Serological responses of dogs to cell wall and internal antigens of Brucella canis (B. canis)

The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 4$\text{1}\text{2}$ years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.     

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.     

Serological evidence of Anaplasma spp., Borrelia burgdorferi and Ehrlichia canis in dogs from the Republic of Korea by rapid diagnostic test kits

BackgroundEmergent and re-emergent canine tick-borne infections are attracting increasing attention worldwide. The rise in pet ownership and the close relationship between dogs and their owners are the most concerning factors because dogs may act as competent reservoirs for human tick-transmitted infectious agents.ObjectivesThis study contributes to the epidemiological surveillance of canine tick-transmitted infections with zoonotic risk in the Republic of Korea (ROK) by investigating the seroprevalence of the pathogens, Anaplasma spp., Borrelia burgdorferi, and Ehrlichia canis.MethodsFour hundred and thirty whole blood samples from domestic dogs were collected in seven metropolitan cities and nine provinces in the ROK and tested using SensPERT Ab test kits (VetAll Laboratories®) to detect seroreactive animals.ResultsThe seroprevalence rates identified were 9.8% (42/430) for Anaplasma spp., 2.8% (12/430) for B. burgdorferi, and 1.4% (6/430) for E. canis. The risk factors evaluated in this study that could be associated with the development of a humoral immune response, such as sex, age, and history of tick exposure, were similar. There was only one exception for dogs seroreactive to Anaplasma spp., where the risk factor “tick exposure” was statistically significant (p = 0.047).ConclusionsThis serological survey exhibited the widespread presence of Anaplasma spp., B. burgdorferi, and E. canis throughout the ROK. Hence, dogs may play a key role as the sentinel animals of multiple zoonotic infectious agents in the country.     

Molecular evidence of vector-borne pathogens in dogs and cats and their ectoparasites in Algiers,Algeria

In Algeria, only limited information is currently available on the prevalence of emergent canine and feline vector-borne diseases. The aim of the present work was to detect by qPCR vector-associated bacteria in stray dogs and cats and their ectoparasites from Algiers.18/117 (15.38%) dogs and 2/107 (1.87%) cats were positive for at least one vector-borne agent. Coxiella burnetii and Bartonella henselae were identified in 1/117 (0.85%) dog individually. Ehrlichia canis DNA was detected in 17/117 (14.52%) dogs. 1/107 (0.93%) cat was positive to C. burnetii and another 1/107 (0.93%) to B. henselae.DNA of Rickettsia massiliae, Rickettsia conorii and E. canis was detected in Rhipicephalus sanguineus. Cat fleas were infected with Rickettsia felis, B. henselae and Bartonella clarridgeiae. B. vinsonii subsp. berkhoffii was identified in Xenopsylla cheopis collected from dogs.The findings of this study indicate that dogs and cats from Algeria are exposed to multiple tick and flea-borne pathogens.     

Conflicting results of serological,PCR and microscopic methods clarify the various risk levels of canine babesiosis in Slovakia: A complex approach to Babesia canis diagnostics

We have performed a survey of Babesia canis prevalence within group of dogs living in Southern and Western Slovakia. Blood samples and sera from 217 dogs, including individuals suspected of having babesiosis, were examined by nested PCR-RFLP, light microscopy and indirect fluorescence antibody test (IFAT). The detection of B. canis DNA revealed the highest number of infected dogs in the region of Nové Zámky, with 23 B. canis-positive blood samples (35.4%, n = 65), followed by an area close to Komárno (both areas of Southern Slovakia), where 1 dog out of 52 collected (1.9%) had detectible B. canis DNA in the blood stream. The serological method revealed an opposing pattern, with only 3 dogs (4.8%, n = 63) sampled at Nové Zámky presenting IgG antibodies against B. canis, while in Komárno region such antibodies were detected in 15 dogs (28.8%, n = 52). This discrepancy may be because the majority of samples from Nové Zámky were dogs suspected of an acute phase of canine babesiosis, whereas dogs at Komárno were sampled during a vaccination campaign, and thus were without any clinical signs of the disease. The latter group contains evidently recovered carriers of IgG against B. canis. Hence, the combination of PCR-based and serological methods enabled us to discover both recently infected as well as recovered dogs, thus obtaining a more realistic view on the epidemiological situation. Remarkably, we did not find any positive samples in the vicinity of Stupava (district Malacky, Western Slovakia), either by PCR-RFLP, microscopy or IFAT (n = 100). Considering the numerous falsely diagnosed cases of canine babesiosis, we suggest that light microscopy as the simplest and most accessible diagnostic test. Southern Slovakia was confirmed as an area of high risk of canine babesiosis, whereas conclusions about B. canis spreading over Western Slovakia should be considered with wariness.     

