Detection of vector-borne pathogens in owned dogs with cranial cruciate ligament rupture living in the Mediterranean area.

BACKGROUND: Cranial cruciate ligament rupture (CCLR) results from a multifactorial degenerative process that leads to rupture of the ligament. Vector-borne pathogens (VBP) in dogs can induce joint disease but their role in CCLR has not been previously investigated. The aim of the present work is to evaluate the prevalence of VBP in dogs with CCLR. METHODS: This was a prospective study that included 46 dogs presented for CCLR surgical treatment and 16 control dogs euthanized for diseases unrelated to the joints. Specimens collected included blood, synovial fluid, and synovial membrane biopsy. Pathogen testing consisted of serology for Leishmania infantum (quantitative ELISA), Ehrlichia canis/ewingii, Borrelia burgdorferi, Anaplasma phagocytophilum/platys, and Dirofilaria immitis (4DX IDEXX test), and PCR for L. infantum, Ehrlichia/Anaplasma spp., Bartonella spp., piroplasms (Babesia spp. and Theileria spp.), and filariae (D. immitis, Dirofilaria repens, Acanthocheilonema dracunculoides, Acanthocheilonema reconditum, and Cercopithifilaria spp.) on both EDTA-whole blood (EB) and synovial fluid (SF) samples. SF cytology and histopathological evaluation of synovial membrane were also performed. RESULTS: The prevalence of VBP was 19.6% in the CCLR group and 18.8% in the control group, with no statistical difference among them. The presence of synovitis was not more frequent in CCLR dogs (45.6%) than in control dogs (43.7%). Lymphoplasmacytic infiltration was the most common inflammatory pattern detected in the joints of both groups of dogs. CONCLUSIONS: This study failed to demonstrate a role of canine VBP in CCLR or the presence or different pattern of joint inflammation in pathogen-positive dogs.

First description of Bartonella koehlerae infection in a Spanish dog with infective endocarditis.

BACKGROUND: Bartonella koehlerae has been recently described as a new cat- and cat fleas-associated agent of culture-negative human endocarditis. It has been also encountered in one dog from Israel and six dogs from the USA, but other clinically relevant reports involving this bacterium are lacking. RESULTS: A 7-year-old intact male mixed dog presented with clinico-pathological signs consistent with mitral endocarditis and cutaneous hemangiosarcoma. Molecular studies revealed the presence of Bartonella koehlerae DNA in samples from blood and mitral valve tissue. CONCLUSIONS: This is the first description of B. koehlerae in Spain, corroborating that it can also be detected in dogs. Bartonella koehlerae infection should also be considered in Spain in humans and dogs presenting with clinical disease suggestive of it, such as culture-negative endocarditis.

Molecular detection of vector-borne pathogens in blood and splenic samples from dogs with splenic disease.

BACKGROUND: The spleen is a highly perfused organ involved in the immunological control and elimination of vector-borne pathogens (VBP), which could have a fundamental role in the pathogenesis of splenic disease. This study aimed to evaluate certain VBP in samples from dogs with splenic lesions. METHODS: Seventy-seven EDTA-blood and 64 splenic tissue samples were collected from 78 dogs with splenic disease in a Mediterranean area. Babesia spp., Bartonella spp., Ehrlichia/Anaplasma spp., Hepatozoon canis, Leishmania infantum, hemotropic Mycoplasma spp. and Rickettsia spp. were targeted using PCR assays. Sixty EDTA-blood samples from dogs without evidence of splenic lesions were included as a control group. RESULTS: More than half (51.56%) of the biopsies (33/64) were consistent with benign lesions and 48.43% (31/64) with malignancy, mostly hemangiosarcoma (25/31). PCR yielded positive results in 13 dogs with spleen alterations (16.67%), for Babesia canis (n = 3), Babesia gibsoni (n = 2), hemotropic Mycoplasma spp. (n = 2), Rickettsia massiliae (n = 1) and "Babesia vulpes" (n = 1), in blood; and for B. canis, B. gibsoni, Ehrlichia canis and L. infantum (n = 1 each), in spleen. Two control dogs (3.3%) were positive for B. gibsoni and H. canis (n = 1 each). Benign lesions were detected in the 61.54% of infected dogs (8/13); the remaining 38.46% were diagnosed with malignancies (5/13). Infection was significantly associated to the presence of splenic disease (P = 0.013). There was no difference in the prevalence of infection between dogs with benign and malignant splenic lesions (P = 0.69); however B. canis was more prevalent in dogs with hemangiosarcoma (P = 0.006). CONCLUSIONS: VBP infection could be involved in the pathogenesis of splenic disease. The immunological role of the spleen could predispose to alterations of this organ in infected dogs. Interestingly, all dogs with B. canis infection were diagnosed with hemangiosarcoma in the present survey. As previously reported, results support that VBP diagnosis could be improved by analysis of samples from different tissues. The sample size included here warrants further investigation.

