Olga Francino

Isolation of Genomic DNA from Feathers

The use of feathers in veterinary clinical practice simplifies the sampling of avian genomic DNA, especially when blood extraction is difficult because of the age or the size of the bird. A rapid and accurate protocol was used to isolate high-quality genomic DNA from feathers. The technique includes a lysis step of the feather quill, which differs in temperature and time of incubation depending on the feather size. Purification of genomic DNA is performed with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. This protocol consistently provided significant amounts of high-quality genomic DNA from more than 800 birds belonging to 120 different species. Genomic DNA isolated with this method was used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing.

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.

Isolation of genomic DNA from milk samples by using Chelex resin

A rapid procedure for isolating genomic DNA from milk samples has been devised, based on the use of Chelex resin. By using this protocol, genomic DNA was extracted from milk samples from 15 cows and 15 goats. The suitability of these DNA preparations as a template for performing the polymerase chain reaction (PCR) was tested by amplifying three different loci of the bovine genome: exon 4 of the kappa-casein gene and the INRA5 and INRA23 microsatellites, together with two others: exon 19 of the alpha s1-casein gene and exon 2 and part of intron 2 of the DRB gene of the caprine genome. No amplification products could be obtained from any sampless at 30 cycles. In contrast, at 45 cycles the number of amplified samples ranged from 86 to 100% and at 65 cycles all the DNA targets were amplified, indicating that the number of cycles was a critical factor to be optimized for obtaining the desired PCR target. These results suggest that this method may be a useful tool for analysing genetic polymorphism at the DNA level by PCR and relating it to milk composition and other traits of economic interest.

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.

Leishmania Infection: Laboratory Diagnosing in the Absence of a “Gold Standard”

There is no gold standard for diagnosing leishmaniases. Our aim was to assess the operative validity of tests used in detecting Leishmania infection using samples from experimental infections, a reliable equivalent to the classic definition of gold standard. Without statistical differences, the highest sensitivity was achieved by protein A (ProtA), immunoglobulin (Ig)G2, indirect fluorescenece antibody test (IFAT), lymphocyte proliferation assay, quantitative real-time polymerase chain reaction of bone marrow (qPCR-BM), qPCR-Blood, and IgG; and the highest specificity by IgG1, IgM, IgA, qPCR-Blood, IgG, IgG2, and qPCR-BM. Maximum positive predictive value was obtained simultaneously by IgG2, qPCR-Blood, and IgG; and maximum negative predictive value by qPCR-BM. Best positive and negative likelihood ratios were obtained by IgG2. The test having the greatest, statistically significant, area under the receiver operating characteristics curve was IgG2 enzyme-linked immunosorbent assay (ELISA). Thus, according to the gold standard used, IFAT and qPCR are far from fulfilling the requirements to be considered gold standards, and the test showing the highest potential to detect Leishmania infection is Leishmania-specific ELISA IgG2.

NESTED PCR ALLOWS THE CHARACTERIZATION OF TAQI AND PSTI RFLPS IN THE SECOND EXON OF THE CAPRINE MHC CLASS II DRB GENE

A nested polymerase chain reaction (PCR) method has been developed to obtain a specific amplification of the second exon of the caprine MHC class II DRB gene. The specificity of this method has been verified by cloning and sequencing the PCR product and comparing its sequence to 21 previously published caprine DRB second exon allelic variants. Nucleotide identity between this sequence (Caae-DRB23) and other caprine DRB alleles ranged between 85.6% (Caae-DRB22) and 96.5% (Caae-DRB5). Caae-DRB5 and Caae-DRB23 sequences diverged in five amino acid substitutions (70, 71, 73, 74, 78), all of them placed at the antigen binding site. Likewise, the restriction polymorphism of the caprine DRB second exon has been analyzed and two different restriction patterns have been found depending on the presence or absence of a TaqI site and a PstI site at positions 122 bp and 241 bp of the PCR product respectively. TaqI and PstI RFLPs were also analyzed in other artiodactyla species. While PstI RFLP was found not only in goats but also in cattle, sheep and pigs, TaqI RFLP was only detected in goats. In all of these species close associations were detected between the presence of TaqI and PstI restriction sites and amino acid substitutions at positions 40 and 78 respectively, suggesting that PCR restriction fragment length polymorphism (RFLP) could be a useful tool in relating amino acid substitutions at critical positions with disease resistance.

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.