Development and characterization of 15 novel polymorphic microsatellite loci for two important bot flies (Diptera, Oestridae) by next-generation sequencing.

Cephenemyia stimulator and Oestrus ovis are two important parasitic bot flies (Oestridae) species causing myiasis, with a potential negative impact on the welfare of the host. Using next-generation sequencing approach and bioinformatics tools, a large panel of possible microsatellites loci was obtained in both species. Primer pairs were designed for 15 selected microsatellite loci in C. stimulator and other 15 loci in O. ovis for PCR amplification. Loci amplification and analysis were performed in four populations of each species. The results demonstrated that all selected loci were polymorphic, with the number of alleles ranging from 2 to 6 per locus in C. stimulator and 3 to 13 per locus in O. ovis. This is the first time to describe these microsatellite loci for C. stimulator and O. ovis. These two sets of microsatellite markers could be further used for biogeographic and population genetics studies.

Noninvasive heavy metal pollution assessment by means of Iberian wolf (Canis lupus signatus) hair from Galicia (NW Spain): a comparison with invasive samples.

The current study represents the first investigation of the suitability of wolf hair as indicator tissue for metal exposure and accumulation within NW Spanish ecosystems. Dead animals, which were not specifically killed for these purposes, were necropsied, and further toxicological analyses were performed in order to establish the heavy metal content (Pb, Cd, and Zn) in liver, kidney, and hair, by means of inductively coupled plasma optical emission spectrometry. Two different factors, gender and age, were considered in order to determine their influence on heavy metal accumulation. Mean liver, kidney and hair concentrations of both toxic element, Cd (0.528, 2.692, and 0.026 ppm) and Pb (4.108, 0.031, and 0.196 ppm) considered on a dry weight basis, were situated below the established as acute toxicity levels for mammals. The highest concentrations were quantified for Zn, with means ranging from a maximum in hair samples (150.9 ppm) to a minimum in kidney samples (25.81 ppm). When the variable of gender and age were considered, female and adult wolves had higher concentrations of Cd in all the considered organs, although differences were only statistically significant for kidney. Neither the sex nor the age had a significant effect on Pb and Zn concentrations. A significant positive correlation (p < 0.05) was identified between hair and liver Pb concentrations, as well as between hair and kidney Cd concentrations, thus suggesting the interest of the noninvasive sample for future ecotoxicological biomonitoring studies.

Seropathotypes, Phylogroups, Stx subtypes, and intimin types of wildlife-carried, shiga toxin-producing escherichia coli strains with the same characteristics as human-pathogenic isolates.

The objectives of this study were to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) strains in wildlife that have spread in Europe, living near human settlements; to analyze their epidemiological role in maintenance and transmission to domestic livestock; and to assess the potential health risk of wildlife-carried strains. STEC strains were recovered from 53% of roe deer, 8.4% of wild boars, and 1.9% of foxes sampled in the northwest of Spain (Galicia). Of the 40 serotypes identified, 21 were classified as seropathotypes associated with human disease, accounting for 81.5% of the wildlife-carried STEC strains, including the enterohemorrhagic serotypes O157:H7-D-eae-γ1, O26:[H11]-B1-eae-ß1, O121:H19-B1-eae-ε1, and O145:[H28]-D-eae-γ1. None of the wildlife-carried strains belonged to the highly pathogenic serotype O104:H4-B1 from the recent Germany outbreak. Forty percent of wildlife-carried STEC strains shared serotypes, phylogroups, intimin types, and Stx profiles with isolates from human patients from the same geographic area. Furthermore, wildlife-carried strains belonging to serotypes O5:HNM-A, O26:[H11]-B1, O76:H19-B1, O145:[H28]-D, O146:H21-B1, and O157:H7-D showed pulsed-field gel electrophoresis (PFGE) profiles with >85% similarity to human-pathogenic STEC strains. We also found a high level of similarity among STEC strains of serotypes O5:HNM-A, O26:[H11]-B1, and O145:HNM-D of bovine (feces and beef) and wildlife origins. Interestingly, O146:H21-B1, the second most frequently detected serotype in this study, is commonly associated with human diarrhea and isolated from beef and vegetables sold in Galicia. Importantly, at least 3 STEC isolates from foxes (O5:HNM-A-eae-ß1, O98:[H21]-B1-eae-ζ1, and O146:[H21]-B1) showed characteristics similar to those of human STEC strains. In conclusion, roe deer, wild boar, and fox in Galicia are confirmed to be carriers of STEC strains potentially pathogenic for humans and seem to play an important role in the maintenance of STEC.

The complete mitochondrial genome of Cephenemyia stimulator (Diptera: Oestridae).