Prevalence and co-infection of haemotropic mycoplasmas in Portuguese cats by real-time polymerase chain reaction.

The diagnosis of feline haemoplasmosis has improved over the years, with several techniques enabling a clear and specific diagnosis, and where polymerase chain reaction (PCR) is considered as the 'gold standard'. The aim of this study was to survey the prevalence of feline haemoplasmas in 320 cats from the north-central region of Portugal by the use of real-time PCR, as well as to evaluate any associations between infection, clinical presentation and risk factors. The overall prevalence of infection by feline haemoplasmas was 43.43% (139/320), where 41.56% (133/320) corresponded to Candidatus Mycoplasma haemominutum (CMhm), 12.81% (41/320) to Mycoplasma haemofelis (Mhf), 4.38% (14/320) to Candidatus Mycoplasma haematoparvum and 1.25% (4/320) to Candidatus Mycoplasma turicensis. Almost 13% (47/320) of the samples were co-infected, with the most common co-infection being CMhm and Mhf (23.74%). Infection was found statistically significant with feline immunodeficiency/feline leukaemia virus status (P = 0.034), but no significant association was found for breed, sex, fertility status (neutered/spayed/entire), age, clinical status, living conditions (in/outdoor), anaemia status, or the presence/absence of ticks or fleas. Cats from north-central Portugal are infected with all the known feline haemoplasma species, with CMhm being the most common one. Prevalence of all feline haemoplasmas was higher than that reported previously in cats from other European countries, but similar to that described in Portugal for dogs. These data provide a better perspective regarding Mycoplasma species infection in Europe, and new information that helps us better understand feline haemoplasmosis.

Small Demodex populations colonize most parts of the skin of healthy dogs.

BACKGROUND: It is unproven that all dogs harbour Demodex mites in their skin. In fact, several microscopic studies have failed to demonstrate mites in healthy dogs. HYPOTHESIS/OBJECTIVES: Demodex canis is a normal inhabitant of the skin of most, if not all, dogs. This hypothesis was tested using a sensitive real-time PCR to detect Demodex DNA in the skin of dogs. ANIMALS: One hundred dogs living in a humane society shelter, 20 privately owned and healthy dogs and eight dogs receiving immunosuppressive or antineoplastic therapy. METHODS: Hair samples (250-300 hairs with their hair bulbs) were taken from five or 20 skin locations. A real-time PCR that amplifies a 166 bp sequence of the D. canis chitin synthase gene was used. RESULTS: The percentage of positive dogs increased with the number of sampling points. When a large canine population was sampled at five cutaneous locations, 18% of dogs were positive for Demodex DNA. When 20 skin locations were sampled, all dogs tested positive for mite DNA. Our study indicates that Demodex colonization of the skin is present in all dogs, independent of age, sex, breed or coat. Nevertheless, the population of mites in a healthy dog appears to be small. Demodex DNA was amplified from all 20 cutaneous points investigated, without statistically significant differences. CONCLUSIONS AND CLINICAL IMPORTANCE: Using a real-time PCR technique, Demodex mites, albeit in very low numbers, were found to be normal inhabitants of haired areas of the skin of healthy dogs.

Canine leishmaniasis: the key points for qPCR result interpretation.

