Clostridioides difficile in Pigs and Dairy Cattle in Northern Italy: Prevalence, Characterization and Comparison between Animal and Human Strains.

It has been observed that novel strains of Clostridioides difficile can rapidly emerge and move between animal and human hosts. The aim of this study was to investigate the prevalence of C. difficile in pigs and dairy cattle in northern Italy and to characterize and compare C. difficile animal strains with those from patients from the same geographical area. The C. difficile strains were isolated from animals from farms and slaughterhouses (cross-sectional studies) and from neonatal animals with enteric disorders in routine diagnostic investigations (passive surveillance). Samples positive for C. difficile were found in 87% of the pig farms and in 40% of the cattle farms involved in the cross-sectional studies, with a 20% prevalence among suckling piglets and 6.7% prevalence in neonatal calves, with no significant difference between animals with and without diarrheal symptoms. The prevalence of C. difficile in older animal categories was significantly lower. This result suggests that young age is an important risk factor for C. difficile colonization. In cross-sectional studies at slaughterhouses, in both the heavy pigs and dairy cows examined, only 2% of the intestinal content samples were positive for C. difficile and no contamination was found on the surface of the carcasses. Considering passive surveillance, the prevalence rates of positive samples were 29% in piglets and 1.4% in calves. Overall, 267 strains of animal origin and 97 from humans were collected. In total, 39 ribotypes (RTs) were identified, with RT 078 and RT 018 being predominant among animals and humans, respectively. Several RTs overlapped between animals and patients. In particular, RT 569 was identified as an emergent type in our country. Resistance to erythromycin and moxifloxacin was widely diffused among C. difficile strains, regardless of origin. This study supports C. difficile as a pathogen of one-health importance and highlights the need for a collaborative approach between physicians and veterinarians to control and prevent infections that are able to cross species and geographical barriers.

Survey on the Presence of Viruses of Economic and Zoonotic Importance in Avifauna in Northern Italy.

Wild birds play an important role in the circulation and spread of pathogens that are potentially zoonotic or of high economic impact on zootechnical production. They include, for example, West Nile virus (WNV), Usutu virus (USUV), avian influenza virus (AIV), and Newcastle disease virus (NDV), which, despite having mostly an asymptomatic course in wild birds, have a strong impact on public health and zootechnical production. This study investigated the presence of these viruses in several wild bird species from North Italy during the biennium 2019-2020. Wild birds derived from 76 different species belonging to 20 orders. Out of 679 birds, 27 were positive for WNV (lineage 2) with a prevalence of 4%; all birds were negative for USUV; one gull was positive for H13N6 influenza virus, and 12 samples were positive for NDV with a prevalence of 2%. Despite the low prevalence observed, the analyses performed on these species provide further data, allowing a better understanding of the diffusion and evolution of diseases of both economic and zoonotic importance.

Detection of a New Genetic Cluster of Influenza D Virus in Italian Cattle.

Influenza D virus (IDV) has been increasingly reported all over the world. Cattle are considered the major viral reservoir. Based on the hemagglutinin-esterase (HEF) gene, three main genetic and antigenic clusters have been identified: D/OK distributed worldwide, D/660 detected only in the USA and D/Japan in Japan. Up to 2017, all the Italian IDV isolates belonged to the D/OK genetic cluster. From January 2018 to May 2019, we performed virological surveillance for IDV from respiratory outbreaks in 725 bovine farms in Northern Italy by RT-PCR. Seventy-four farms were positive for IDV. A full or partial genome sequence was obtained from 29 samples. Unexpectedly, a phylogenetic analysis of the HEF gene showed the presence of 12 strains belonging to the D/660 cluster, previously unreported in Europe. The earliest D/660 strain was collected in March 2018 from cattle imported from France. Moreover, we detected one viral strain with a reassortant genetic pattern (PB2, PB1, P42, HEF and NP segments in the D/660 cluster, whilst P3 and NS segments in the D/OK cluster). These results confirm the circulation of IDV in the Italian cattle population and highlight the need to monitor the development of the spreading of this influenza virus in order to get more information about the epidemiology and the ecology of IDV viruses.

Influenza D in Italy: towards a better understanding of an emerging viral infection in swine.

Influenza D virus (IDV), a new member of the Orthomyxoviridae family, was first reported in 2011 in swine in Oklahoma, and consequently found in cattle across North America and Eurasia. To investigate the circulation of IDV among pigs in Italy, in the period between June 2015 and May 2016, biomolecular and virological tests were performed on 845 clinical samples collected from 448 pig farms affected by respiratory distress located in the Po Valley. Serological tests were conducted on 3698 swine sera, including archive sera collected in 2009, as well as samples collected in 2015 from the same region. Viral genome was detected in 21 (2.3%) samples from 9 herds (2%), while virus was successfully isolated from 3 samples. Genetic analysis highlighted that Italian swine IDVs are closely related to the D/swine/Oklahoma/1334/2011 cluster. Sera collected in 2015 showed a high prevalence of IDV antibody titers (11.7%), while archive sera from 2009 showed statistically significant lower positivity rates (0.6%). Our results indicate an increasing epidemiological relevance of the pathogen and the need for in-depth investigations towards understanding its pathogenesis, epidemiology and possible zoonotic potential of this emerging virus.

Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus.

The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains. A new Real-Time PCR, specific for influenza D virus (IDV), was developed and evaluated. The target sequences of primers and probe are highly conserved among IDV strains currently known. The specificity of the method was demonstrated in silico by BLAST, and in vitro with a huge panel of common swine and bovine respiratory pathogens. The analytical sensitivity of the Real-Time PCR was estimated through synthetic RNA molecules and the limit of detection was about 20 copies/µL. The assay was assessed in field and proved to be a valuable tool for the detection of IDV strains.

Effects of DNA extraction method on Porcine circovirus-2 real-time polymerase chain reaction quantification in swine lymph node samples.

Quantitative real-time polymerase chain reaction (PCR) has become an important tool for Porcine circovirus-2 (PCV-2) research and diagnosis. However, significant differences in detection limit and quantification data, among laboratories and quantitative real-time PCR methods, have been demonstrated. New efforts are required for providing more accurate and comparable results. The current study is an evaluation of the effects of DNA extraction procedures on PCV-2 quantification in lymph node samples. Differences, greater than 1 log(10) copies/g, were shown among PCV-2 loads detected after different extraction procedures. The work highlighted the critical role of the DNA extraction method in PCV-2 quantification by quantitative real-time PCR. This important aspect should be evaluated when comparing data from different laboratories or different studies. The PCV-2 quantification data should not be considered comparable before demonstrating the equivalence of the DNA extraction methods performed.