Development of a PCR technique specific for Demodex injai in biological specimens.

The identification of Demodex injai as a second Demodex species of dog opened new questions and challenges in the understanding on the Demodex-host relationships. In this paper, we describe the development of a conventional PCR technique based on published genome sequences of D. injai from GenBank that specifically detects DNA from D. injai. This technique amplifies a 238-bp fragment corresponding to a region of the mitochondrial 16S rDNA of D. injai. The PCR was positive in DNA samples obtained from mites identified morphologically as D. injai, which served as positive controls, as well as in samples from three cases of demodicosis associated with proliferation of mites identified as D. injai. Furthermore, the PCR was positive in 2 out of 19 healthy dogs. Samples of Demodex canis and Demodex folliculorum were consistently negative. Skin samples from seven dogs with generalized demodicosis caused by D. canis were all negative in the D. injai-specific PCR, demonstrating that in generalized canine demodicosis, mite proliferation is species-specific. This technique can be a useful tool in the diagnosis and in epidemiologic and pathogenic studies.

PCR survey of vectorborne pathogens in dogs living in and around Barcelona, an area endemic for leishmaniasis.

Blood samples from 153 dogs living in and around Barcelona were assayed for Leishmania infantum and Ehrlichia, Anaplasma, Rickettsia, Bartonella, Hepatozoon, Babesia and Theileria species by PCR amplification of DNA, and the amplicons obtained were sequenced. The prevalence of the infectious agents was L infantum (29.4 per cent), Ehrlichia and Anaplasma species (4.0 per cent), Hepatozoon canis (3.3 per cent), Babesia canis vogeli (2.0 per cent), Babesia gibsoni (2.0 per cent), Babesia canis canis (1.3 per cent) and Theileria annae (0.7 per cent). Coinfections were present in seven of the dogs and they were significantly associated with L infantum infection (P=0.024). There was a significant correlation between clinical signs of illness and the load of L infantum.

Endosymbionts carried by ticks feeding on dogs in Spain.

Studies on tick microbial communities historically focused on tick-borne pathogens. However, there is an increasing interest in capturing relationships among non-pathogenic endosymbionts and exploring their relevance for tick biology. The present study included a total of 1600 adult ticks collected from domestic dogs in 4 different biogeographical regions of Spain. Each pool formed by 1 to 10 halves of individuals representing one specific ticks species was examined by PCR for the presence of Coxiellaceae, Rickettsia spp., Rickettsiales, Wolbachia spp., and other bacterial DNA. Of the pools analyzed, 92% tested positive for endosymbiont-derived DNA. Coxiella spp. endosymbionts were the most prevalent microorganisms, being always present in Rhipicephalus sanguineus sensu lato (s.l.) pools. Rickettsia spp. DNA was detected in 60% of Dermacentor reticulatus pools and 40% of R. sanguineus s.l. pools, with a higher diversity of Rickettsia species in R. sanguineus s.l. pools. Our study reveals a negative relationship of Rickettsia massiliae with the presence of tick-borne pathogens in the same pool of ticks. An additional endosymbiont, 'Candidatus Rickettsiella isopodorum', was only detected in D. reticulatus pools. Data from this study indicate that dogs in Spain are exposed to several endosymbionts. Due to the importance of tick-borne pathogens, characterizing the role of endosymbionts for tick physiology and prevalence, may lead to novel control strategies.

Longitudinal analysis of cytokine gene expression and parasite load in PBMC in Leishmania infantum experimentally infected dogs.

Canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, an intracellular protozoan parasite that causes a severe infectious disease. To evaluate the gene expression profile associated to CVL in vivo, we have measured monthly by real-time PCR over one year the IL-4, IL-10, IL-12, IL-13, IFN-gamma, TGF-beta and TNF-alpha mRNA levels in peripheral blood mononuclear cells in 6 experimentally infected dogs that exhibited different progressions of the illness. While in two dogs no parasite, or a very low number of parasites, was detected and the two dogs did not show any clinico-pathological abnormalities at the end of the study (L dogs), for the remaining dogs high parasite loads were detected and they developed clinical leishmaniasis (H dogs). The L dogs have null expression of both IL-4 and IL-13 for the first 4 months after the infection, whereas an early IL-4 and IL-13 expression occurs in this period of infection in most of the dogs that developed clinical leishmaniasis (H dogs). Furthermore, a higher IFN-gamma expression was associated with the increase of parasite load and clinical status in these dogs. Moreover, the high variability of expression at the pre-infection stage causes us to reject the possibility that the basal levels of these cytokines indicate the prognosis of the subsequent response against infection.

Detection of Leishmania infantum by real-time PCR in a canine blood bank.

