Characterization of a new protective antigen of <Emphasis Type="Italic">Streptococcus canis</Emphasis>

Streptococcus canis (S. canis), a lancefield group G streptococcus, is an opportunistic pathogen mainly found in dogs and cats. The study on pathogenesis and protective immune mechanism of S. canis is not clear. A new streptococcal protective antigen (SPA) was first identified from a genomic library of S. canis. SPA of S. canis (SPASc) contained a 1224-bp open reading frame which encoded a 407aa protein and a 34-aa signal sequence with a deduced molecular mass of 46.368 kDa. Protein analysis and BLAST result showed that SPASc was homologous to the SPA of Streptococcus. equi subsp. zooepidemicus, M protein Streptococcus. equi., and SPA of Streptococcus pyogenes. The protective response of SPASc antiserum was demonstrated by passive mouse protection. These studies suggested that SPASc might be an important component of vaccines to prevent S. canis infections.     

Sequence and phylogenetic analysis of the gp200 protein of Ehrlichia canis from dogs in Taiwan

Ehrlichia (E.) canis is a Gram-negative obligate intracellular bacterium responsible for canine monocytic ehrlichiosis. Currently, the genetic diversity of E. canis strains worldwide is poorly defined. In the present study, sequence analysis of the nearly full-length 16S rDNA (1,620 bp) and the complete coding region (4,269 bp) of the gp200 gene, which encodes the largest major immunoreactive protein in E. canis, from 17 Taiwanese samples was conducted. The resultant 16S rDNA sequences were found to be identical to each other and have very high homology (99.4~100%) with previously reported E. canis sequences. Additionally, phylogenetic analysis of gp200 demonstrated that the E. canis Taiwanese genotype was genetically distinct from other reported isolates obtained from the United States, Brazil, and Israel, and that it formed a separate clade. Remarkable variations unique to the Taiwanese genotype were found throughout the deduced amino acid sequence of gp200, including 15 substitutions occurring in two of five known species-specific epitopes. The gp200 amino acid sequences of the Taiwanese genotype bore 94.4~94.6 identities with those of the isolates from the United States and Brazil, and 93.7% homology with that of the Israeli isolate. Taken together, these results suggest that the Taiwanese genotype represents a novel strain of E. canis that has not yet been characterized.     

A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

Study of ehrlichiosis in kennel dogs under treatment and prevention during seven months in Dakar (Senegal)

In Dakar kennels where morbidity and mortality attributed to diseases transmitted by ticks were high, we conducted a field study to assess the prevalence of Ehrlichia canis, Anaplasma platys and Babesia spp. infections in two kennels (n = 34 dogs) and to study the impact of tick protection. The first day of the study, the E. canis PCR were positive in 18 dogs (53%). A. platys was found in one dog and all dogs were negative for Babesia spp. After one month of doxycycline treatment, the number of PCR positive dogs decreased significantly to 2 (5.9%). During seven months, all dogs were treated monthly topically with a novel combination (Certifect^®, Merial) delivering at least 6.7 mg fipronil/kg body weight, 8.0 mg amitraz/kg and 6 mg (S)-methoprene/kg. The number of PCR positive dogs remained stable all over the seven months, with 4 dogs being positive at Day 90 and 2 at Day 210. The combination of treatment and monthly prevention had a significant effect in the two kennels. All dogs remained healthy, which was not the case in previous years.     

Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island

Streptococcus sp. is gram-positive coccus that causes streptococcal infections in fish due to intensification of aquaculture and caused significant economic losses in fish farm industry. A streptococcal infection occurred from cultured diseased olive flounder (Paralichthys olivaceus) in May, 2005 at a fish farm in Jeju Island, Korea. The diseased flounder exhibited bilateral exophthalmic eyes and rotten gills; water temperature was 16~18℃ when samples were collected. Of the 22 fish samples collected, 3 samples were identified as Lactococcus garvieae and 18 samples were identified as Streptococcus parauberis by culture-based, biochemical test. Serological methods such as slide agglutination, hemolysis and antimicrobial susceptibility test were also used as well as multiplex PCR-based method to simultaneously detect and confirm the pathogens involved in the infection. S. parauberis and L. garvieae have a target region of 700 and 1100 bp., respectively. One fish sample was not identified because of the difference in the different biochemical and serological tests and was negative in PCR assay. In the present study, it showed that S. parauberis was the dominant species that caused streptococcosis in the cultured diseased flounder.     