Molecular evaluation of the incidence of Ehrlichia canis, Anaplasma platys and Babesia spp. in dogs from Ribeirão Preto, Brazil

Canine monocytic ehrlichiosis caused by Ehrlichia canis is endemic in many regions of Brazil. Since thrombocytopenia is a common finding in infected dogs, many clinicians tend to use it as an indication for antibiotic treatment. Polymerase chain reaction (PCR) and nested PCR were used to study the presence of E. canis, Anaplasma platys and Babesia spp. in thrombocytopenic and non-thrombocytopenic dogs from Ribeirão Preto, Brazil. Despite the high prevalence of E. canis infection among thrombocytopenic dogs, 46.7% of the thrombocytopenic dogs studied were either infected with Babesia spp. or A. platys or not infected with any of the three pathogens. There was a high incidence (25.4%) of E. canis infection in non-thrombocytopenic dogs. Although infection with E. canis should be considered in thrombocytopenic dogs, the final diagnosis needs to be confirmed by complementary tests such as blood smears and PCR to avoid the unnecessary use of antibiotics.     

Faecal excretion of intestinal spirochaetes by urban dogs,and their pathogenicity in a chick model of intestinal spirochaetosis

This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled “Brachyspira pulli”, nine clustered with “Brachyspira canis” and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, “B. canis” and “B. pulli”, and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P < 0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with “B. pulli”. Canine “B. canis”, “B. pulli” and B. pilosicoli isolates may have pathogenic potential.     

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.     

Molecular detection of Anaplasma platys and Ehrlichia canis in dogs from the North of Portugal

Tick-transmitted rickettsial pathogens belonging to the Ehrlichia and Anaplasma genera can infect dogs and humans. In this study, four dogs from the North of Portugal, in which an ehrlichial disease was suspected clinically, were tested by molecular methods. After DNA extraction from blood on filter paper, a 345 bp fragment of the Ehrlichia/Anaplasma 16S rRNA gene was amplified by the polymerase chain reaction (PCR). Sequence analysis of PCR products revealed one dog infected with Ehrlichia canis and three with Anaplasma platys. One of these latter animals was co-infected with Babesia canis subspecies vogeli. This is the first report of the genetic characterisation of both A. platys and E. canis in naturally infected dogs from the North of Portugal.     

Identification of Babesia species infecting dogs using reverse line blot hybridization for six canine piroplasms,and evaluation of co-infection by other vector-borne pathogens

Canine infection by vector-borne hemoparasites is frequent in tropical and sub-tropical areas where exposure to hematophageous ectoparasites is intensive. A reverse line blot (RLB) assay was designed to improve the simultaneous detection of all named canine piroplasm species combined with other vector-borne pathogens of dogs including Ehrlichia canis, Hepatozoon canis and Leishmania infantum common in the Mediterranean basin. Blood samples of 110 dogs from Spain (n = 21), Portugal (n = 14) and Israel (n = 75) were analyzed. The study evaluated 2 groups of dogs, 49 dogs with piroplasm infection detected by blood smear microscopy from Portugal, Spain and Israel, and 61 dogs surveyed from rural areas in Israel, for which infection status with vector-borne pathogens was unknown. Among the dogs previously diagnosed with piroplasmosis, infection with Babesia canis, Babesia vogeli, Babesia gibsoni and Theileria annae was detected in the Iberian dogs while only B. vogeli was found in Israeli dogs. These differences are attributed to the absence of tick vectors for some piroplasm species such as Dermacentor reticulatus in Israel. Eleven (79%) of the Babesia-positive dogs from Portugal were co-infected with other pathogens including L. infantum, H. canis and E. canis. Eight of 61 (13%) rural Israeli dogs were co-infected with two or more pathogens including B. vogeli, L. infantum, E. canis, and H. canis. Triple infections were demonstrated in 2 dogs. The RLB detection limit for Babesia was 50-fold lower than that of PCR. This study presents a RLB to simultaneously detect and separate the major vector-borne dog pathogens in southern Europe and the Middle East.     

