite challenging to identify Brucella species in a routine diagnostic laboratory and to conduct epidemiological investigations to unveil possible transmission routes.

Clinical investigation and management of Brucella suis seropositive dogs: A longitudinal case series.

BACKGROUND: Brucellosis in dogs caused by Brucella suis is an emerging zoonotic disease. OBJECTIVES: To document clinical characteristics, serology, microbiology, and clinical response to treatment in B. suis-seropositive dogs. ANIMALS: Longitudinal study of 27 privately-owned dogs. Dogs that tested positive by serology, culture, or real-time polymerase chain reaction (qPCR) were included in the study. METHODS: Clinical (physical examination and imaging) and laboratory (serology, hematology, serum biochemistry, and qPCR or culture) assessments were made at baseline and after approximately 3, 6, 12, and 18 months. RESULTS: Dogs were followed for 10 895 dog days, with 17/27 dogs completing the 18-month follow-up. Ten dogs had signs consistent with brucellosis before enrollment (n = 4), at baseline (n = 2) or during follow-up (n = 6), with 2 dogs experiencing relapse of historical signs. Antibody titers persisted for the duration of follow-up in 15/17 dogs (88%). Radiographic (n = 5) and ultrasound (n = 11) findings, of variable clinical relevance, were observed. Brucella DNA and organisms were detected in 3 dogs, all of which had clinical signs, including in the milk of a bitch around the time of whelping. Brucella DNA was not detected in blood (n = 92 samples), urine (n = 80), saliva (n = 95) or preputial swabs (n = 78) at any time during follow-up. Six dogs underwent treatment, all of which achieved clinical remission although remission was not reflected by decreasing antibody titers. CONCLUSIONS AND CLINICAL IMPORTANCE: Most dogs with B. suis infections have subclinical infections. Serology is poorly associated with clinical disease. Excretion of organisms appears rare except in whelping bitches. Clinical management using antibiotics with or without surgery is recommended.

A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species.

Canine brucellosis is primarily caused by Brucella canis, but other Brucella species can also cause the disease. Identifying sequences specific to B. canis and establishing PCR assays that can distinguish between B. canis and other Brucella species is essential to determine the etiology of canine brucellosis and the source of infection and to achieve effective control. We analyzed the gaps and SNPs of genomes I and II from B. canis strain RM6/66 and B. melitensis strain 16M using the Mauve genome alignment software, and the specificity of each of these differential regions was analyzed by BLAST. A 132 bp specific sequence was found between the DK60_915 (glycosyl hydrolase 108 family protein) and DK60_917 (aldose 1-epimerase) loci in B. canis chromosome 1. Further comparative analysis revealed that this is a reverse complement sequence between B. canis and other Brucella species. Then, three primers were designed based on the sequence that could detect B. canis with a 310 bp amplification product or other Brucella species with a 413 bp product. The PCR based on these primers had reasonable specificity and a sensitivity of 100 copies of Brucella DNA. The detection results for the blood samples of the aborted dogs showed a favorable accordance with the Bruce-ladder multiplex PCR assay. In conclusion, we found a specific reverse complement sequence between B. canis and other Brucella and developed a PCR method that allows a more comprehensive identification of the pathogen involved in canine brucellosis. These findings provide an effective means for preventing and controlling brucellosis.

Comparison of BP26, Omp25 and Omp31 and a Multiepitope-Based Fusion Protein in the Serological Detection of Canine Brucellosis.

Background: Brucellosis is one of the most important zoonotic diseases in the world. Canine brucellosis, caused mainly by Brucella canis, is seriously neglected, and there is a lack of accurate diagnostic tools. Methods: In this study, to compare BP26, Omp25, Omp31 and a multiepitope-based fusion protein in the serological detection of canine brucellosis, using 34 brucellosis-positive dog sera and 62 negative control sera, the Brucella outer membrane proteins Omp31, BP26, Omp25 and a multiepitope-based fusion protein were evaluated by iELISA for their potential use as antigens in the serological diagnosis of canine brucellosis. Results: The results showed that the multiepitope-based fusion protein performed best in distinguishing brucellosis-positive and brucellosis-negative dog sera, with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 98.41%. BP26 and Omp31 showed excellent sensitivity in detecting brucellosis-positive dog sera, but their cross reaction to sera infected with Vibrio parahaemolyticus and Listeria monocytogenes may hinder their application as diagnostic reagents. Omp25 lacked sufficient sensitivity and showed limited ability in distinguishing positive and negative dog sera. Conclusion: The multiepitope-based fusion protein can be used as an ideal antigen for serologically diagnosing canine brucellosis currently prevalent worldwide.

