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.     

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.     

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.     

The 23S rRNA gene PCR-RFLP used for characterization of porcine intestinal spirochete isolates

Using three reference strains of Brachyspira hyodysenteriae (B204, B234, B169), one B. pilosicoli (P43/6/78), one B. murdochii (56-150), one B. intermedia (PWS/A), one B. innocens (B256) and ten Korean isolates, PCR-RFLP analysis of DNA encoding 23S rRNA was performed to establish a rapid and accurate method for characterizing porcine intestinal spirochetes. Consequently, B. hyodysenteriae and B. pilosicoli revealed different restriction patterns; however, the other three species shared the same pattern. These findings are not consistent with a prior report. Differences in 23S rRNA gene sequences, between two B. murdochii strains, 56-150 and 155-20, were observed. These results indicate that 23S rRNA PCR-RFLP could be used as an identification method for pathogenic Brachyspira spp. (B. hyodysenteriae and B. pilosicoli) as well as an epidemiological tool for characterizing spirochetes isolated from swine.     

“Brachyspira hampsonii” clade I isolated from Belgian pigs imported to Germany

This report describes the detection of “Brachyspira (B.) hampsonii” clade I in Belgian pigs imported to Germany. Two of seventeen pigs from one herd were reported positive for Brachyspira hyodysenteriae by culture in a Belgian diagnostic laboratory, but negative for this Brachyspira species by specific PCR. In this study, from 22 fecal samples and 2 colon contents of these animals various Brachyspira species were cultured and identified by nox-RFLP as Brachyspira murdochii, Brachyspira innocens and Brachyspira intermedia. Albeit the six B. intermedia isolates proved to be negative in a species specific PCR. Sequencing of the nox-gene of three of these isolates revealed that the sequences were 99% identical to published sequences of “B. hampsonii” clade I. From one pig which was positive for “B. hampsonii” clade I histopathology was done and showed moderate lesions consistent with brachyspiral disease.     

Colonisation of pet shop puppies with Brachyspira pilosicoli

Anaerobic intestinal spirochaetes of the genus Brachyspira are known to colonise dogs, but relatively little is known about their prevalence, distribution or pathogenic potential. One species, Brachyspira pilosicoli, is thought to cause diarrhoea in dogs, as well as in other animals and humans. To investigate the prevalence and distribution of infection, faecal samples from 49 puppies from six pet shops in the suburbs of Perth, Western Australia were subjected to selective culture for anaerobic intestinal spirochaetes. Growth from the primary plates was also harvested, the DNA extracted and a polymerase chain reaction (PCR) amplification of a portion of the 16S rRNA gene of B. pilosicoli applied. Weakly beta-haemolytic intestinal spirochaetes (WBHIS) grew on plates from 20 of the dogs (40.8%). Seven plates (14.2%) yielded PCR positive amplification for B. pilosicoli. Seven WBHIS isolates were obtained in pure culture, and two of these were shown to be B. pilosicoli by PCR. Application of multilocus enzyme electrophoresis to the seven isolates confirmed that the two PCR positive isolates were B. pilosicoli, whilst the other five belonged to a group previously designated "Brachyspira canis". All the "B. canis" isolates came from healthy puppies, suggesting that this WBHIS is a commensal. Three of the seven puppies with PCR evidence of B. pilosicoli had diarrhoea, but the sample size was small and the association between colonisation and diarrhoea was not statistically significant. Pet shop puppies are commonly infected with intestinal spirochaetes, and may act as a reservoir of B. pilosicoli for other animals and humans.     

Butyrylcholinesterase as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis

The aim of this study was to evaluate the role of butyrylcholinesterase (BChE) as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis. Forty-two serum samples of dogs naturally infected with Ehrlichia canis were used, of which 24 were from animals with the acute phase of the disease and 18 with subclinical disease. In addition, sera from 17 healthy dogs were used as negative controls. The hematocrit, BChE activity, hepatic injury (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), nitric oxide, and cytokines levels were evaluated. The BChE activity was significantly elevated (P < 0.05) in dogs with the acute phase of the disease when compared to healthy animals. However, there was a reduction on BChE activity on dogs with subclinical disease compared to the other two groups. AST and ALT levels were significantly higher (P < 0.05) in the acute phase, as well as the inflammatory mediators (NO_x, TNF-α, INF-γ, IL-4, IL-6) when compared to the control group. On the other hand, IL-10 levels were lower in the acute phase. Based on these results, we are able to conclude that the acute infection caused by E. canis in dogs leads to an increase on seric BChE activity and some inflammatory mediators. Therefore, this enzyme might be used as a marker of acute inflammatory response in dogs naturally infected by this bacterium.     