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.     

Pathogenicity of different strains of Histophilus somni in the experimental induction of ovine epididymitis

The purpose of this study was to determine any differences in pathogenicity when sheep are experimentally infected with different Histophilus somni isolates: a) 2336 bovine origin strain; b) an isolate from ram orchitis and epididymitis; c) an isolate from the brain of a sheep with neurological signs; d) an isolate from the vagina of a clinically healthy ewe. A total of 20 rams divided in groups of 5 animals each were inoculated in the epididymis with 1 × 10⁷ CFU/mL of H. somni; a negative control group of 5 rams was used. All groups inoculated with H. somni showed some epididymitis, but the most pathology was caused by the epididymitis isolate, followed by the vaginal isolate. It was demonstrated that there is a difference in experimental infection capacity among isolates from different origins, as epididymitis occurred and the bacteria was recovered only from groups inoculated with isolates originating from epididymitis and vaginal exudate.     

Prevalence and molecular characterization of Hepatozoon canis in dogs from urban and rural areas in Southeast Brazil

The objective of this survey was to investigate the prevalence of Hepatozoon infection in dogs in the rural and urban areas of Uberlândia, Brazil by PCR and molecular characterization. DNA was obtained from blood samples collected from 346 local dogs from both genders and various ages. Seventeen PCR products from positive blood samples of urban dogs and 13 from the rural dogs were sequenced. Partial sequences of the 18S rRNA gene indicated that all 30 dogs were infected with Hepatozoon canis similar in sequence to H. canis from southern Europe. Four local dog sequences were submitted to GenBank (accessions JN835188; KF692038; KF692039; KF692040). This study indicates that H. canis is the cause of canine hepatozoonosis in Uberlândia and that infection is similarly widespread in rural and urban dogs.     

Evidence for Unapparent Brucella canis Infections among Adults with Occupational Exposure to Dogs

Human serological assays designed to detect brucellosis will miss infections caused by Brucella canis, and low levels of periodic bacteremia limit diagnosis by blood culture. Recent B. canis outbreaks in dogs and concomitant illnesses in caretakers suggest that unapparent human infections may be occurring. With more than a quarter of a million persons in occupations involving dogs, and nearly 80 million dog owners in the United States, this pathogen is an under‐recognized human health threat. To investigate occupational exposure to B. canis, we adapted a commercial canine serological assay and present the first controlled seroepidemiological study of human B. canis infections in recent years. 306 adults with occupational exposure to dogs and 101 non‐matched, non‐canine‐exposed subjects were enrolled. Antibodies were detected using the canine D‐Tec^® CB rapid slide agglutination test (RSAT) kit with a secondary 2‐mercaptoethanol (ME)‐RSAT. Results were validated on a blinded subset of sera with an additional RSAT and indirect enzyme‐linked immunoassay at the National Administration of Laboratories and Health Institutes (ANLIS) in Argentina. Seroprevalence ranged from 10.8% (RSAT) to 3.6% (ME‐RSAT) among canine‐exposed subjects. Kennel employees were more likely to test RSAT seropositive compared with other canine exposures (OR = 2.7; 95% CI, 1.3–5.8); however, low seroprevalence limited meaningful occupational risk factor analyses. Two seropositive participants reported experiencing symptoms consistent with brucellosis and having exposure to B. canis‐infected dogs; however, temporality of symptom onset with reported exposure could not be determined. D‐Tec^® CB results had substantial agreement with ANLIS assays (Cohen's kappa = 0.60–0.68). These data add to a growing body of literature suggesting that people occupationally exposed to dogs may be at risk of unapparent B. canis infection. It seems prudent to consider B. canis as an occupational public health concern and encourage the development of serological assays to detect human B. canis infections.     

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.     