Genetic and molecular Omp25 analyses from worldwide Brucella canis strains: Possible mutational influences in protein function.

Brucella canis is responsible for canine brucellosis, a neglected zoonotic disease. The omp25 gene has been described as an important marker for Brucella intra-species differentiation, in addition to the ability to interact with the host immune system. Therefore, this study investigated the omp25 sequence from B. canis strains associated to a phylogenetic characterization and the unveiling of the molecular structure. In vitro analyses comprised DNA extraction, PCR, and sequencing of omp25 from 19 B. canis strains. Moreover, in silico analyses were performed at nucleotide level for phylogenetic characterization and evolutionary history of B. canis omp25 gene; and in amino acid level including modeling, dynamics, and epitope prediction of B. canis Omp25 protein. Here, we identified a new mutation, L109P, which diverges the worldwide omp25 sequences in two large branches. Interestingly, this mutation appears to have epidemiology importance, based on a geographical distribution of B. canis strains. Structural and molecular dynamics analyses of Omp25 revealed that Omp25L109P does not sustain its native ß-barrel. Likewise, the conformation of B-cell epitope on the mutated region was changed in Omp25L109P protein. Even without an evolutive marker, the new identified mutation appears to affect the basic function of B. canis Omp25 protein, which could indicate virulence adaptation for some B. canis strains in a context of geographical disposition.

Transboundary Spread of Brucella canis through Import of Infected Dogs, the Netherlands, November 2016-December 2018.

Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notified to the authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis-endemic areas, posing a threat to the naive autochthonous dog population and humans.

First genome sequence of Chilean Brucella canis SCL strain provides insights on the epidemiology and virulence factors, explaining differences between geographical origins

BACKGROUND: Brucella canis is the etiological agent of canine brucellosis, a worldwide neglected zoonosis that constitutes one of the major infectious causes of infertility and reproductive failure in dogs. Although genomic information available for this pathogen has increased in recent years, here we report the first genome sequencing of a B. canis strain in Chile, and the differences in virulence genes with other B. canis strains. RESULTS: Genome assembly produced a total length of 3,289,216 bp, N50 of 95,163 and GC% of 57.27, organized in 54 contigs in chromosome I, and 21 contigs in chromosome II. The genome annotation identified a total of 1981 CDS, 3 rRNA and 36 tRNA in chromosome I, and 1113 CDS and 10 tRNA in chromosome II. There is little variation between the different strains and the SCL isolate. Phylogenetic analysis showed that the Chilean SCL strain is closely related to B. canis and B. suis strains. Small differences were found when compared to the Serbian isolate, but all strains shared the same recent common ancestor. Finally, changes in the sequence of some virulence factors showed that the SCL strain is similar to other South American B. canis strains. CONCLUSIONS: This work sequenced and characterized the complete genome of B. canis strain SCL, evidencing the complete presence of all the genes of the virB operon, and minor changes in outer membrane proteins and in the urease operon. Our data suggest that B. canis was introduced from North America and then spread throughout the South American continent.

Prevalence and Genomic Characterization of Brucella canis Strains Isolated from Kennels, Household, and Stray Dogs in Chile.