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

Multiplex PCR-based identification of Streptococcus canis,Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs

Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are β-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.     

Perspectives on canine and feline hepatozoonosis

Two species of Hepatozoon are currently known to infect dogs and cause distinct diseases. Hepatozoon canis prevalent in Africa, Asia, southern Europe, South America and recently shown to be present also in the USA causes infection mainly of hemolymphoid organs, whereas Hepatozoon americanum prevalent in the southeastern USA causes myositis and severe lameness. H. americanum is transmitted by ingestion of the Gulf Coast tick Amblyomma maculatum and also by predation on infected prey. H. canis is transmitted by Rhipicephalus sanguineus, in South America also by Amblyomma ovale, and has also been shown to be transmitted transplacentally. Hepatozoonosis of domestic cats has been described mostly from the same areas where canine infection is present and the exact identity of the species which infect cats, their pathogenicity and vectors have not been elucidated. The diagnosis of hepatozoonosis is made by observation of gamonts in blood smears, histopathology, PCR or serology. The main treatment for H. canis is with imidocarb dipropionate whereas H. americanum infection is treated with an initial combination of trimethoprim-sulfadiazine, pyrimethamine and clindamycin followed by maintenance with decoquinate. Treatment for both diseases has not been reported to facilitate complete parasite elimination and new effective drugs are needed for the management of these infections. Prevention of hepatozoonosis should be based on avoidance of oral ingestion of infected tick vectors and infected prey.     

Genetic diversity of Ehrlichia canis in dogs from Turkey inferred by TRP36 sequence analysis and phylogeny

Canine monocytic ehrlichiosis, an important tick-borne disease caused by Ehrlichia canis, is cosmopolitan but particularly prevalent in tropical and subtropical regions. In Turkey, the genetic diversity of E. canis remains undefined. The aim of this study was to characterize E. canis in naturally infected dogs from Turkey by sequencing and phylogenetic analysis of the Tandem Repeat Protein 36 (TRP36) encoded by the trp36 gene. A total of 167 archived blood samples randomly collected from municipal shelter dogs in three distinct geographic regions were analyzed for E. canis. Only ten samples (5.98%) were found positive by PCR assays target regions of the trp36 and 16S rRNA genes. Sequence analysis of Turkish E. canis TRP36 revealed five Tanden Repeat sequences (TRs) resulting to three TR genotypes: i) the previously reported US genotype composed exclusively from TRs of “TEDSVSAPA” sequence (14 or 8 TRs), ii) the previously Brazilian genotype composed exclusively from TRs of ASVVPEAE sequence (13 TRs), and iii) a novel genotype. In addition, phylogenetic analysis based on the entire sequences of TRP36 revealed that these genotypes correspond to four distinct genogroups (US genogroups I and II, Brazilian genogroup and Costa Rica-Turkey genogroup), all containing Turkish genotypes amongst other geographically distant E. canisgenotypes.     

Ehrlichia canis in dogs of Mexico: Prevalence,incidence, co–infection and factors associated

Rickettsial infections in dogs of Mexico were investigated. A total of 246 dogs were blood sampled and initially screened to detect Ehrlichia canis, E. chaffeensis, E. ewingii, Anaplasma phagocytophilum and Rickettsia rickettsii by a quantitative real–time PCR (qPCR) assay. Sixty–five dogs were monitored and sampled twice 7–8 months apart. Using the qPCR, 72 positive dogs to E. canis were detected (prevalence of 29.26%). These dogs were also tested by nested PCR to detect the same pathogens. None of the studied dogs were positive to E. chaffeensis, E. ewingii, R. rickettsii nor A. phagocytophilum by both PCR assays. The cumulative incidence of E. canis infection was 38.46%. Sequencing analysis of the nested PCR products revealed 100% and 98.1% identity of E. canis and R. parkeri, respectively. We found a dog co–infected with E. canis and R. parkeri.     

Prevalence,molecular characterization and risk factor analysis of Ehrlichia canis and Anaplasma platys in domestic dogs from Paraguay

This is the first study to investigate the prevalence and risk factors associated with Ehrlichia canis and Anaplasma platys positivity in dogs from Paraguay. Conventional PCR assays for the E. canis 16SrRNA gene and A. platys p44 gene were carried out in blood samples from 384 dogs from Asunción city, Paraguay. Sequencing and phylogenetic analysis were performed in selected positive E. canis and (16SrRNA gene) and A. platys (16S and p44 genes) samples. The overall prevalence of E. canis and A. platys in dogs in Paraguay was 10.41% (40/384) and 10.67% (41/384), respectively. Older dogs without veterinary care had higher odds for E. canis positivity and a higher number of dogs in the same household, as well as absence of anti-tick treatment were considered risk factors for A. platys. Ehrlichia canis and A. platys circulate in the dog population from Asunción, and are described for the first time in Paraguay.     

