Whole-Genome Sequencing and De Novo Assembly of Malassezia pachydermatis Isolated from the Ear Canal of a Dog with Otitis.

We have de novo assembled the genome sequence of Malassezia pachydermatis isolated from a canine otitis sample with Nanopore-only long reads. With 99× coverage and 8.23 Mbp, the genome sequence was assembled in 10 contigs, with 6 of them corresponding to chromosomes, improving the scaffolding of previous genome assemblies for the species.

Whole-Genome Sequencing and De Novo Assembly of 61 Staphylococcus pseudintermedius Isolates from Healthy Dogs and Dogs with Pyoderma.

We have de novo assembled and polished 61 Staphylococcus pseudintermedius genome sequences with Nanopore-only long reads. Completeness was 99.25%. The average genome size was 2.70 Mbp, comprising 2,506 coding sequences, 19 complete rRNAs, 56 to 59 tRNAs, and 4 noncoding RNAs (ncRNAs), as well as CRISPR arrays.

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.

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.

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.

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.

Biting rates of Culicoides midges (Diptera: Ceratopogonidae) on sheep in northeastern Spain in relation to midge capture using UV light and carbon dioxide-baited traps.

Biting midges in the genus Culicoides (Diptera: Ceratopogonidae) were collected near sunset by direct aspiration from sheep in northeastern Spain to determine species-specific biting rates and crepuscular activity. Midges were also collected by UV-baited light traps and CO2-baited traps over the same period to compare species diversity and abundance using these common surveillance methods to actual sheep attack rates. Culicoides aspirated from sheep included C. obsoletus, C. parroti, C. scoticus, C. punctatus, and C. imicola. Peak host-seeking activity during the time period examined for the two most commonly collected species (C. obsoletus and C. parroti) occurred just before sunset and activity ceased within 1 h after sunset. Host attack rates near sunset averaged 0.9 midges/min for both species with maximum attack rates of 3/min for C. obsoletus and 4/min for C. parroti. For both species, approximately 35% of midges collected from the sheep were engorged, giving a maximum biting rate of 1.1/min for C. obsoletus and 1.5/min for C. parroti. Traps baited with CO2 collected fewer midges of each species relative to other collection methods. Traps baited with UV light provided a good indication of species richness but significantly underestimated the host attack rate of C. obsoletus and C. parroti while overestimating the host attack rate of C. imicola. Animal-baited collecting is critical to interpret the epidemiological significance of light trap collections used for surveillance of the midge vectors of bluetongue virus and African horse sickness virus.

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.

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.

Lack of PrP(sc) immunostaining in intracranial ectopic lymphoid follicles in a sheep with concomitant non-suppurative encephalitis and Nor98-like atypical scrapie: a case report.

During active surveillance for transmissible spongiform encephalopathies (TSEs) in sheep, an initial reactor was detected using a rapid test on a brain sample. Immunohistochemistry confirmed an atypical TSE presentation that closely resembled the previously described Nor98 cases. Sequencing of the prnp gene confirmed the ARQ/AHQ genotype with the L141F mutation at codon 141 associated with this phenotype. The head, including the brain and cranial lymphoid tissues, was sampled and examined thoroughly. Non-purulent encephalitis, with ectopic lymphoid follicle formation within the brain, was diagnosed concomitant to the TSE. When scrapie-associated prion protein (PrP(sc)) deposition was studied by immunohistochemistry there was a noticeable lack of lymphotropism. The distribution of PrP(sc) in the brain differed considerably from that of classical scrapie cases. Astrogliosis and microgliosis were demonstrated by histochemical procedures.

Canine DLA diversity: 1. New alleles and haplotypes.

The aim of this component was to establish the range of DLA diversity in as many dog breeds as possible. In particular, we wanted to collect breeds that had not previously been studied. Data were submitted of 937 dogs of over 80 different breeds, and these included 17 'new' breeds. Twenty-eight new alleles were identified including 21 DLA-DRB1, 2 DLA-DQA1 and 5 DLA-DQB1 alleles. These occurred in many new haplotype combinations. One haplotype was identified that appeared to lack DQB1. Two other haplotypes carry two DQB1 genes. It was clear that each dog breed has a restricted range of DLA alleles and haplotypes, and no breed had all 88 haplotypes identified in this study.

Association analyses between the prion protein locus and reproductive and lamb weight traits in Ripollesa sheep.

The objective of this study was to analyze the association between the haplotypes of the prion protein (PrP) locus and several reproductive and lamb weight traits in Ripollesa sheep. Prion protein genotypes were available for a total of 310 sheep (7 rams, 114 ewes, and 189 lambs), all of them belonging to the purebred Ripollesa flock of the Universitat Autònoma of Barcelona, for which all sheep had a known pedigree. In addition, the genotype of 24 historical descendants of the previously genotyped adult individuals was reconstructed, provided that both parents were homozygous for PrP haplotypes. Only 3 haplotypes (ARR, ARQ, and ARH) were observed in the PrP locus of the sheep sampled. Reproductive traits included conception rate and litter size, whereas birth BW and 90-d BW were the lamb weight traits studied. The additive effect of PrP haplotypes was analyzed through Bayesian animal threshold and linear models, for reproduction and weight traits, respectively. Ewe reproductive data belonged to 89 ewes that gave 492 conception rate records and 440 litter size records. Analyses of BW at birth and at 90 d of age were made on 323 and 164 lamb records, respectively. No associations between PrP haplotypes and conception rate and BW traits were observed. For litter size, the effect of the ARH haplotype was greater than that of the ARQ haplotype. Differences between ARH and ARR haplotypes also suggested an advantage for the ARH. As a whole, our results indicated that the selection favorable to increase litter size in Ripollesa ewes may also increase the ARH haplotype frequency, which contradicts the recommendations of the current European Union legislation aiming to increase the genetic resistance to scrapie. As a consequence, scrapie genotyping needs to be included as a new selection criterion in the breed.

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.

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.

Safety and efficacy of antimicrobial peptides against naturally acquired leishmaniasis.

Leishmaniases, which are important causes of morbidity and mortality in humans and dogs, are extremely difficult to treat. Antimicrobial peptides are rarely used as alternative treatments for naturally acquired parasitic diseases. Here we report that the acylated synthetic antimicrobial peptide Oct-CA(1-7)M(2-9) is safe and effective for treating naturally acquired canine leishmaniasis.

Nomenclature for factors of the dog major histocompatibility system (DLA), 2000: Second report of the ISAG DLA Nomenclature Committee.

The ISAG DLA Nomenclature Committee met during the "Comparative Evolution of the Mammalian MHC" meeting in Manchester, England on 10th September 2000. The main points discussed were the naming of class I genes and alleles, and the inclusion of alleles from other canidae.

Nomenclature for factors of the dog major histocompatibility system (DLA), 2000: second report of the ISAG DLA Nomenclature Committee.

The International Society for Animal Genetics (ISAG) Dog Leukocyte Antigen (DLA) Nomenclature Committee met during the "Comparative Evolution of the Mammalian major Histocompatibility Complex (MHC)" meeting in Manchester, UK on 10 September 2000. The main points discussed were the naming of class I genes and alleles, and the inclusion of alleles from other canidae.

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.