Early Detection of Brucella Canis via Quantitative Polymerase Chain Reaction Analysis

Canine brucellosis is a reportable zoonotic disease that can lead to canine reproductive losses and human infection through contact with infected urine or other genitourinary secretions. Although many locations require testing and euthanasia of positive dogs, current diagnosis is limited by the time required for seroconversion, for example, presence of B. canis‐specific antibodies. The goal of this study was to determine the diagnostic ability of Brucella canis‐specific quantitative polymerase chain reaction (qPCR) assay to detect B. canis in field samples prior to serological positivity for faster diagnosis and prevention of transmission within kennels or in households. Two kennels, one of which was located in the owner's home, were sampled following observation of suggestive clinical signs and positive serology of at least one dog. Specimens obtained were comparatively analysed via serology and qPCR analysis. 107 dogs were analysed for B. canis infection via qPCR: 105 via whole‐blood samples, 65 via vaginal swab, six via urine and seven via genitourinary tract tissue taken at necropsy. Forty‐five dogs were found to be infected with canine brucellosis via qPCR, of which 22 (48.89%) were seropositive. A statistically significant number (P = 0.0228) of qPCR‐positive dogs, 5/25 (20.00%), seroconverted within a 30‐day interval after initial serologic testing. As compared to serology, qPCR analysis of DNA from vaginal swabs had a sensitivity of 92.31% and specificity of 51.92%, and qPCR analysis of DNA from whole‐blood samples had a sensitivity of 16.67% and specificity of 100%. B. canis outer membrane protein 25 DNA qPCR from non‐invasive vaginal swab and urine samples provided early detection of B. canis infection in dogs prior to detection of antibodies. This assay provides a critical tool to decrease zoonotic spread of canine brucellosis, its associated clinical presentation(s), and emotional and economic repercussions.     

Molecular detection of Hepatozoon canis in dogs and ticks in Shaanxi province,China

Hepatozoon canis, transmitted by Rhipicephalus sanguineus, is a tick-borne pathogen and causes canine hepatozoonosis. Until now, only limited previous studies were conducted on the molecular detection and characterization of Hepatozoon sp. in dogs in China. Blood samples were collected from 93 sick dogs that were clinically diagnosed as babesiosis but tested negative for Babesia, and 103 apparently healthy dogs, as well as their infesting ticks in Xi’an and Hanzhong cities, Shaanxi province of China. PCR amplifying partial 18S rRNA gene was used to detect the DNA of Hepatozoon sp. Genetic and phylogenetic analysis were performed to determine the Hepatozoon species. Our results demonstrated that H. canis was identified from the sick dogs and the infested ticks in Hanzhong, with no significant differences of prevalence between both genders and ages. No positive blood or tick samples were found in Xi’an. Moreover, all the 18S rRNA gene sequences recovered from both dogs and the infested ticks showed a high genetic similarity with each other, and also presented a close relationship with other known sequences in and outside China. In conclusion, H. canis was identified in babesiosis-suspected dogs and ticks infesting them in Shaanxi, China, although the association between clinical signs and H. canis need further study.     

Osmotic fragility of erythrocytes in clinically normal dogs and dogs infected with parasites

The osmotic fragility of the erythrocytes was measured in blood samples collected from randomly selected healthy and infected dogs at a dogs' rescue shelter. The dogs were classified into six groups on the basis of the final diagnoses from clinical, post mortem and laboratory findings. The minimum (less than 5 per cent) and maximum (more than 90 per cent) haemolysis of the erythrocytes of the clinically normal dogs (group 1), occurred in 0·60 per cent and 0·30 per cent solutions of sodium chloride (NaCI). For the non-anaemic hookworm-infected dogs (group 2a) the respective values were 0·8 per cent and 0·4 per cent NaCl, and for the anaemic hookworm-infected dogs (group 2b) they were 0·85 per cent and 0·5 per cent NaCl, respectively. The erythrocytes from dogs with Babesia canis (group 3), concurrent hookworm and B canis (group 4) and Ehrlichia canis infections (group 5) had minimum haemolysis in 0·75 per cent NaCl and maximum haemolysis at between 0·20 per cent and 0·35 per cent NaCI solutions. The derivative fragiligrams for groups 2a, 2b, 3 and 4 were shifted to the left, whereas the fragiligram for group 5 was similar to that for the clinically normal dogs (group 1). The left shift for the hookworm-infected dogs was due to the increased osmotic fragility of a minor sub-population of the erythrocytes, but for the dogs, infected with B canis major sub-populations of the erythrocytes had an increased osmotic fragility.