Canine brucellosis caused by Brucella canis is a zoonotic disease that causes reproductive alterations in dogs, such as infertility, abortion, and epididymitis. This pathogen is especially prevalent in South America, and due to the lack of official control programs and the growing trend of adopting dogs it constitutes a public health risk that must be addressed. The aim of this study was to determine the prevalence of B. canis infection in kennel, shelter, and household dogs and to characterize the genomic properties of circulating strains, including ure and virB operons and omp25/31 genes. Samples from 771 dogs were obtained, and the infection was detected by blood culture and/or serology in 7.0% of the animals. The complete ure and virB operons and the omp25/31 genes were detected. Interestingly, we found different single-nucleotide polymorphisms (SNPs) in some of the analyzed genes, which could mean a change in the fitness or virulence of these strains. This study provides further evidence about dogs as a source of B. canis strains that can infect people. This also highlights the need to implement official control programs, including the mandatory testing of dogs, especially stray dogs, before adoption.

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.

Brucella melitensis infection in dog: a critical issue in the control of brucellosis in ruminant farms.

Canine brucellosis is a contagious disease associated with health implications for humans as well as for a wide range of wild and domesticated animals. In this study, 173 dog blood specimens were sampled from herding dogs in three different provinces including Tehran (n = 127), Qom (n = 40) and Alborz (n = 6) provinces. The presence of Brucella antibodies was determined using Rose Bengal plate test (RBPT), slow agglutination test (SAT) and 2-mercaptoethanol (2-ME), respectively. The seropositive samples were further screened using blood culture and PCR tests to identify and differentiate the implicated Brucella species. According to our results, 24.3% (42/173), 13.8% (24/173) and 6.3% (11/173) of blood samples were tested positive using RBPT, SAT and 2-ME, respectively. However, among 42 seropositive samples, only 38.1% (16/42) and 14.2% (6/42) were positive by PCR and culture, respectively. Brucella melitensis biovar 1 and biovar 2 was isolated from the bacterial cultures of 6 blood samples and confirmed by biotyping, AMOS PCR and Bruce-ladder PCR assays. These findings highlight the potential risk of Brucella transmission from dog to humans along with other livestock and reflect the critical role of infected dogs in the persistence of Brucella infections among ruminant farms. This study stresses the need for further epidemiological investigations on canine brucellosis among herding dogs and suggests the systematic screening of the disease among companion animals such as dogs in order to improve brucellosis surveillance and control programs.

Seroprevalence and Risk Factors of Brucellosis in Dogs in Enugu and Anambra States, Nigeria / Seroprevalencia y factores de riesgo de la brucelosis en perros de los Estados Enugu y Anambra, Nigeria

Abstract Seroprevalence of the antibodies of Brucella canis and Brucella abortus in dogs was assessed using a cross-sectional survey in Anambra and Enugu States, Nigeria. A total of 123 Companion dogs made up of 65 clinic dogs, 34 slaughter dogs and 24 household dogs were screened. For B. abortus antibody assay, the collected serum was used for Rose Bengal plate test (RBPT), Serum agglutination test (SAT) and Solid Phase Immunoassay technique with Immunocomb® Canine Brucellosis Antibody Test Kit was used. Out of the 123 dogs screened, none was positive for Brucella abortus antibodies while 34 (27.7%) of the dogs screened were positive for B. canis antibodies. There was a significant association (P<0.05) between infection and sex, the infection was significantly higher (P<0.05) in female than male dogs. Prevalence was significantly higher (P<0.05) in Exotic breeds than in mixed and local dog breeds. There was no association (P>0.05) between infection and antibody titre levels in the different categories of dogs. However, there was significant association (P<0.05) between the presence of Brucella canis antibodies and free roaming of dogs. This study provides the first serological evidence of B. canis infection in dogs in Enugu and Anambra States. This shows that B. canis is endemic in both states, underscoring the need for further studies. Female dogs, exotic breeds and freely roaming dogs are at a higher risk of Brucella infection in the study area; therefore, preventive and control measures are strongly recommended.

