Raffaella Iorio

Diagnosis and risk factors of Aelurostrongylus abstrusus (Nematoda, Strongylida) infection in cats from Italy

Aelurostrongylus abstrusus (Nematoda, Strongylida) in cats and associated risk factors have been investigated by classical coprology in central (site A) and southern (site B) Italy, i.e. by floatations with sugar and zinc sulphate solutions and a Baermann technique. Overall, 227 individual stool samples (i.e. 162 from site A and 65 from site B) were processed and risk factors were related to infection by binary logistic multiple-regression models. The presence of A. abstrusus was used as the dependent variable and the epidemiological data as independent variables. Twenty-eight (17.3%) and 12 (18.5%) cats were positive for A. abstrusus in sites A and B, respectively, both using the Baermann and flotation with zinc sulphate solution methods. Six and three (from site A and B, respectively) of the infected cats were negative for A. abstrusus when faeces were processed with sugar solution. Stray and free-ranging (P=0.357), young cats (P=0.008) and the presence of respiratory symptoms (P=0.000) were risk factors for A. abstrusus infection. The results suggest that A. abstrusus is spread in Italy. Furthermore, these results possibly include infection in the differential diagnosis of feline respiratory diseases and offer a correct diagnostic approach by using either a Baermann method or flotation with zinc sulphate solution in suspected cases.

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization

ABSTRACT The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species.

Prevalence and molecular characterization of Giardia duodenalis from sheep in central Italy

Giardiosis in domestic ruminants is an important parasitic disease and it has been shown to impair growth in lambs, thus constituting a disease of economic concern. In Europe, surveys on the prevalence of giardiosis in sheep are limited. In order to obtain additional information on the presence of giardiosis and on the potential zoonotic role of Giardia duodenalis affecting sheep in central Italy, faecal samples of 325 native sheep from 20 farms in Abruzzo region (Italy) were examined for the presence of Giardia and the isolates were genotyped and sequenced. G. duodenalis cysts were detected in five of the 325 sheep (1.5%) (mean of 450 cysts/g) coming from two farms. The 770-bp fragment of the glutamate dehydrogenase gene and the 753-bp fragment of the β-giardin gene showed 100% homology with the Assemblage AI. This work suggests for the first time in Italy that sheep carry a Giardia genotype, which can be a potential public health hazard.

Aelurostrongylus abstrusus in a feline colony from central Italy : clinical features, diagnostic procedures and molecular characterization

Clinical features and conventional and molecular diagnostic procedures have been investigated and evaluated for the infection caused by the lungworm Aelurostrongylus abstrusus (Nematoda, Strongylida). Individual fecal samples from all cats living in a colony with suspected lungworm infection underwent coprological flotation with sugar and zinc sulfate solution and the Baermann migration method. Also, pharyngeal swabs collected for each animal were subjected to a diagnostic nested PCR assay specific for a region internal to the ribosomal Internal Transcribed Spacer 2 of A. abstrusus. Eighteen animals were positive at the Baermann method, while 12 and ten out of them were negative when feces were subjected to the flotation with sugar and zinc sulfate solution, respectively. The nested PCR assay yielded positive results when using the pharyngeal swabs from the 18 coprologically positive cats and from six more cats which were coprologically negative, thus indicating an overall infection rate of 24.4%. Twenty-two out of 24 infected cats showed clinical respiratory symptoms and the most common were general respiratory distress, cough, wheezing, sneezing, and nasal discharge. These results indicate that cat aelurostrongylosis is of clinical importance and, thus, needs to be included in differential diagnosis of feline respiratory diseases. The importance of the disease is discussed together with pros and cons of different conventional and innovative diagnostic approaches.

Diagnostic and Clinical Implications of a Nested PCR Specific for Ribosomal DNA of the Feline Lungworm Aelurostrongylus abstrusus (Nematoda, Strongylida)

ABSTRACT Aelurostrongylus abstrusus (Nematoda, Strongylida, Metastrongyloidea) is a cosmopolitan parasite of cats and causes severe respiratory distress. Information on the biology and epidemiology of feline aelurostrongylosis is fragmentary, mainly due to the limits inherent in the classical diagnosis. In the present work, a two-step nested PCR based on the use of genetic markers in the second internal transcribed spacer (ITS2) of ribosomal DNA was established for A. abstrusus in different biological samples. Characterization of the ITS2 (321 bp of length) revealed a G+C content of 39.5%. To exploit the sequence difference between the ITS2 of A. abstrusus and those of other common feline endoparasites, specific primers were designed and tested by PCR for their specificities and sensitivities. The PCR assay was validated on a panel of fecal (i.e., feces, flotation supernatant, and Baermann sediment) and pharyngeal swab samples from cats with known histories of lungworm infections, and it showed a specificity of 100% and a sensitivity of up to 96.6%. Also, the nested PCR was able to identify cats that were actually infected but that tested negative by the classical diagnostic methods. This PCR method was shown to be a powerful tool for the molecular diagnosis of feline aelurostrongylosis, overcoming the constraints of the classical diagnosis. The implications of such a molecular tool for further bioepidemiological studies of both intermediate and definitive hosts have been discussed.