BACKGROUND: Diagnosis and follow up of CanL is difficult since the range of clinical signs is varied and seroprevalence is high in endemic areas. The aims of this study were: i) demonstrate the advantages of Leishmania qPCR to diagnose and control CanL and highlight its prognostic value and ii) propose guidelines for tissue selection and infection monitoring. FINDINGS: This study included 710 dogs living in an endemic area of leishmaniasis. Forty percent (285/710) exhibited clinical signs consistent with CanL. Infection was detected in 36.3% (258/710) of the dogs of which 4.5% (32/710) were detected by qPCR, 16.2% (115/710) detected by ELISA and 15.6% (111/710) tested positive for both tests. Only 17.9% (127/710) of the dogs were classified sick (affected) with CanL. All symptomatic dogs with medium or high ELISA titers were qPCR-positive in blood samples. All dogs with inconclusive or low ELISA results with high or medium qPCR parasitemia values developed the disease. Seventy one percent of asymptomatic ELISA-positive dogs confirmed by qPCR (medium to high parasitemia) developed the disease. Bone marrow or lymph node aspirate should be selected to ensure the absence of the parasite in asymptomatic dogs: 100-1,000 parasites/ml in bone marrow are detectable in blood, whereas lower parasite loads are usually negative. Almost 10% of negative samples in blood were positive in conjunctival swabs. CONCLUSIONS: Because qPCR allows parasite quantification, it is an effective tool to confirm a diagnosis of CanL in (i) cases of inconclusive ELISA results, (ii) when the dog has not yet seroconverted, or (iii) for treatment monitoring.

Mycoplasma and herpesvirus PCR detection in tortoises with rhinitis-stomatitis complex in Spain.

Chelonid herpesvirus (ChHV) and mycoplasmal infections cause similar clinical signs in terrestrial tortoises and may be the most important causative agents of rhinitis-stomatitis complex, a common disease in captive tortoises worldwide. Currently, diagnosis of ChHV and Mycoplasma spp. infections is most often based on serologic testing. However, serologic results only detect past exposure, and the specificity of these tests can be reduced due to antigenic cross-reactions with other pathogens. Molecular-based techniques could help to define the causative agent and to better manage infected tortoises. Using polymerase chain reaction, we analyzed 63 tortoises (59 spur-thighed tortoise, Testudo graeca; three Greek tortoise, Testudo ibera; and one Russian tortoise, Agryonemys horsfieldii) with clinical signs of rhinitis-stomatitis complex to identify the causative agent. Molecular evidence of ChHV type I (24%), type II (3%), and Mycoplasma agassizii (6%) infections, as well as coinfection of Mycoplasma-ChHV and both types of ChHV, were detected. Both ChHV and M. agassizii are considered pathogenic in captive tortoises and both are a threat to wild populations. However, neither agent was detected from most of the symptomatic tortoises we evaluated, indicating that other agents could be involved in the rhinitis-stomatitis complex.

Development of a real-time PCR to detect Demodex canis DNA in different tissue samples.

The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed paraffin embedded skin biopsies. The real-time PCR amplified on the hairs of all 14 dogs with a firm diagnosis of demodicosis and consistently failed to amplify on negative controls. Eleven of 12 skin biopsies with a morphologic diagnosis of canine demodicosis were also positive. Sampling hairs on two skin points (lateral face and interdigital skin), D. canis DNA was detected on nine of 51 healthy dogs (17.6%) a much higher percentage than previously reported with microscopic studies. Furthermore, it is foreseen that if the number of samples were increased, the percentage of positive dogs would probably also grow. Moreover, in four of the six dogs with demodicosis, the samples taken from non-lesioned skin were positive. This finding, if confirmed in further studies, suggests that demodicosis is a generalized phenomenon in canine skin, due to proliferation of local mite populations, even though macroscopic lesions only appear in certain areas. The real-time PCR technique to detect D. canis DNA described in this work is a useful tool to advance our understanding of canine demodicosis.

Prevalence of hemotropic mycoplasmas in healthy and unhealthy cats and dogs in Spain.