Resumen Se evaluó la seroprevalencia de los anticuerpos de Brucella canis y Brucella abortus en perros usando un sondeo transversal en los Estados Anambra and Enugu, Nigeria. Se examinó un total de 123 perros de compañía, de los cuales 65 eran perros de clínica, 34 perros de matadero y 24 perros caseros. Para el ensayo de anticuerpos de B. abortus, el suero muestreado se usó para la prueba de Rosa de Bengala (RBPT), prueba de aglutinación del suero (SAT) y se usó la técnica de inmunoensayo en fase sólida con el kit de prueba de anticuerpos para brucelosis canina Immunocomb®. De los 123 perros analizados, ninguno dio positivo para los anticuerpos de Brucella abortus mientras que 34 (27.7%) de los perros analizados dieron positivo para los anticuerpos de B. canis. Hubo una asociación significativa (P<0.05) entre infección y género; la infección fue significativamente más alta (P<0.05) en las hembras que en los machos. La prevalencia fue significativamente más alta (P<0.05) en las razas exóticas que en las razas cruzadas y las razas locales. No hubo ninguna relación (P>0.05) entre la infección y los niveles de titulación de anticuerpos en las diferentes categorías de perros. Sin embargo, hubo una relación significativa (P<0.05) entre la presencia de anticuerpos Brucella canis y los perros que andan libremente por doquier. Este estudio provee la primera evidencia serológica de infección con B. canis en perros de los Estados Enugu y Anambra. Esto muestra que la B. canis es endémica en ambos estados, enfatizando la necesidad de hacer más estudios. Las hembras, las razas exóticas y los animales que deambulan libremente se encuentran en el riesgo más alto de infección con Brucella en el área de estudio; por consiguiente, se recomienda enormemente tomar medidas preventivas y de control.

Canine Brucellosis: Insights Into the Epidemiologic Situation in Europe.

Brucella canis is one of many responsible pathogens of discospondylitis in dogs and infections require specific management. Little is known about the epidemiologic situation in Europe. The purpose of the study was to get insights into the occurrence of brucellosis in dogs in Europe. The database of a European veterinary laboratory was screened for Brucella positive samples. Additionally, medical records of a veterinary hospital in Germany were screened for diagnosis of discospondylitis and brucellosis. The laboratory received samples from 20 European countries for Brucella testing in dogs: 3.7% of submitted samples were Brucella spp. PCR-positive (61/1,657), and Brucella canis antibodies were identified in 5.4% of submitted samples (150/2,764). Brucella spp. PCR-positive samples originated from Spain (11.1% of submitted samples), Poland (6.7% of submitted samples) and rarely from Italy and France. Samples with Brucella canis antibodies originated from 13 European countries (Sweden, Belgium, Austria, Switzerland, Italy, Finland, Germany, Denmark, Hungary, Norway, Poland, France, Netherlands). Young dogs (0-24 months) had a 5.4-fold increased risk of PCR positive samples. The supplementary medical records search identified four young female dogs (7-30 months) with Brucella canis discospondylitis in Germany. The four dogs had been imported to Germany from Eastern European countries (Moldavia, Romania, Macedonia). In conclusion, infection with Brucella canis needs to be considered in dogs in Europe and diagnostics for Brucella canis infection appear indicated in young dogs with discospondylitis.

Seroprevalence of Brucella canis antibodies in dogs entering a Minnesota humane society, Minnesota, 2016-2017.