Risk factors associated with Neospora caninum seropositivity in randomly sampled Canadian dairy cows and herds

Our objective was to determine cow- and herd-level risk factors associated with seropositivity for Neospora caninum in a large number of randomly selected Canadian dairy herds, controlling for important confounding variables and co-infections with bovine leukemia virus (BLV), bovine viral diarrhea virus (BVDV) and Mycobacterium avium subspecies paratuberculosis (MAP). Serum samples were obtained from 30 randomly selected cows, where available, in 240 herds using monthly milk testing, within 6 of 10 provinces, and these samples were tested for antibodies against BLV, MAP and N. caninum using commercially available ELISA test kits. Five unvaccinated cattle >6 months old from each herd were tested for antibodies to BVDV using virus neutralization. Most herd-level predictors were obtained through personal interviews with questionnaires administrated to each farm manager. A mixed logistic-regression model was built using N. caninum serostatus at the cow-level as the outcome variable, with herd as a random effect and province as a fixed effect. A BLV seropositive cow was 1.50 times more likely to be seropositive for N. caninum than a BLV-seronegative cow, and this was the only cow-level variable to remain in the final model. Regarding herd-level variables, with “no on-farm dogs” as the baseline, “presence of dogs but not known to eat placentas and/or fetuses” increased the odds of seropositivity for N. caninum by a factor of 1.66. For “presence of dogs known to eat placentas and/or fetuses”, the odds ratio (OR) was 2.75, demonstrating a dose–response relationship. “Using embryo transfer” (OR = 0.69), “asking for a BVDV-negative test before introducing an animal” (OR = 0.30), “using monensin in dry cows” (OR = 0.71), and “heifers having nose-to-nose contact with calves” (OR = 0.73) were all dichotomous variables negatively associated with seropositivity for N. caninum. “Number of milk cows on the farm” (OR = 0.99), and “area (acres) used for forage production” (OR = 0.99) were continuous variables negatively associated with N. caninum seropositivity.     

Diagnosis of canine brucellosis by ELISA using an antigen obtained from wild Brucella canis

An indirect ELISA test was developed for the diagnosis of Brucella canis infection in dogs. A bacterial whole cell extract was used as a solid phase antigen, using B. canis isolated from an infected animal. Sera from culture-positive and healthy negative animals were used as internal reference controls. The cut-off point was determined by a mathematical formula for a statistically valid value, which defined the upper prediction limit, based on the upper tail of the t-distribution of 21 negative control sera readings, for the confidence level of 99.5%. The sensitivity and specificity of the ELISA test were 95% and 91%, respectively. The ELISA test showed a significant concordance index (K = 0.84) with the agar gel immunodiffusion test. The reliability of the ELISA for the detection of infected animals was established by a double blind study testing 280 sera provided by serum banks from different diagnostic and research institutions and analyzed by ROC Curve.     

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.     

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.     

Ability of an oral formulation of afoxolaner to protect dogs from Borrelia burgdorferi infection transmitted by wild Ixodes scapularis ticks

A randomized, blinded, negative controlled study was conducted to determine whether treatment with afoxolaner (NexGard^®, Merial, Inc.) would prevent the transmission of Borrelia burgdorferi to dogs by wild caught Ixodes scapularis ticks. Twenty healthy dogs were randomly assigned to two groups of ten dogs each. Ten dogs were treated orally on Day 0 at a dose near the minimum recommended dose of afoxolaner of 2.5 mg/kg (actual doses 2.5–3.1 mg/kg) and ten control dogs were not treated. On Day 28, each dog was infested with approximately 50 adult unfed wild caught I. scapularis that had a 67% B. burgdorferi infection rate (determined by polymerase chain reaction). On Day 33, live ticks were counted and removed. No ticks were found on treated dogs while control dogs had an average of 21.4 ticks. To detect infection, the B. burgdorferi-specific C6 antibody SNAP^® 4Dx^® test (IDEXX) was performed on serum collected before infestation (all dogs seronegative on Days -6 and 27) and on Days 48, 63, 77 and 92. The ten treated dogs remained seronegative through the end of the study (Day 92), while nine out of the ten control dogs were infected, as demonstrated by their seroconversion to being positive for the presence of the B. burgdorferi-specific C6 antibody starting on Day 48. In this study, all dogs treated with NexGard^® 28 days prior to challenge with wild caught I. scapularis ticks were protected from B. burgdorferi infection, while nine out of the ten untreated control dogs were infected.     

