Investigation on the occurrence of Echinococcus multilocularis in Central Italy

<p>Abstract</p> <p>Background</p> <p>Recent studies on geographic distribution of <it>Echinococcus multilocularis </it>in Europe show that it has a wider range than previously thought. It is unclear, however, if the wider distribution is due to its recent spreading or to a lack of previous data from the new areas. Italy, previously considered <it>E. multilocularis</it>-free, is now part of these new areas: infected foxes (the main definitive host of the tapeworm) have been observed in a Northern Alpine territory. Thus, more surveys need to be done in other Italian regions in order to monitor the spreading of <it>E. multilocularis</it>. The aim of the present study was to look for this parasite in 283 foxes collected in an Apennine area of Central Italy by different diagnostic methods.</p> <p>Results</p> <p>The foxes were heavily parasitized by 11 helminthic genera, but none of the animals was infected by <it>E. multilocularis </it>neither by <it>E. granulosus </it>(harboured adult worms or their DNA). Low specificity was observed in commercially available ELISA kits for the detection of <it>E. multilocularis </it>antigens in the faeces. Molecular diagnostics were sensitive and specific for the detection and identification of tapeworm eggs in faeces, but less sensitive, although specific, to adult tapeworms in the intestinal content.</p> <p>Conclusion</p> <p>Preliminarily, we can say that no <it>E. multilocularis </it>could be found in the study area. These data will enable us to follow temporal changes of the spatial distribution of the parasite in the study area of the Central Apennines. Due to its low specificity the ELISA kit for <it>E. multilocularis </it>coproantigens is not suitable for epidemiological surveys, whereas molecular diagnostics applied to faecal samples give useful results. Finally, absence of <it>E. granulosus </it>in foxes living in the endemic areas studied confirms the thought that this tapeworm prefers a different definitive host.</p

Selection of Biomarkers from Differentially Expressed Genes in Leukocytes of Buffalos Treated with Recombinant Bovine Somatotropin: The Importance of Sample Size for Reliable Discriminating Systems

The research on biomarkers to detect livestock treated with recombinant bovine somatotropin (rbST) is still an open issue. In fact, beyond undertaking confirmation methods, there is the need to develop simple and inexpensive screening tests. In this direction, some proposals have been forwarded, mostly involving the measurement of circulating molecules, whereas the possibility of using biomarkers related to gene expression is a field under investigation. The present study was carried out on sixteen buffalos, eight of which treated with rbST. Blood samples were collected six times during the treatment to investigate on the presence of differentially expressed genes in leukocytes. Analysis with the microarray technique was performed on two sampling moments, in order to obtain a first selection of genes. Further analysis was carried out by real time RT-PCR, in order to create a discriminating linear system. A study on the variation of the error related to the number of samples included in statistics was also performed. Results showed that, including an increasing number of samples to build the discriminating algorithm, the b-error grows and tends to stabilize on 6.5%. This study clearly shows the paramount importance of including a proper number of samples to obtain reliable algorithms

Natural and recombinant bovine somatotropin: immunodetection with a sandwich ELISA

Bovine Somatotropin (bST) is a peptide hormone secreted by the anterior pituitary gland and its recombinant form (rbST) is used for artificially boosting milk yield in cows. Identification of rbST is difficult in that there is little difference from the pituitary bST (pbST). In this work, we further studied the possibility of immunologically discriminating between rbST and pbST. With this purpose, we produced mouse monoclonal antibodies using, as antigen, a peptide mimicking the N-terminus of rbST from Monsanto (rbST-M) conjugated to keyhole limpet haemocyanin (KLH) and polyclonal antibodies in rabbits immunized with the whole bST or rbST-M. Hence, we developed a sandwich ELISA with the obtained antibodies for detection and quantification of bST in serum and compared its performance on the two worldwide commercialized rbSTs: rbST-M and rbST from LG Life Science (rbST-LG). The lowest detection limit of the assay was 0.05 ng/ml for rbST-M, 0.10 ng/ml for rbST-LG and 0.15 ng/ml for pbST. Furthermore, the assay showed the capability to amplify the signal in the presence of rbSTs, recognizing more efficiently rbST-M and rbST-LG than pbST (ECn pbST/ECn rbST: 3 and 1.6 respectively). Its employment for measuring bST levels in sera from bovines administered with rbST LG allowed us to detect exceptional values due to the treatment itself and probably further increased as a consequence of the higher affinity for rbSTs of our monoclonal antibod

Strongyloides stercoralis in a dog litter: Evidence suggesting a transmammary transmission

Strongyloides stercoralis is a soil-transmitted helminth with an unusual life cycle, causing canine and human strongyloidiasis, mainly endemic in tropical and subtropical areas. Following percutaneous or oral transmission of infective third-stage larvae in the vertebrate host, the parasite can cause autoinfection, leading to life-long infection. At present, the transmammary transmission was only assessed in experimentally infested dogs. Here, we provide observational evidence of S. stercoralis transmammary transmission in puppies suckling from a truffle dog from Central Italy, from where its presence was neglected