Canine vector-borne disease in travelled dogs in Germany--a retrospective evaluation of laboratory data from the years 2004-2008

When importing dogs from various Mediterranean countries into Western Europe canine vector-borne infections are often considered as a major issue. Several diseases including babesiosis, leishmaniosis, hepatozoonosis, canine heartworm disease or ehrlichiosis can potentially be endemic in this region and pose a potential health risk for travelling dogs. Information on such infections in travelled dogs is scarce and therefore this study has been undertaken to examine the frequency of vector-borne infections in travelled dogs from the years 2004-2008. A total of 997 samples were screened by direct and/or indirect methods. Total seroprevalence was 7.5% with individual seroprevalence for the 3 species Leishmania spp., Ehrlichia canis and Babesia canis spp. ranging from 3.1 to 4.9%. Total detection rate for pathogens by direct methods was 3.5%. Ninteen Giemsa-stained blood smears were positive for large Babesia. None of the samples screened for microfilariae by Knott's test or for Dirofilaria immitis antigen by DiroChek^® were positive. Using PCR methods Leishmania-DNA was detected in 1/42 samples but none of 59 animals screened for E. canis-DNA was positive. The prevalence values as established by indirect and direct pathogen detection are considered as rather low.     

Intraoperative Bleeding in Dogs from Grenada Seroreactive to Anaplasma platys and Ehrlichia canis

Background

Frequent exposure of Grenadian dogs to Rhipicephalus sanguineus results in Anaplasma platys, and Ehrlichia canis seroreactivity. During elective surgeries, substantial intraoperative hemorrhage occurs in some seroreactive dogs.

Objectives

To assess hemostatic parameters and bleeding tendencies as well as prevalence of PCR positivity in apparently healthy A. platys and E. canis seroreactive and seronegative free‐roaming dogs from Grenada.

Animals

Forty‐seven elective surgery dogs allocated to 4 groups: Seronegative control (n = 12), A. platys (n = 10), E. canis (n = 14) and A. platys, and E. canis (n = 11) seroreactive.

Methods

Preoperatively, hemostasis was assessed by platelet count, prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time. Intra‐ and postoperative bleeding scores were subjectively assigned. Blood, spleen, bone marrow, and lymph node aspirates were tested by PCR.

Results

Bleeding scores in dogs coseroreactive for A. platys and E. canis were higher (P = .015) than those of seronegative dogs. A. platys DNA was amplified from 7/21 (33%) A. platys seroreactive dogs and from 1 E. canis seroreactive dog; E. canis DNA was amplified from 21/25 (84%) E. canis seroreactive dogs. E. canis DNA was amplified most often from blood, whereas A. platys DNA was amplified most often from bone marrow.

Conclusions and Clinical Importance

Apparently healthy, free‐roaming dogs coseropositive for A. platys and E. canis may have increased intraoperative bleeding tendencies despite normal hemostatic parameters. Future investigations should explore the potential for vascular injury as a cause for bleeding in these dogs. Improved tick control is needed for dogs in Grenada.     

Brucella canis induces canine CD4+ T cells multi-cytokine Th1/Th17 production via dendritic cell activation

Brucella canis is a small intracellular Gram-negative bacterium that frequently leads to chronic infections highly resistant to antibiotic therapy in dogs. Also, it causes mild human brucellosis compared to other zoonotic Brucella spp. Herein we characterize the cellular immune response elicited by B. canis by analysing human and canine CD4⁺ T cells after stimulation with autologous monocyte-derived dendritic cells (MoDCs). Human and canine B. canis-primed MoDCs stimulated autologous CD4⁺ T cells; however, a Th1 response was triggered by human MoDCs, whereas canine MoDCs induced Th1/Th17 responses, with increased CD4⁺ T cells producing IFN-γ and IL-17A simultaneously. Each pattern of cellular response may contribute to host susceptibility, helping to understand the differences in B. canis virulence between these two hosts. In addition, other aspects of canine immunology are unveiled by highlighting the participation of IL-17A-producing canine MoDCs and CD4⁺ T cells producing IFN-γ and IL-17A.     