BACKGROUND: Canine brucellosis, caused by the bacterium Brucella canis, is a zoonotic and largely reproductive disease of dogs. The disease is a recognized problem in canine breeding populations, and the risk to individuals assisting with birthing is well described. Prior to 2015, all cases of canine brucellosis reported to the Minnesota Board of Animal Health were in dogs used for breeding. In 2015, canine brucellosis was identified in eight Minnesota rescue dogs, all originating from specific geographic areas in South Dakota. Our objective was to measure the seroprevalence of B. canis in stray and previously owned dogs entering a large Minnesota animal rescue organization to determine if our observations represented a localized or generalized disease issue among rescue dogs. METHODS: A stratified random sample of stray and previously owned dogs entering the largest Minnesota animal rescue organization between November 1, 2016 and November 7, 2017, was tested for B. canis antibodies by the 2-Mercaptoethanol Rapid Slide Agglutination Test (2ME-RSAT) (Zoetis d-TEC® CB kit). Sample sizes for each strata were calculated using previously published seroprevalence estimates. Blood from selected dogs was collected, serum harvested, and transported to the Minnesota Veterinary Diagnostic Laboratory for testing. Positive samples in the 2ME-RSAT were shipped to Cornell University for confirmation by Agarose Gel Immunodiffusion (AGID) testing. Demographics, state and setting of origin, and health status were collected on study-dogs. RESULTS: Of the 10,654 dogs accepted by AHS during the study period, 943 (8.9%) were selected for testing. Most study dogs arrived from Oklahoma (28%), Alabama (18%), and Minnesota (12%). The median age of study dogs was 1.5 years; 303 (32%) were intact males and 294 (31%) were intact females. Most study dogs were strays (n = 716, 76%). Of the total, 22 (3.1%) stray and eight (3.5%) owner-surrendered dogs were presumptively positive by RSAT; one (0.11%) of the stray dogs was positive by 2ME-RSAT and confirmed by AGID. The positive dog was a healthy-appearing 1 year-old neutered male beagle from Texas. CONCLUSIONS: The seroprevalence of canine brucellosis in dogs entering Minnesota for adoption from multiple states was low. Never-the-less, care must to be taken to consider all potential risks and outcomes of interstate and international dog trade, including the spread of infectious diseases such as canine brucellosis.

Prevalence of presentation of some zoonotic agents transmitted by canines and felines in Medellín, Colombia / Prevalencia de presentación de algunos agentes zoonóticos transmitidos por caninos y felinos en Medellín, Colombia

ABSTRACT Objective. To determine in Medellín, Colombia, the prevalence of zoonotic agents in canines and felines. Materials and methods. 1501 individuals were sampled for the analysis of zoonotic gastrointestinal parasites by direct coprology and flotation. 500 canine sera were examined by PARP-2ME and MAT for the diagnosis of Brucella canis and Leptospira sp, respectively. 500 feline sera were processed by IFI for the diagnosis of Toxoplasma gondii. The frequency for each zoonosis and the statistical significance for the different variables were established (p≤0.05; OR≥1; 95% CI). Results. 23.6% of canines and 16.3% of felines were positive for gastrointestinal parasites; Ancylostomids and D. caninum were the most prevalent; species, age, sex, sector, socioeconomic level and the month of sampling showed associations with gastrointestinal parasitism in pets. Canines showed a seroprevalence of 6.6% in B. canis and 8.4%, Leptospira sp; in felines 56.2% for T. gondii. All of the above associated with the commune, month of sampling, age and stratum. Conclusions. Pets located in different communes and socioeconomic strata with lower quality of life conditions represent a risk of zoonotic transmission.

RESUMEN Objetivo. Determinar la prevalencia de agentes zoonóticos en caninos y felinos en Medellín, Colombia. Materiales y métodos. Se muestrearon 1501 individuos para el análisis de parásitos gastrointestinales zoonóticos por medio de coprología directa y flotación. Se examinaron 500 sueros caninos por medio de PARP-2ME y MAT para el diagnóstico de Brucella canis y Leptospira sp, respectivamente. Se procesaron 500 sueros felinos por medio de IFI para el diagnóstico de Toxoplasma gondii. Se estableció la frecuencia para cada zoonosis y la significancia estadística para las diferentes variables (p≤0.05; OR≥1; IC 95%). Resultados. El 23.6% de los caninos y 16.3% de los felinos fueron positivos a parásitos gastrointestinales, siendo los Ancylostomideos y D. caninum los más prevalente, respectivamente; la especie, edad, sexo, sector, estrato socioeconómico y el mes de muestreo presentaron asociaciones con el parasitismo gastrointestinal en mascotas. En caninos se evidenció una seroprevalencia del 6.6% para B. canis y 8.4% para Leptospira sp; en felinos del 56.2% para T. gondii. Todas las anteriores asociadas con la zona de muestreo, mes, edad y estrato. Conclusiones. Las mascotas ubicadas en diferentes comunas y estratos socioeconómicos con condiciones de calidad de vida menores representan un riesgo de transmisión zoonótica.