A LC-MS-MS method to detect recombinant bovine somatotropin misuse in buffalos

Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the fair trade and the law prescriptions. For this reason, efficient and reliable analytical techniques are required to contrast rbST misuse. A few LC-MS-MS methods have been developed to detect, in cow serum, methyonil-rbST, one of the two main rbST forms available on the market. The other form, which is widespread, is identical to the most abundant variant of bovine somatotropin (bST) and differs from the buffalo somatotropin for one amino acid in the N-terminus. For this reason, it is technically possible to distinguish both rbST forms in serum of buffalos. In this work, we describe a novel LC-MS-MS-based method, capable to quantify, with a high sensitivity and selectivity, the methyonil-rbST and the other bST-identical recombinant form in buffalo serum, previously purified using a solid-phase extraction procedure. The method was internally validated and used to analyse 152 serum samples, collected from eight buffalos administered with rbST for a period of 3 months, according to conventional protocols. The obtained results confirmed the suitability of the method in the detection of illegal hormonal treatments

A Canine Distemper Virus Retrospective Study Conducted from 2011 to 2019 in Central Italy (Latium and Tuscany Regions)

Canine distemper virus (CDV) is a highly lethal contagious viral pathogen mainly found in domestic and wild canids and mustelids. Although, in Italy, circulating strains of Europe 1, Europe wildlife and Arctic type are reported, data relating to Latium and Tuscany regions are limited. In view of this, through passive surveillance, we investigated the presence of CDV and which strains were circulating in these Regions. From March 2017 to October 2019, a group of 122 subjects were tested for CDV using a PCR protocol described in the literature, with 12 detected positive; analyses were carried out on a set of target samples (brain and lung, conjunctival, nasal and rectal swabs, urine or swab from bladder and intracardiac clot) that was defined for the detection of CDV in both live and dead animals. The rectal swab, easily collected also from live animals, represented the most suitable sample for CDV diagnosis, with 9 positive of the 11 (81.82%) tested. In addition, brain and lung of 15 subjects out of 181 susceptible animals collected between 2011 and 2018, during post mortem investigations in routine diagnostic activity, were CDV positive. Molecular analyses of all positive samples, using a 287 bp fragment located within the conserved N terminus of the morbillivirus nucleoprotein gene, detected the circulation of strain CDV599/2016 (KX545421.1) belonging to the “Europe wildlife” lineage, and of strain CDV12254/2015 (KX024709.1), belonging to the Arctic-lineage, thus confirming the co-circulation of the two lineages, as already noted in previous studies

Angiostrongylus vasorum in wolves in Italy: prevalence and pathological findings

Abstract Background Angiostrongylus vasorum is a nematode residing in the heart and pulmonary vessels of dogs and wild carnivores. In Europe the red fox is its reservoir, while only three records from wolves have been published. Angiostrongylus vasorum has a worldwide distribution, and many pieces of evidence demonstrate that it is spreading from endemic areas to new ones. In Italy, A. vasorum was reported with increasing frequency in dogs and foxes in the last decades, and now it is considered endemic throughout the country. Angiostrongylus vasorum can be asymptomatic or cause respiratory and circulatory disorders, at times causing severe disseminated infections. Methods Between February 2012 and December 2016, 25 wolves found dead in central Italy were submitted to the Istituto Zooprofilattico del Lazio e della Toscana for post-mortem examination. Samples of lungs, heart, liver, spleen, kidneys, mediastinic lymph nodes and brain were collected from each animal for histological examination. When adult and larval nematodes were microscopically seen in lungs, the other organs were processed, and five histological sections for each organ were examined. To confirm parasite identification, lung samples were submitted to a PCR-sequencing protocol targeting the ITS2 region of A. vasorum. Results Seven wolves (28.0%) harboured nematode larvae in lung sections. In two of the positive wolves, adult nematodes were visible in pulmonary arteries, in four animals larvae were also detected in other organs. DNA sequencing reactions confirmed parasite identification as A. vasorum in all the cases. Conclusions As a result of the high prevalence of A. vasorum reported in wolves in the present study, a focus of high circulation could be hypothesised in central Italy. Nevertheless, the similarly high prevalence in foxes originating from the same areas were reported in previous papers. Histopathological evidence highlights the pathogenic potential of A. vasorum in the wolf, especially in juvenile animals

Ethylene glycol toxicity: A retrospective pathological study in cats

Ethylene glycol (EG) is a well known toxic compound, the assumption of which can be fatal to pet animals as well as to humans. Limited information is available on the pathological features of EG poisoning in pet animals, with special emphasis on cats. Twenty-five cats with histologically confirmed EG intoxication were retrospectively investigated, in order to define more precisely the gross pathological findings and improve the diagnostic process. Furthermore, a brief comparison with the lesions reported in EG-poisoned human patients and dogs was also made