Infection by Eucoleus aerophilus in dogs and cats: is another extra-intestinal parasitic nematode of pets emerging in Italy?

The occurrence of the infection by the lungworm Eucoleus aerophilus (syn. Capillaria aerophila) in dogs and cats from Italy has been evaluated with conventional diagnostic procedures. Individual faecal samples from 569 dogs and 200 cats were undertaken to faecal flotation with sugar and zinc sulphate solution. Sixteen dogs (2.8%) and 11 cats (5.5%) scored positive for eggs of E. aerophilus when samples were processed with either of the two flotation solutions. Overall 14 of 16 dogs and eight of 11 cats infected by E. aerophilus showed respiratory symptoms and the most common were general respiratory distress, dry cough, wheezing and sneezing. These results indicate that E. aerophilus is not uncommon and that canine and feline capillariosis is of clinical importance. Given the impact that E. aerophilus infections may have upon animal health and its zoonotic potential, it is strongly advisable to routinely include this disease in the differential diagnosis of (cardio)-respiratory diseases of dogs and cats.

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.

Efficacy and Safety of Imidacloprid 10%/Moxidectin 1% Spot-on Formulation in the Treatment of Feline Aelurostrongylosis

The objective of the present study was to evaluate the efficacy and safety of the antiparasitic spot-on formulation containing imidacloprid 10%/moxidectin 1% (Advocate®, Bayer) in the treatment of natural feline infection with the lungworm Aelurostrongylus abstrusus (Nematoda, Strongylida). The efficacy of Advocate® administered once was tested in comparison to a control oral formulation containing fenbendazole 18.75% (Panacur® Intervet) administered over three consecutive days based on larvae per gramme of faeces (LPG), measured on days 28 ± 2 following treatment and compared to counts on days −6 to −2. In total 24 cats treated either with Advocate® (n = 12) or with Panacur® (n = 12) were included. Mean LPG postbaseline (days 28 ± 2) were low in both treatment groups, i.e., 0 LPG for Advocate® and 1.3 LPG for Panacur®. Reduction of post-baseline larval counts showed Advocate® (100% reduction) to be superior in efficacy compared to the control product (99.29% reduction). No treated animals showed adverse events. This trial demonstrated that both Advocate® spot-on formulation and Panacur® oral paste are safe and effective in the treatment of aelurostrongylosis in cats. Future practical perspectives in feline medicine and the major advantages of the spot-on product compared to the oral paste are discussed.

Dermatophytoses in cats and humans in central Italy: epidemiological aspects

Two hundred hair/skin samples were collected from 2002 to 2004 from two groups of cats (privately owned and stray cats from a shelter) and 165 samples were obtained during the same period from persons in whom dermatophyte infection was highly suspected. The epidemiological data were statistically evaluated. Thirteen of the 100 privately owned cats (13%) and 100% of the stray cats were positive; of the 165 human samples examined 109 (66%) were positive for dermatophytes. Microsporum canis was the most common dermatophyte isolated in both cat groups while Trichophyton mentagrophytes was the most common in humans. Interestingly, a geophylic dermatophyte species (Microsporum gypseum) was found to be present and associated with clinical signs. Living in the countryside proved to be a risk factor for dermatophytoses in privately owned cats while in humans the main risk factor for M. canis was contact with animals followed by young age. None of the variables considered was associated with positivity for T. mentagrophytes while positivity for other fungi was correlated with life in the countryside.

Molecular evidence for Cryptosporidium infection in dogs in Central Italy

Two hundred and forty kennel and privately owned dogs were tested for a molecular epidemiological study on Cryptosporidium infection. Genomic DNA was extracted from individual faecal samples. All the DNA extracts were analysed with a PCR assay specific for a ∼400-bp fragment internal to the gene encoding for the Cryptosporidium oocyst wall protein. The prevalence was 3.3% and it was higher in kennel dogs and in dogs with gastrointestinal symptoms. Cryptosporidiumparvum was detected by sequencing analysis in six kennel dogs and one privately owned dog, and Cryptosporidiumcanis was detected in one kennel dog. This is the first investigation on the prevalence of Cryptosporidium in Italian dogs and has pointed to the existence of genotypes that may be of public health significance.