MALDI-TOF MS and genomic analysis can make the difference in the clarification of canine brucellosis outbreaks.
ABSTRACT: Brucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of canine brucellosis.
Project description:Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.
Project description:BACKGROUND/OBJECTIVES:Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ?abcBA (Bo?abcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. METHODS:Intracellular growth of the vaccine strain Bo?abcBA was assessed in canine and ovine macrophages. Protection induced by Bo?abcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain Bo?abcBA was assessed in experimentally inoculated dogs. RESULTS:Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The Bo?abcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated Bo?abcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-?, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated Bo?abcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, Bo?abcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. Bo?abcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. CONCLUSION:Encapsulated Bo?abcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ?abcBA has potential as a vaccine candidate for canine brucellosis prevention.
Project description:Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 ?vjbR strain. Mice vaccinated with a dose of 109 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 ?vjbR was capable of activating canine dendritic cells in vitro These data demonstrate that the B. canis RM6/66 ?vjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies.IMPORTANCE Canine brucellosis, caused by Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ?vjbR vaccine strain capable of protecting mice against challenge.
Project description:Background:Brucella canis is a pathogenic bacterium that causes brucellosis in dogs, and its zoonotic potential has been increasing in recent years. B. canis is a rare source of human brucellosis in China, where Brucella melitensis has been the major pathogen associated with human brucellosis outbreaks. In late 2011, a case of a B. canis infection was detected in a human patient in Zhejiang Province, China. To compare the genotypes between strains of B. canis isolated from the patient and from dogs, a multiple-locus variable-number tandem-repeat analysis (MLVA-16) was performed. In addition, the lipopolysaccharide-synthesis-related genes were analyzed with the B. canis reference strain RM6/66. Results:32 B. canis strains were divided into 26 genotypes using MLVA-16 [Hunter-Gaston Diversity Index (HGDI)?=?0.976]. The HGDI indexes for various loci ranged between 0.000 and 0.865. All four Hangzhou isolates were indistinguishable using panel 1 (genotype 3) and panel 2A (genotype 28). However, these strains were distinctly different from other isolates from Beijing, Jiangsu, Liaoning, and Inner Mongolia at Bruce 09. The emergence of a human B. canis infection was limited to an area. Comparative analysis indicated B. canis from canines and humans have no differences in lipopolysaccharide-synthesis locus. Conclusion:The comprehensive approaches have been used to analyze human and canine B. canis isolates, including molecular epidemiological and LPS genetic characteristics. Further detailed analysis of the whole genomic sequencing will contribute to understanding of the pathogenicity of B. canis in humans.
Project description:Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.
Project description:Two clustered clinical cases of canine babesiosis were diagnosed by veterinary practitioners in two areas of northeastern Italy close to natural parks. This study aimed to determine the seroprevalence of babesial infection in dogs, the etiological agents that cause canine babesiosis and the potential tick vector for the involved Babesia spp.The study area was represented by two parks in northeastern Italy: Groane Regional Park (Site A) and the Ticino Valley Lombard Park (Site B). From March to May 2015 ticks were collected from the vegetation in three transects in each site. In the same period, blood samples were collected from 80 dogs randomly chosen from veterinary clinics and kennel located in the two areas. Morphological identification of the ticks was performed and six specimens were molecularly characterised by the amplification and sequencing of partial mitochondrial 12S rRNA, 16S rRNA and cox1 genes. For phylogenetic analyses, sequences herein obtained for all genes and those available from GenBank for other Dermacentor spp. were included. Dog serum samples were analysed with a commercial indirect fluorescent antibody test to detect the presence of IgG antibodies against Babesia canis. Ticks and blood samples were tested by PCR amplification using primers targeting 18S rRNA gene of Babesia spp.Ticks collected (n = 34) were morphologically identified as adults of D. reticulatus. Twenty-eight ticks were found in all transects from Site A and the remaining six were collected in Site B. Blast analysis of mitochondrial sequences confirmed the morphological identification of processed tick specimens by revealing a highest nucleotide similarity (99-100%) with those of D. reticulatus available in the GenBank database. The phylogenetic trees were concordant in clustering D. reticulatus in a monophyletic clade. Seven dogs (8.8%) had antibodies against B. canis, most of which (n = 6) came from Site A. Analysis of nucleotide sequences obtained from one tick and from one dog identified B. canis displayed a 100% similarity to those available in GenBank.This study morphologically and molecularly confirms the presence of D. reticulatus in Italy and links it, for the first time, with the occurrence of B. canis infection in dogs in this country.