Babesiosis in dogs and cats--expanding parasitological and clinical spectra

Canine babesiosis caused by different Babesia species is a protozoal tick-borne disease with worldwide distribution and global significance. Historically, Babesia infection in dogs was identified based on the morphologic appearance of the parasite in the erythrocyte. All large forms of Babesia were designated Babesia canis, whereas all small forms of Babesia were considered to be Babesia gibsoni. However, the development of molecular methods has demonstrated that other Babesia species such as Babesia conradae, Babesia microti like piroplasm, Theileria spp. and a yet unnamed large form Babesia spp. infect dogs and cause distinct diseases. Babesia rossi, B. canis and Babesia vogeli previously considered as subspecies are identical morphologically but differ in the severity of clinical manifestations which they induce, their tick vectors, genetic characteristics, and geographic distributions, and are therefore currently considered separate species. The geographic distribution of the causative agent and thus the occurrence of babesiosis are largely dependent on the habitat of relevant tick vector species, with the exception of B. gibsoni where evidence for dog to dog transmission indicates that infection can be transmitted among fighting dog breeds independently of the limitations of vector tick infestation. Knowledge of the prevalence and clinicopathological aspects of Babesia species infecting dogs around the world is of epidemiologic and medical interest. Babesiosis in domestic cats is less common and has mostly been reported from South Africa where infection is mainly due to Babesia felis, a small Babesia that causes anemia and icterus. In addition, Babesia cati was reported from India and sporadic cases of B. canis infection in domestic cats have been reported in Europe, B. canis presentii in Israel and B. vogeli in Thailand. Babesiosis caused by large Babesia spp. is commonly treated with imidocarb dipropionate with good clinical response while small Babesia spp. are more resistant to anti-babesial therapy. Clinical and parasitological cure are often not achieved in the treatment of small Babesia species infections and clinical relapses are frequent. The spectrum of Babesia pathogens that infect dogs and cats is gradually being elucidated with the aid of molecular techniques and meticulous clinical investigation. Accurate detection and species recognition are important for the selection of the correct therapy and prediction of the course of disease.     

Staphylococcus spp., Streptococcus canis,and Arcanobacterium phocae of healthy Canadian farmed mink and mink with pododermatitis

Pododermatitis is a disease of concern for mink breeders in Canada and worldwide, as it causes discomfort and lowers the breeding rates on farms affected by the disease. Unfortunately, the etiology and pathogenesis of pododermatitis are still unknown. In this study, we compared Staphylococcus spp. and Streptococcus canis isolates from healthy mink with isolates from animals with pododermatitis on 2 farms in Ontario. Almost all hemolytic Staphylococcus spp. isolated were shown to be Staphylococcus delphini Group A by 16S ribosomal ribonucleic acid (rRNA) sequence analysis and polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) did not reveal any S. delphini or S. canis clonal lineages specifically associated with pododermatitis, which suggests that these bacteria do not act as primary pathogens, but does not dismiss their potential roles as opportunistic pathogens. While S. delphini and S. canis were the most prevalent bacterial pathogens in mink pododermatitis, they were also present in samples from healthy mink. Arcanobacterium phocae is occasionally isolated from pododermatitis cases, but is difficult to recover with conventional culture methods due to its slow growth. A quantitative real-time PCR was developed for the detection of A. phocae and was tested on 138 samples of footpad tissues from 14 farms. The bacterium was detected only in pododermatitis-endemic farms in Canada and was at higher concentrations in tissues from infected footpads than in healthy tissues. This finding suggests that A. phocae is involved in the pathogenesis of pododermatitis.     

The incidence of canine haematozoa in peninsular Malaysia

In 3 urban areas in Selangor, Peninsular Malaysia between 1973 and 1981, blood from 4080 dogs was examined for haematozoa. The following frequencies were found: Babesia gibsoni 17.7%; microfilariae of Dirofilaria immitis 9.6%; Hepatozoon canis 1.2%; B. canis 1.1%; Ehrlichia canis 0.2%; Trypanosoma evansi 0.1%. A detailed examination of B. gibsoni infections and microfilariasis due to D. immitis with regards to monthly distribution, breed frequency, sex and age, revealed that pedigree and non-pedigree dogs were equally susceptible to Babesia and microfilariae infections.