OBJECTIVES: Risk for transmission of Leishmania infantum from blood products has been largely demonstrated in human and veterinary literature. Appropriate screening of canine blood donors is important especially in an endemic area such as Barcelona (Spain). The purpose of this study was to evaluate the presence of L infantum DNA parasites by real-time quantitative PCR in our canine blood bank. METHODS: Samples from blood products obtained from 92 canine blood donors were assayed for L infantum by means of real-time PCR amplification and quantification. RESULTS: The prevalence of quantitative PCR-positive blood samples among healthy seronegative blood donors was 19.6 per cent. CLINICAL SIGNIFICANCE: The results of this study show that L infantum infection is common in canine blood donors and their blood products in an endemic area, despite a negative commercial serological screening for infectious diseases. Therefore, screening by PCR should be included in an integrated approach to evaluate L infantum infection among potential blood donors.

Polymerase chain reaction detection of Bartonella spp. in dogs from Spain with blood culture-negative infectious endocarditis.

OBJECTIVES: The presence of Bartonella spp. was detected by polymerase chain reaction (PCR) in dogs from Spain with blood culture-negative endocarditis. The aim of this study is to add information about canine infectious endocarditis in Europe. ANIMALS: Thirty dogs with naturally occurring blood culture-negative endocarditis were examined from 2010 to 2017 at three veterinary referral hospitals, located in northwest, northeast, and southeast of Spain. METHODS: It is a retrospective study. Medical records were reviewed to extract relevant data. Frozen or paraffin-embedded cardiac valve tissue and/or ethylenediamine tetraacetic acid blood samples were evaluated by PCR for the presence of Bartonella DNA. Positive results were sequenced to confirm the species. RESULTS: Polymerase chain reaction was positive for eight out of 30 dogs included (26.6%). Bartonella rochalimae, Bartonella vinsonii subsp. berkhoffii, and Bartonella koehlerae were detected in valve tissue or blood. CONCLUSIONS: Bartonella could be an important cause of blood culture-negative infectious endocarditis in dogs from Spain. The outcome for those dogs affected with Bartonella spp. was grave. Prompt empirical treatment with amoxicillin-clavulanate plus fluoroquinolones could be of value in cases of blood culture-negative endocarditis.

PCR evaluation of selected vector-borne pathogens in dogs with pericardial effusion.

OBJECTIVES: To investigate evidence for selected vector-borne pathogen infections in dogs with pericardial effusion living in a Mediterranean area in which several canine vector-borne diseases are endemic. MATERIALS AND METHODS: Archived EDTA blood (n=68) and pericardial fluid samples (n=58) from dogs with pericardial effusion (n=68) were included. Dogs without pericardial effusion examined for other reasons were included as controls (n=60). Pericardial effusion was classified as neoplastic in 40 dogs, idiopathic in 23 dogs and of unknown aetiology in 5 dogs. Real-time PCR was performed for Leishmania infantum, Ehrlichia/Anaplasma species, Hepatozoon canis, Babesia species, Rickettsia species and Bartonella species, and sequencing of PCR products from positive samples was used to confirm species specificity. RESULTS: Vector-borne pathogens were found in 18 dogs: 16 of 68 dogs with pericardial effusion (23·5%) and two of 60 control dogs (3·3%). Positive dogs demonstrated DNA of Leishmania infantum (n=7), Anaplasma platys (n=2, one dog coinfected with Leishmania infantum), Babesia canis (n=5), Babesia gibsoni (n=3) and Hepatozoon canis (n=2). Vector-borne pathogens were more commonly detected among dogs with pericardial effusion than controls (P=0·001). There was no relationship between aetiology of the pericardial effusion and evidence of vector-borne pathogens (P=0·932). CLINICAL SIGNIFICANCE: Vector-borne pathogens are often detected in dogs with pericardial effusion and require further investigation, especially in dogs with idiopathic pericardial effusion. PCR can provide additional information about the potential role of vector-borne pathogens in dogs with pericardial effusion living in endemic areas.

Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis.

The aim of the present study is to highlight the advantages of real-time quantitative PCR intended to aid in the diagnosis and monitoring of canine leishmaniosis. Diagnosis of canine leishmaniosis is extremely challenging, especially in endemic areas, due to the diverse and non-specific clinical manifestations, and due to the high seroprevalence rate in sub-clinical dogs. Veterinarian clinicians are usually confronted with cases that are compatible with the disease, and with several diagnostic tests, sometimes with contradictory results. We have developed a new TaqMan assay, targeting the kinetoplast, applied to 44 samples of bone marrow aspirate or peripheral blood. The dynamic range of detection of Leishmania DNA was established in 7 logs and the limit of detection is 0.001 parasites in the PCR reaction. At the time of diagnosis parasitemia ranges from less than 1 to 10(7)parasites/ml. The ability to quantify the parasite burden allowed: (i) to elucidate the status of positive dogs by conventional PCR, although larger studies are necessary to clarify the dividing line between infection and disease, (ii) to estimate the kinetics of the parasite load and the different response to the treatment in a follow-up and (iii) to validate blood as less invasive sample for qPCR. The continuous data provided by real-time qPCR could solve the dilemma for the clinician managing cases of canine leishmaniosis by differentiating between Leishmania-infected dogs or dogs with active disease of leishmaniosis.

Wolbachia, filariae and Leishmania coinfection in dogs from a Mediterranean area.

