Barbara Paoletti

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.

Molecular detection of Anaplasma platys in dogs using polymerase chain reaction and reverse line blot hybridization

Several polymerase chain reactions (PCRs) and a reverse line blot hybridization (RLB) method were used to identify Anaplasma platys in dogs held in a kennel in Italy. Whereas PCR techniques confirmed the presence of A. platys, the RLB method not only correlated the results obtained by PCR but also ruled out the presence of other species such as Ehrlichia canis or E. chaffeensis. There was no correlation between infection status and age or breed of the dogs. Polymerase chain reaction performed on the Rhipicephalus sanguineus ticks collected from those dogs showed that they were also infected with A. platys. Sequences obtained from some samples and compared with those within the GenBank also confirmed the presence of A. platys.

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.

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.

First report of multiple drug resistance in trichostrongyles affecting sheep under field conditions in Italy

Drug resistance in sheep gastrointestinal trichostrongyles is a cosmopolitan major constraint to small ruminant production. Despite reports that anthelmintic drug resistance has become common, there are limited information on the presence of drug resistance in Italy. The purpose of this study was to determine the effect of four anthelmintics to control infection in sheep in central Italy. Fifty sheep with fecal egg counts (FEC) ≥150 eggs per gram were selected on each of three farms (n = 150 total sheep) which were randomly allocated to one of five groups. Groups were treated with febantel, levamisole, ivermectin, or moxidectin while the fifth group acted as the control group. A FEC reduction test (FECRT) was conducted on each animal and the mean FEC of each treatment group was compared to that of the control group within farm. Resistance was declared when percentage reduction (R) <95% and the lower 95% confidence interval was <90%. Levamisole (mean R = 89%) resistance was found on all farms and ivermectin (mean R = 93%) resistance was found on two of the three farms. Posttreatment larval cultures showed the presence of Teladorsagia (Ostertagia) circumcincta and Trichostrongylus spp. larvae. Febantel (mean R = 96%) and moxidectin (mean R = 100%) remained effective. This study suggests that drug resistance in sheep gastrointestinal trichostrongyles is present in central Italy and a potential problem which would justify a broader nationwide geographical investigation.

Autochthonous foci of canine and feline infections by Dirofilaria immitis and Dirofilaria repens in central Italy

This survey was conducted to investigate the presence of Dirofilaria immitis and Dirofilaria repens in native dogs and cats living in an area of Italy where animal and human dirofilarioses have been sporadically reported in the past few years. From May 2008 to March 2009 individual blood samples were collected from 300 native cats and 300 native dogs in the Abruzzo region of central Italy. All 600 samples were subjected to a Knott modified method, to a test for the detection of circulating D. immitis antigen and to two species-specific PCR-coupled sequencing protocols. Epidemiological data were collected and then correlated with the filarial prevalence by univariate and multivariate statistical analyses. Twenty-five dogs (8.3%) and six cats (2%) were positive for filariae in at least one diagnostic test, with an overall prevalence rate for D. immitis and D. repens of 2.3% and 5.6% in dogs and 0.3% and 1.6% in cats, respectively. Male and large sized dogs resulted more likely to be infected by Dirofilaria spp., possibly due to the fact that animals living outdoor and of large size are more exposed to mosquito bites. However these factors were not confirmed as convincing risk factors by multivariate analysis. The results are discussed, together with the origin of these new autochthonous foci in central Italy and the present expansion southward of canine and feline filariae in Italy.

Molecular Detection of Capillaria aerophila, an Agent of Canine and Feline Pulmonary Capillariosis

ABSTRACT Capillaria aerophila, a trichuroid nematode causing pulmonary infections in wild and domestic carnivores, is occasionally and potentially poorly recognized in infections of humans due to clinicopathological mimicry and a lack of accurate, robust laboratory diagnostics. The present work evaluated the efficiency of a DNA-based assay amplifying a partial cytochrome c oxidase subunit 1 (cox1) gene of C. aerophila in the diagnosis of lung capillariosis. Fecal samples from 34 dogs and 10 cats positive at parasitological examination for C. aerophila and other endoparasites (i.e., other lungworms, whipworms, roundworms, hookworms, tapeworms, and/or coccidia) and from 44 animals negative for C. aerophila but positive for other endoparasites were molecularly examined. Of the 44 samples positive for C. aerophila at copromicroscopy, 43 scored positive (i.e., 33/34 dogs and 10/10 cats) in seminested PCR, resulting in a sensitivity of 97 to 100%. Samples that were copromicroscopy negative for C. aerophila although positive for other endoparasites never produced a PCR product or nonspecific amplicons. The specific PCR amplification of C. aerophila (i.e., specificity of 100%) was confirmed by a nucleotide sequence analysis of the cox1 amplicons. The potential implications of the molecular diagnosis of lung capillariosis are discussed.

Prevalence of Giardia spp. in dogs and humans in northern and central Italy.

Despite the public health importance and the possible zoonotic role of Giardia, epidemiological survey on the prevalence of giardiosis in Italy are lacking and fragmentary, compared to other countries. A cross-sectional coprological study on giardiosis was carried out in central and northern Italy in dogs and humans with the aim to providing data about prevalence and potential risk factors. A total of 616 dogs (436 from central and 180 from northern Italy) and 300 humans (central Italy) were examined. The overall prevalence was 21.3% in dogs and 2% in humans. No differences were noted between dog’s prevalence in central and northern Italy (22.2% and 19.4% respectively). The major risk factors for giardiosis in dogs were represented by density of dogs and age, i.e. dogs kept in kennel (PR=2.13, APe=53%) and younger than 1 year (PR=1.99, APe=50%) showed higher prevalence than others. Higher prevalence was noted also in dogs with gastrointestinal symptoms (PR=1.86%, APe=46%). Looking forward to obtain information on these Giardia genotypes, all the measures for the prevention of giardiosis, screening of dogs for giardiosis, particularly puppies, even those with no symptoms, and their treatment to prevent eventual human infection is highly recommended.

Immunoenzymatic analysis and genetic detection of Cryptosporidium parvum in lambs from Italy.

Cryptosporidiosis is a worldwide-diffused protozoan disease causing important economic losses to animal husbandry and livestock production. Additionally, several species/genotypes of Cryptosporidium have a relevant zoonotic potential and ruminants may be important sources of infection for human beings. Nonetheless, in Europe, little is known of the presence of Cryptosporidium in sheep nor on the species/genotypes involved. To obtain information on the occurrence of cryptosporidiosis in lambs and the potential zoonotic role of the Cryptosporidium isolates, one hundred and forty-nine faecal samples individually collected from lambs in central Italy have been examined for the presence of Cryptosporidium. All faecal specimens were processed with a commercial ELISA kit immunoassay and all ELISA-positive samples were further analyzed genetically. Twenty-six ELISA-positive samples scored positive at the PCR and the sequences obtained displayed 100% identity with the zoonotic Cryptosporidum parvum. This work suggests for the first time that lambs in Italy may shed C. parvum, thus representing a potential public health hazard.