Theileria sp. OT3 and other tick-borne pathogens in sheep and ticks in Italy: molecular characterization and phylogeny.

PCR Reverse Line Blot (RLB) hybridization and sequencing were used to determine the dynamics of infection with tick-borne pathogens in one hundred apparently healthy sheep in Italy. Blood samples were tested once prior to the onset of the grazing season (June 2010) and once after the end of the grazing season (August 2010). Ticks collected from sheep and from the vegetation were also tested by PCR/RLB. Before grazing, 56% of the sheep harbored several tick-borne pathogens: Anaplasma ovis was the most prevalent (41%), followed by A. ovis co-infected with Theileria sp. OT3 (14%). After grazing, 87% of sheep were positive for A. ovis alone (41%), co-infected with Theileria sp. OT3 (8%) or co-infected with Babesia motasi (5%). Other sheep were infected with Anaplasma phagocytophilum alone (20%), co-infected with B. motasi (7%) or with Theileria sp. OT3 (5%) (p<0.001). After grazing, sheep were significantly more infected with tick-borne pathogens than before grazing. Ticks collected were all Haemaphysalis punctata (n-89) and 36% were positive for A. ovis, Ehrlichia ovina and A. ovis combined with A. phagocytophilum. Phylogenetic analysis including isolates from countries in the Mediterranean Basin show circulation of the same variants of Theileria sp. OT3, whereas two different geographical origins for the isolates of A. ovis and A. phagocytophilum were identified. This is the first report from Italy of Theileria sp. OT3 in sheep, whereas the detection of Ehrlichia ovina in ticks is worth noting, and the presence of A. phagocytophilum in sheep and in ticks poses a potential public health risk.

Occurrence and molecular typing of Giardia isolates in pet rabbits, chinchillas, guinea pigs and ferrets collected in Europe during 2006-2012.

A total of 1180 faecal samples (528 from rabbits, 531 from chinchillas and 121 from guinea pigs) collected during 2006-2012 by veterinarians in Germany and in other European countries were submitted to a diagnostic laboratory for Giardia testing by means of coproantigen ELISA. Of these samples, 40 rabbits (7.6 per cent), 326 chinchillas (61.4 per cent) and five guinea pigs (4.1 per cent ) were found to be positive. To gain insights into the genetic identity of Giardia in small mammals, ELISA-positive samples from 23 chinchillas, five ferrets, a rabbit, and a Desmarest's hutia were investigated by PCR and sequencing of fragments of the small subunit ribosomal DNA (ssu), the triose phosphate isomerase (tpi) and the ß-giardin (bg) genes. At the ssu locus, assemblage B was identified in 28 of 30 isolates, whereas assemblage A and D were each detected in one sample. The majority of isolates from chinchillas and those from ferrets had Giardia duodenalis sequences identical to sub-assemblages AI or BIV, based on either a single locus (tpi or bg) or multiple loci (tpi and bg). As sub-assemblages AI or BIV are associated with human infection, these results indicate that small mammals can act as reservoirs of cysts potentially infectious to humans.

Fatal bronchopneumonia in a Metastrongylus elongatus and Porcine circovirus type 2 co-infected pig.

Porcine circovirus type 2 (PCV2) infection is distributed worldwide and PCV2-associated disease (PCVAD) is considered among the most economically relevant ones to the global swine industry. PCV2 is known to play a causal role in the porcine respiratory disease complex, usually in close association with a large plethora of other biologic agents. We describe herein a case of fatal parasitic bronchopneumonia by Metastrongylus elongatus in a PCV2-infected pig. Metastrongylosis may still represent a major concern for outdoor herds. Our recent experience suggests that a concurrent PCVAD condition may trigger metastrongylosis, which may subsequently result, at its turn, in severe, sometimes fatal, pulmonary disease.

Identification of the intermediate hosts of Habronema microstoma and Habronema muscae under field conditions.

A polymerase chain reaction (PCR)-based assay was used for the specific detection of Habronema microstoma and Habronema muscae (Nematoda, Spirurida) in order to identify the intermediate hosts of both nematode species under field conditions. A total of 1087 netted and 165 laboratory-bred flies were tested. Flies were identified as Musca domestica Linnaeus 1758, Musca autumnalis De Geer 1776, Haematobia irritans (Linnaeus 1758), Haematobia titillans (De Geer 1907) and Stomoxys calcitrans (Linnaeus 1758) (Muscidae). Genomic DNA was extracted from pools of fly heads, thoraces and abdomens, and 703 samples were subjected to a duplex two-step semi-nested PCR assay to specifically detect diagnostic regions within the ribosomal ITS2 sequence of both H. microstoma and H. muscae. Stomoxys calcitrans specimens were positive for H. microstoma DNA and M. domestica specimens were positive for H. muscae DNA. In particular, PCR-positive samples derived from both farm-netted and laboratory-bred flies. The present study represents the first evidence of the vectorial competence of different fly species as intermediate hosts of Habronema stomachworms under field conditions. We discuss the roles of S. calcitrans and M. domestica in transmitting H. microstoma and H. muscae.

Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces.

Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.