Project description:Canine brucellosis, caused by Brucella canis, is a disease of dogs and represents a public health concern as it can be transmitted to humans. Canine brucellosis is on the rise in the United States and there is currently no vaccine for use in dogs. Mice have been extensively utilized to investigate host-pathogen interactions and vaccine candidates for smooth Brucella species and could serve a similar role for studying B. canis. However, comparatively little is known about B. canis infection in mice. The objective of this study was to characterize the kinetics of colonization and pathogenicity of B. canis in mice in order to evaluate the mouse as a model for studying this pathogen. C57BL/6 mice were inoculated intraperitoneally with 105, 107, or 109 CFU of Brucella canis RM6/66 and euthanized 1-, 2-, 4-, 6-, 9-, and 12-weeks post-inoculation. B. canis induced splenomegaly in mice infected with 109 CFU at 1- and 2 weeks post-inoculation while no gross lesions were observed in other dose groups. Infection at the two higher doses resulted in dose-dependent granulomatous hepatitis and histiocytic infiltration of the spleen and mesenteric lymph nodes by 1-2 weeks. B. canis was cultured from the liver, spleen, uterus, bone marrow, lung, and kidney in all groups with colonization declining at a slow but steady rate throughout the experiment. Clearance was achieved by 9 weeks 105 CFU group and by 12 weeks in the 107 CFU group, while B. canis persisted in the spleen until 12 weeks in the highest dose group. Although B. canis does not demonstrate significant replication in C57BL/6 mice, it has the ability to establish an infection, induce splenomegaly, and persist for several weeks in multiple organs. Moreover, 1 x 107 CFU appears to be a suitable challenge dose for investigating vaccine safety.
Project description:An outbreak of norovirus (NoV) infection was identified in a kennel. Sequence analysis of a short fragment in the polymerase complex indicated the clonal origin of the strains, which were similar to the prototype canine NoV strain GIV.2/Bari/170/07-4/ITA (94.7% nucleotide identity). The findings demonstrate that canine NoV circulates in dogs in Greece and that it can spread easily across a group of animals.
Project description:Cryptosporidium is a common intestinal protozoan that can lead to diarrhea in humans and dogs. The predominant species of infection are C. hominis and C. parvum in humans, and C. canis in dogs. However, C. canis can infect immunocompromised humans. Considering the close contact with humans, dogs have the potential to be reservoirs for human cryptosporidiosis. Breeding kennels are the major supply source of puppies for pet shops. The present study is to determine the molecular prevalence and characteristics of Cryptosporidium spp. found in breeding kennel dogs. A total of 314 fecal samples were collected from young and adult dogs kept in 5 breeding kennels. A polymerase chain reaction targeting the small subunit rRNA gene was employed for the detection of Cryptosporidium spp. To determine the species, the DNA sequences were compared to GenBank data. Overall, 21.0% of the fecal samples were positive for Cryptosporidium spp. infection. Cryptosporidium spp. was detected in all 5 facilities. A sequencing analysis demonstrated that all isolates shared 99-100% similarity with C. canis. The results suggest that Cryptosporidium spp. infection is present at a high-level in breeding kennel dogs. However, because dominant species in this survey was C. canis, the importance of breeding kennel dogs as reservoirs for Cryptosporidium spp. transmission to humans is likely to be low in Japan.
Project description:BACKGROUND:Human brucellosis has become a severe public health problem in China's Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. RESULTS:A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012-2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. CONCLUSIONS:Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.