OBJECTIVES: In an endemic area for leishmaniosis and filariasis, coinfection can occur and the immunomodulation triggered by Wolbachia infection might influence the clinical signs and progression of both diseases. The aims of this study were to determine the prevalence of Wolbachia in dogs infected with Dirofilaria immitis and other filarial nematodes, to evaluate the prevalence of coinfection of Leishmania infantum, filariae and Wolbachia and their association with clinical presentation. METHODS: Polymerase chain reaction assays were performed to detect filarial species, Wolbachia species and Leishmania in 118 samples of dogs from southeastern Spain with leishmaniosis and/or filariasis. RESULTS: Ninety-eight dogs were infected with Leishmania and 49 had filarial infection (29 were coinfected with both). Wolbachia DNA was detected in 30·6% of filariae-positive dogs (15/49). Dogs coinfected with Leishmania and filaria had more severe clinical signs. Wolbachia infection was significantly (P=0·026) more frequent in dogs that were not infected with Leishmania. There was no correlation between outcome and coinfection with these pathogens. CLINICAL SIGNIFICANCE: This study highlights the increased sensitivity of polymerase chain reaction in the diagnosis of filariasis, confirms the presence of Wolbachia in dogs from the Mediterranean basin, shows the increased severity of clinical signs when Leishmania-filarial coinfection is present and suggests a protective role of Wolbachia in leishmaniosis.

Gammopathy in a Spanish dog infected with Bartonella henselae.

Generalised pyogranulomatous disease and hyperviscosity syndrome associated with a presumed monoclonal gammopathy was diagnosed in a three-year-old intact female Pomeranian. The Bartonella henselae antibody titer was 1:64 and Bartonella species DNA was amplified from the splenic tissue. Monoclonal gammopathies in dogs are typically associated with plasma cell and lymphoid dyscrasias and other inflammatory or infectious diseases such as ehrlichiosis and leishmaniosis. Based on this case report, infection with Bartonella species should also be added to the differential diagnoses for gammopathy in dogs. To the authors' knowledge, this is the first report of molecular evidence of Bartonella species infection in a sick dog in Spain.

Polymorphism of Slc11a1 (Nramp1) gene and canine leishmaniasis in a case-control study.

The prevalence of canine leishmaniasis infection in an endemic area such as the Mediterranean basin (67%) is higher than the prevalence of the disease (10%), suggesting a role of host genetics related to the outcome of the disease. Because Slc11a1 gene affects susceptibility and clinical outcome of autoimmune and infectious diseases, we analyzed five polymorphisms of the Slc11a1 gene in a case-control study with 97 dogs: three new single nucleotide polymorphisms and a G-stretch in the promoter and a microsatellite in intron 1. Haplotype frequency distributions showed significant differences between case and control populations (P = .01), most likely owing to the single nucleotide polymorphisms in the promoter region that were associated to case dogs. The most frequent haplotypes included TAG-8-141, which was present in all the breeds, in both case and control animals; and TAG-9-145, which was overrepresented in the control population and mostly found in boxer dogs. Within the boxer breed, 81% of the healthy dogs were homozygous TAG-9-145, whereas TAG-8-141 was significantly associated to case boxers (P = .02). The special genotype distribution for the Slc11a1 polymorphism associated with the prevalence of the illness in the boxer breed emphasizes the potential importance that breed genetic background has in canine leishmaniasis susceptibility.

Microsatellite polymorphism in closely related dogs.

The effectiveness of microsatellites in parentage testing and individual identification has been proven in many species, including dogs. However, the use of these markers has not been extended to control for pedigrees in large populations of closely related animals. We have analyzed polymorphism in a set of 10 microsatellites over three generations of 360 pedigree rottweilers. Results were compared with two pure-bred populations of unrelated animals and with one population constituted by unrelated dogs of mixed breeds to measure polymorphism variation. We optimized this set of microsatellites to be analyzed by a semiautomated capillary electrophoresis method after amplification in two multiplex polymerase chain reactions (PCRs). The mean polymorphism information content (PIC) value in the rottweiler pedigree is 0.401 and the combined paternity exclusion probability (CPE) is 95.6%. These values are similar to those obtained in pure-bred populations of unrelated animals, and although polymorphism is reduced in relation to the pool population, we solved all paternity exclusions. In only a few cases did we have to use two additional microsatellites to solve individual identification of full-sib dogs.

Nomenclature for factors of the dog major histocompatibility system (DLA), 1998: first report of the ISAG DLA Nomenclature Committee.

A Nomenclature committee for Factors of the Dog Major Histocompatibility System or Dog Leukocyte Antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence based nomenclature for the genes of the DLA system. The remit of this committee includes: assignment of gene names rules for naming alleles assignment of names to published alleles assignment of names to new alleles rules for acceptance of new alleles DLA Nomenclature Committee, rules for acceptance, DLA genes and alleles, sequence based nomenclature.

Nomenclature for factors of the dog major histocompatibility system (DLA), 1998. First report of the ISAG DLA Nomenclature Committee. International Society for Animals Genetics.

A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles.