Laura Altet

Mapping and sequencing of the canine NRAMP1 gene and identification of mutations in leishmaniasis-susceptible dogs

ABSTRACT The NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, including Leishmania. Our aims were to sequence and map the canine Slc11a1 gene and to identify mutations that may be associated with resistance or susceptibility to Leishmania infection. The canine Slc11a1 gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected with Leishmania infantum to search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility.

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.

Feline vector-borne pathogens in the north and centre of Portugal

BackgroundIn recent years, several clinical cases and epidemiological studies of feline vector-borne diseases (FVBD) have been reported worldwide. Nonetheless, information on FVBD agents and their prevalence in Portugal is scarce.MethodsThree-hundred and twenty domestic cats presented to 30 veterinary medical centres in the north and centre regions of Portugal were randomly sampled. Blood was assayed by real-time polymerase chain reaction (PCR) for genera Anaplasma/Ehrlichia, genus Babesia, Hepatozoon canis, Hepatozoon felis, Leishmania infantum and the genus Rickettsia. Babesia-positive samples were further tested for Babesia canis and Babesia vogeli.ResultsEighty (25.0%) out of the 320 cats were positive to at least one vector-borne agent, including seven (2.2%) cats co-infected with two agents. Two cats (0.6%) were infected with Anaplasma/Ehrlichia spp., four (1.3%) with B. canis, 26 (8.1%) with B. vogeli, 50 (15.6%) with H. felis, one (0.3%) with L. infantum and four (1.3%) with Rickettsia spp. No cat tested positive for H. canis. One cat (0.3%) was co-infected with B. canis and B. vogeli, three (0.9%) with B. vogeli and H. felis, one (0.3%) with H. felis and L. infantum, and two (0.6%) with H. felis and Rickettsia spp.ConclusionsA considerable prevalence of infection with vector-borne pathogens among the domestic feline population of the north and centre of Portugal has been revealed by the present study. Additionally, this is the first detection of B. vogeli in cats from Europe and of H. felis in cats from Portugal.

Canine leishmaniasis: the key points for qPCR result interpretation

BackgroundDiagnosis 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.FindingsThis 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.ConclusionsBecause 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.

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

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.

Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program

AbstractThe main goal of ex situ conservation programs is to improve the chances of long term survival of natural populations by founding and managing captive colonies that can serve as a source of individuals for future reintroductions or to reinforce existing populations. The degree in which a captive breeding program has captured the genetic diversity existing in the source wild population has seldom been evaluated. In this study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison. Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indicating that no substantial loss of diversity has occurred in the captive-breeding program. The effective number of founders of the program was estimated as ∼ ∼16, suggesting that all founders in the studbook pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to distinguish wolves, dogs and their hybrids.

Slc11a1 (formerly Nramp1) and susceptibility to canine visceral leishmaniasis.

Visceral leishmaniasis is the most important zoonosis in Europe and it is caused by Leishmania infantum, a protozoan intracellular parasite. Canine visceral leishmaniasis (CVL) is endemic in the Mediterranean basin, Middle East, and South America, and is emerging within non endemic areas such as the United Kingdom and North America. We have analyzed 24 polymorphisms in the canine Slc11a1 (formerly NRAMP1) gene: 19 new polymorphisms characterized by direct sequencing from 40 dogs of different breeds and five polymorphisms previously described. Data analysis in a case-control study including 164 dogs of 19 different breeds revealed that two of the 24 polymorphisms were associated with increased risk for CVL: one intronic single nucleotide polymorphism (SNP) (A4549G in intron 6: odds ratio (OR) = 6.78, P = 0.001) and one silent SNP in exon 8 (C4859T: OR = 13.44, P = 0.004). In silico analysis of the significant SNP revealed that SNP in the promoter region affect putative transcription binding sites and SNP C4859T in exon 8 disrupts a putative exonic splicing enhancer (ESE). These results corroborate that Slc11a1 polymorphisms are associated with increased risk for CVL.

Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control

A novel triple lines lateral-flow assay (LFA) with enhanced sensitivity for the detection of Leishmania infantum DNA in dog blood samples was designed and successfully applied. The enhanced LFA methodology takes advantage of the gold nanoparticle tags (AuNPs) conjugated to polyclonal secondary antibodies, which recognize anti-FITC antibodies. The polyclonal nature of the secondary antibodies allows for multiple binding to primary antibodies, leading to enhanced AuNP plasmonics signal. Furthermore, endogenous control consisting of the amplified dog 18S rRNA gene was introduced to avoid false negatives. Using this strategy, 0.038 spiked Leishmania parasites per DNA amplification reaction (1 parasite/100 μL of DNA sample) were detected. Detection limit of LFA was found to be lower than that of the conventional techniques. In summary, our novel LFA design is a universal and simple sensing alternative that can be extended to several other biosensing scenarios.

A selective sweep of >8 Mb on chromosome 26 in the Boxer genome.

BackgroundModern dog breeds display traits that are either breed-specific or shared by a few breeds as a result of genetic bottlenecks during the breed creation process and artificial selection for breed standards. Selective sweeps in the genome result from strong selection and can be detected as a reduction or elimination of polymorphism in a given region of the genome.ResultsExtended regions of homozygosity, indicative of selective sweeps, were identified in a genome-wide scan dataset of 25 Boxers from the United Kingdom genotyped at ~20,000 single-nucleotide polymorphisms (SNPs). These regions were further examined in a second dataset of Boxers collected from a different geographical location and genotyped using higher density SNP arrays (~170,000 SNPs). A selective sweep previously associated with canine brachycephaly was detected on chromosome 1. A novel selective sweep of over 8 Mb was observed on chromosome 26 in Boxer and for a shorter region in English and French bulldogs. It was absent in 171 samples from eight other dog breeds and 7 Iberian wolf samples. A region of extended increased heterozygosity on chromosome 9 overlapped with a previously reported copy number variant (CNV) which was polymorphic in multiple dog breeds.ConclusionA selective sweep of more than 8 Mb on chromosome 26 was identified in the Boxer genome. This sweep is likely caused by strong artificial selection for a trait of interest and could have inadvertently led to undesired health implications for this breed. Furthermore, we provide supporting evidence for two previously described regions: a selective sweep on chromosome 1 associated with canine brachycephaly and a CNV on chromosome 9 polymorphic in multiple dog breeds.

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.