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.

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs.

AIM: This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog's clinical samples. MATERIALS AND METHODS: A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711. RESULTS: The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89). CONCLUSION: We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples.

Development of improved enzyme-based and lateral flow immunoassays for rapid and accurate serodiagnosis of canine brucellosis.

Brucellosis is a widespread zoonotic disease caused by Brucella spp. Brucella canis is the etiological agent of canine brucellosis, a disease that can lead to sterility in bitches and dogs causing important economic losses in breeding kennels. Early and accurate diagnosis of canine brucellosis is central to control the disease and lower the risk of transmission to humans. Here, we develop and validate enzyme and lateral flow immunoassays for improved serodiagnosis of canine brucellosis using as antigen the B. canis rough lipopolysaccharide (rLPS). The method used to obtain the rLPS allowed us to produce more homogeneous batches of the antigen that facilitated the standardization of the assays. To validate the assays, 284 serum samples obtained from naturally infected dogs and healthy animals were analyzed. For the B. canis-iELISA and B. canis-LFIA the diagnostic sensitivity was of 98.6%, and the specificity 99.5% and 100%, respectively. We propose the implementation of the B. canis-LFIA as a screening test in combination with the highly accurate laboratory g-iELISA. The B. canis-LFIA is a rapid, accurate and easy to use test, characteristics that make it ideal for the serological surveillance of canine brucellosis in the field or veterinary laboratories. Finally, a blind study including 1040 serum samples obtained from urban dogs showed a prevalence higher than 5% highlighting the need of new diagnostic tools for a more effective control of the disease in dogs and therefore to reduce the risk of transmission of this zoonotic pathogen to humans.

Polymeric antigen BLSOmp31 in aluminium hydroxide induces serum bactericidal and opsonic antibodies against Brucella canis in dogs.

Polymeric antigen BLSOmp31 is an immunogenic vaccine candidate that confers protection against Brucella canis in mice. In this preliminary study, the immunogenicity and safety of BLSOmp31 adsorbed to aluminum hydroxide gel (BLSOmp31-AH) were evaluated in Beagle dogs. In addition, the potential to elicit serum antibodies with complement-dependent bactericidal activity and/or to enhance phagocytosis by neutrophils were analyzed. Dogs were immunized three times with BLSOmp31-AH by subcutaneous route, followed by an annual booster. The vaccine elicited specific antibodies 3 weeks after the first immunization. Annual booster induced comparable antibody response as the primary series. Humoral immune response stimulated by BLSOmp31-AH did not interfere with routine agglutination test for canine brucellosis. Antibodies demonstrated a high complement-dependent bactericidal activity against B. canis. Moreover, opsonization by immune serum not only stimulated binding and uptake of the bacteria by neutrophils but effectively enhanced the destruction of B. canis. Specific IgG was detected in 3/4 immunized dogs in preputial secretions. The antibody profile corresponded to a marked Th2 response, since IgG1 prevailed over IgG2 and cellular immune response was not detected in vitro or in vivo. These results require further evaluation in larger field studies to establish the full prophylactic activity of BLSOmp31 against canine brucellosis.

Human Brucella canis Infection and Subsequent Laboratory Exposures Associated with a Puppy, New York City, 2012.

Human Brucella canis infection incidence is unknown. Most identified cases are associated with pet dogs. Laboratory-acquired infections can occur following contact with Brucella spp. We identified a paediatric B. canis case, the source and other exposed persons. A 3-year-old New York City child with fever and dyspnoea was hospitalized for 48 h for bronchiolitis. After her admission, blood culture grew B. canis, she was prescribed anti-microbials and recovered. B. canis was also isolated from blood of the child's pet dog; these isolates were genetically similar. The dog originated from an Iowa breeding facility which was quarantined after identification of the dog's infection. Additionally, 31 laboratory workers were exposed and subsequently monitored for symptoms; 15 completed post-exposure prophylaxis. To our knowledge, this is the first report strongly suggesting B. canis zoonotic transmission to a child in the United States, and highlights the need for coordinated control policies to minimize human illness.