Detection of Lawsonia intracellularis,Serpulina hyodysenteriae,weakly beta-haemolytic intestinal spirochaetes,Salmonella enterica,and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs

A polymerase chain reaction (PCR) was optimized to detect Lawsonia intracellularis in faeces from naturally infected pigs. By combining a boiling procedure to extract DNA and a nested PCR procedure, a detection limit at 2×10² bacterial cells per gram of faeces was achieved. The optimized PCR was used together with conventional culture techniques to detect Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes (WBHIS), Salmonella enterica, and haemolytic Escherichia coli, in a case control study to examine selected risk factors for the development of diarrhoea in growing pigs. Herds with diarrhoea were selected as cases and randomly chosen herds without diarrhoea were chosen as controls. Infection with L. intracellularis significantly enhanced the chance of diarrhoea. S. hyodysenteriae, WBHIS group IV (Serpulina pilosicoli), and S. enterica were isolated only from case herds which indicate that these species may influence the development of diarrhoea. In addition, herd-type had a significant impact, that is specific pathogen-free herds showed an odds ratio at 0.2 relative to conventional herds for the development of diarrhoea.     

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10⁸ CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.     

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.     

Follow-up of 100 dogs with acute diarrhoea in a primary care practice

This study aimed to examine the aetiology of acute diarrhoea and the relapse rate in 100 client-owned dogs presented to a first-opinion clinic. History, physical examination, faecal testing and owner questionnaire data were collected at initial presentation (T₀) and at either the time of relapse or at a recheck performed within 3 months. All dogs received treatment according to their clinical signs. Of 96 dogs that completed the study, 37 (38.5%) relapsed during the study period, 21 (21.9%) relapsed within 3 months, and 16 others (16.6%) at 3 months to 1 year after initial examination. Dogs that had undergone a change in housing location within 1 month prior to presentation and dogs <1 year old were significantly more likely to have positive parasitological analyses (P = 0.02 and P = 0.001, respectively). Pica was a risk factor for relapse (P = 0.0002).     

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.     

Computed tomographic morphology and clinical features of extrahepatic portosystemic shunts in 172 dogs in Japan

Canine extrahepatic congenital portosystemic shunts (EH-cPSS) are classified into several anatomical types, depending on the origin and termination of the shunt vessel. The aim of this retrospective study was to determine the proportion and clinical features of each anatomical shunt type in a population of dogs presented to a veterinary teaching hospital in Japan. Dogs diagnosed with EH-cPSS using computed tomographic (CT) portography were included (n = 172) and shunts were classified based on previous reports. Clinical data were collected from case records and analysed statistically. The most common anatomical type was the spleno-phrenic shunt (n = 64), followed by the spleno-azygos (n = 38), right gastric-caval (n = 29), spleno-caval (n = 21), right gastric-caval with caudal loop (n = 9), right gastric-phrenic (n = 6), colono-caval (n = 3), spleno-phrenic and azygos (n = 1), and porto-caval (n = 1) shunts. Spleno-phrenic and spleno-azygos shunts were diagnosed more frequently in older dogs than right gastric-caval and spleno-caval shunts (P < 0.05). The portal vein/aortic (PV/Ao) ratio was significantly larger in dogs with spleno-phrenic shunts than in dogs with spleno-azygos, right gastric-caval or spleno-caval shunts (P < 0.05). The PV/Ao ratio was significantly larger in dogs with spleno-azygos shunts than in dogs with right gastric-caval shunts. Dogs with spleno-phrenic shunts had significantly lower serum alkaline phosphatase activities than those with right gastric-caval or spleno-caval shunts. Dogs with spleno-phrenic shunts had significantly lower fasting ammonia concentrations than those with spleno-caval shunts.