The aims of the present study were to determine the prevalence of hemoplasmas in cats and dogs from the Barcelona area of Spain with the use of species-specific quantitative polymerase chain reaction (qPCR) assays and to evaluate any associations between hemoplasma infection, clinical presentation, and vector-borne infections. Blood samples from cats (191) and dogs (182) were included and were classified as healthy (149) or unhealthy (224). Ethylenediamine tetra-acetic acid blood samples underwent DNA extraction and qPCR analysis. Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis' were detected in cats, whereas Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' were detected in dogs, with prevalences of 3.7%, 9.9%, 0.5%, 14.3%, and 0.6%, respectively. In cats, no association between hemoplasma infection and health status, age, breed, presence of anemia, Feline leukemia virus status, and other vector-borne infections was found, but outdoor access (P = 0.009), male sex (P = 0.01), and Feline immunodeficiency virus status (P < 0.001) were significantly associated with hemoplasma infection. In dogs, sex, age, health status, presence of anemia, and breed were not significantly associated with hemoplasma infection, but a significant association was found between hemoplasma infection and vector-borne infections (P < 0.001). The present report documents the occurrence of feline 'Candidatus M. turicensis' and canine 'Candidatus M. haematoparvum' infections in Spain.

Association of CA repeat polymorphism at intron 1 of insulin-like growth factor (IGF-I) gene with circulating IGF-I concentration, growth, and fatness in swine.

Evidence is accumulating that intronic polymorphic cytosine-adenosine (CA) repeats may play a role in gene expression. In this work, we investigated whether a polymorphic CA short tandem repeat (STR) located at the first intron of the pig insulin-like growth factor I (IGF-I) gene influences plasma IGF-I concentration in pigs as well as phenotypic variation in growth and fatness traits. We measured plasma IGF-I levels at one to four time points from 35 to 215 days of age in 340 performance-tested Landrace and Duroc pigs previously genotyped for the IGF-I STR. Data were analyzed within breed with a linear mixed model with the number of CA repeats as a covariate. At least five alleles were segregating in each breed, differing in one to seven repeats. The results showed that in each breed, circulating IGF-I at 160 days of age increased with the length of the shortest allele, accounting for an average trend of 4.38 +/- 1.28 ng/ml of IGF-I per additional repeat (P = 0.001). Longer repeats were associated with early growth in Landrace boars (1.92 +/- 0.92 kg per CA at 160 days; P = 0.038) and with back fat thickness (-0.57 +/- 0.20 mm per CA; P = 0.005) and lean content (7.52 +/- 3.00 g/kg per CA at 105 kg; P = 0.013) adjusted for carcass weight in Duroc barrows, as expected from the effect of circulating IGF-I on these traits. The consistency of the results across populations supports the hypothesis that the length of the CA repeats at intron 1 of the IGF-I gene is associated with circulating IGF-I levels, and that this effect is not neutral with respect to growth and fatness.

A long term experimental study of canine visceral leishmaniasis.

Previous studies on Leishmania infantum and the canine immune response are derived mainly from short-term studies. To date, there have been no longitudinal studies that perform a serial analysis of the intensity of infection in conjunction with immunological parameters and clinical signs in Leishmania-infected dogs. For this purpose, six dogs were infected experimentally by the i.v. route and were monitored for 1 year. Clinical, immunological (humoral and cellular response) and parasitological (parasitaemia) parameters were evaluated monthly. Four dogs developed clinico-pathological signs compatible with leishmaniasis, whereas two dogs showed few abnormalities during the study. Evaluation of clinical, immunological and parasitological parameters showed that the intensity of Leishmania infection in blood samples, as indicated by the amount of Leishmania DNA, was correlated significantly with IgG, IgG1, IgG2, IgA, and IgM concentrations and with clinical signs. Parasitaemia and Leishmania-specific cell-mediated immunity were inversely correlated. Moreover, higher quantities of Leishmania DNA were detected in the liver, spleen, lymph node, skin and bone marrow of dogs exhibiting clinical signs than those exhibiting few such signs. These findings suggest that progressive disease in experimental canine leishmaniasis is associated with specific T-cell unresponsiveness and unprotective humoral responses which allow the dissemination and multiplication of L. infantum in different tissues.