Cephenemyia stimulator parasitizes roe deer (Capreolus capreolus) throughout its geographical distribution. The complete circular C. stimulator mitogenome was assembled, which is 16,407 bp in length, and encodes 13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes. A phylogenetic tree was built with mitogenome sequences, including C. stimulator and 13 related Oestridae species, using Sarcophaga tuberosa as an outgroup.

Characterization of New Molecular Markers of Three Botflies Parasitizing Cervid Hosts.

Specific identification of oestrid larvae is usually problematic not only when using morphobiometric features, but also when applying molecular criteria, since very few molecular markers have been described for this group of flies. New molecular markers for oestrid are needed for more reliable species identification, diagnostic purposes, and epidemiological surveys; moreover, they can help in phylogenetic reconstruction. Here, we report the characterization of COI, 28S rDNA, ITS1, and ITS2 in Cephenemyia stimulator from roe deer and in Cephenemyia auribarbis and Pharyngomyia picta from red deer. The COI and 28S rDNA are very uniform in length, while the ITSs sequences are highly variable at both intraspecific and interspecific levels. The described ITSs sequences were longer than those described for other dipteran species by the presence of simple repeats and tandem repeat sequences. In C. auribarbis both ITS1 and ITS2 appeared as two variants, one short and the other long. In general, the analyzed markers present low intraspecific genetic variation and high interspecific variation. ITSs showed the greatest amount of intraspecific and interspecific variation. Phylogenetic analysis demonstrated that the characterized sequences differentiate the species and genera of Oestridae.

Xanthine nephrolithiasis in a galician blond beef calf.

A six-month-old female Galician Blond beef calf presented signs of apathy, anorexia and weight loss. The analysis of a blood sample confirmed renal failure. Bilateral nephrolithiasis was diagnosed at necropsy. Quantitative analysis revealed the nephroliths to be composed of 100 per cent xanthine. In cattle, xanthinuria has only been described in the Japanese Black breed, but never before in other breeds. Clinical history suggested a naturally occurring xanthinuria.

The red fox (Vulpes vulpes) as a potential natural reservoir of human cryptosporidiosis by Cryptosporidium hominis in Northwest Spain.

Giardia duodenalis and Cryptosporidium spp. are ubiquitous intestinal protozoa that parasitize domestic and wild animals, as well as human beings. Due to their zoonotic potential, the objective of the present study was to determine the presence of these pathogens in the fox population (Vulpes vulpes) located in Northwest Spain. A total of 197 faecal samples from legally hunted foxes were collected in the autonomous region of Galicia. The presence of G. duodenalis and Cryptosporidium spp. was investigated by PCR-based methods amplifying the small subunit ribosomal RNA (ssu rRNA) gene of the parasites. Attempts to genotype obtained positive samples were subsequently conducted at the glutamate dehydrogenase (gdh) and ß-giardin (bg) genes of G. duodenalis, and the 60 kDa glycoprotein (gp60) gene of Cryptosporidium. Giardia duodenalis and Cryptosporidium spp. were identified in 19 (9.6%) and 12 (6.1%) of the investigated samples, respectively. However, five Cryptosporidium species were detected at the ssu rRNA locus: C. hominis (33.4%, 4/12), C. canis (25.0%, 3/12), C. parvum (16.7%, 2/12), C. ubiquitum (8.3%, 1/12) and C. suis (8.3%, 1/12). An additional Cryptosporidium-positive sample was identified at the genus level only. Typing and subtyping of Giardia- and Cryptosporidium-positive samples were unsuccessful. The detection of C. hominis in wild foxes indicates the probable overlapping of sylvatic and domestic cycles of this parasite in rural settings. Besides, this finding raises the question of whether red foxes may act as natural reservoirs of C. hominis. The detection of C. parvum and C. suis is suggestive of active transmission events between farm and wild animals, opening up the possibility of transmission to human beings.

Clinico-pathological findings and coagulation disorders in 45 cases of canine babesiosis in Spain.

A retrospective study of clinical cases of babesiosis in dogs examined at the Veterinary Teaching Hospital, Rof Codina, from January 2003 to October 2004 is presented. The diagnosis was confirmed by direct observation of large piroplasms in stained blood smears. Dogs with concurrent diseases were excluded from the study. Clinical signs, complete blood count, serum biochemistry and hemostasis profiles were obtained. The observed clinical signs were due to hemolytic anemia and inflammatory responses but the most relevant clinico-pathological findings were related to alterations in hemostasis. All dogs presented with thrombocytopenia and 20% had disseminated intravascular coagulation syndrome. Anemia of variable severity was observed in most of the dogs.