Comparative Analysis of the Methanogen Microbiota Associated to Pasture and Stall Housing in Kazakh Cattle.

The microbial community of cattle rumen (archaea) are the key players in methane emissions. Methane pollutes the atmosphere and leads to the loss of feed energy. The aim of this study was to comparatively investigate the cattle microbiota, with a particular focus on archaea, in relation to the type of housing: pasture versus stall. A 16S metabarcoding analysis of the intestinal contents of cattle was carried out. Аlpha - diversity of grazing animals showed to be higher compared to animals in the stall period (p=0.002). Beta - diversity confirmed a difference in methanogens and microbiota between animals kept on pasture and those in a stall. Differential abundance analysis showed that the relative abundance of the Methanobacteriacea family in animals in the pasture period was significantly higher compared to animals in the stall period (FDR p = 0.00122). In conclusion, this study demonstrates that the concentration of methanogens in the fecal contents of animals during pasture period was higher than that in animals during the stall period. We recommend feeding grazing animals with concentrates in the evening in order to mitigate methane emissions.

Draft genome of three isolates of Listeria monocytogenes isolated from Stenella coeruleoalba in Italy.

Listeria monocytogenes is the etiological agent of the listeriosis. Here, we described three draft genome sequences of L. monocytogenes isolated in Italy from stranded individuals of the striped dolphin Stenella coeruleoalba. All the genomes have been molecular typed through the multilocus sequence typing to identify the phylogenetic lineage, clonal complex, sublineage, and serogroup.

Multi-Omics Approaches for Freshness Estimation and Detection of Illicit Conservation Treatments in Sea Bass (Dicentrarchus Labrax): Data Fusion Applications.

Fish freshness consists of complex endogenous and exogenous processes; therefore, the use of a few parameters to unravel illicit practices could be insufficient. Moreover, the development of strategies for the identification of such practices based on additives known to prevent and/or delay fish spoilage is still limited. The paper deals with the identification of the effect played by a Cafodos solution on the conservation state of sea bass at both short-term (3 h) and long-term (24 h). Controls and treated samples were characterized by a multi-omic approach involving proteomics, lipidomics, metabolomics, and metagenomics. Different parts of the fish samples were studied (muscle, skin, eye, and gills) and sampled through a non-invasive procedure based on EVA strips functionalized by ionic exchange resins. Data fusion methods were then applied to build models able to discriminate between controls and treated samples and identify the possible markers of the applied treatment. The approach was effective in the identification of the effect played by Cafodos that proved to be different in the short- and long-term and complex, involving proteins, lipids, and small molecules to a different extent.

First Swedish case of fatal equine parasitic encephalitis by Halicephalobus gingivalis.

BACKGROUND: Halicephalobus gingivalis is a nematode with zoonotic potential which can cause fatal opportunistic infections in various mammals. The parasite has never been diagnosed in Sweden, in any species, prior to the presented case. CASE PRESENTATION: An imported 21-year-old Icelandic mare developed severe neurological signs. The horse was eventually euthanized and submitted for post-mortem examination where severe lesions in the kidneys were noted. Histopathology revealed the presence of H. gingivalis in both kidneys and the brain. Phylogenetic analysis of the parasite determined it to belong to Lineage 1. CONCLUSIONS: With the occurrence of H. gingivalis in Sweden, the disease should be added to the list of differential diagnoses in cases with acute onset of neurological disease in both horses and other mammals including humans.

Application of Microsatellites to Trace the Dairy Products Back to the Farm of Origin.

The increasing number of food frauds, mainly targeting high quality products, is a rising concern among producers and authorities appointed to food controls. Therefore, the development or implementation of methods to reveal frauds is desired. The genetic traceability of traditional or high-quality dairy products (i.e., products of protected designation of origin, PDO) represents a challenging issue due to the technical problems that arise. The aim of the study was to set up a genetic tool for the origin traceability of dairy products. We investigated the use of Short Tandem Repeats (STRs) to assign milk and cheese to the corresponding producer. Two farms were included in the study, and the blood of the cows, bulk milk, and derived cheese were sampled monthly for one year. Twenty STRs were selected and Polymerase Chain Reactions for each locus were carried out. The results showed that bulk milk and derived cheese express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study allowed the identification of a panel of 20 markers useful for the traceability of milk and cheeses, and its effectiveness in the traceability of dairy products obtained from small producers was demonstrated.

Epidemiological and genetic analysis of Cetacean Morbillivirus circulating on the Italian coast between 2018 and 2021.

Cetacean morbillivirus (CeMV) has caused several outbreaks, unusual mortality events, and interepidemic single-lethal disease episodes in the Mediterranean Sea. Since 2012, a new strain with a northeast (NE) Atlantic origin has been circulating among Mediterranean cetaceans, causing numerous deaths. The objective of this study was to determine the prevalence of CeMV in cetaceans stranded in Italy between 2018 and 2021 and characterize the strain of CeMV circulating. Out of the 354 stranded cetaceans along the Italian coastlines, 113 were CeMV-positive. This prevalence (31.9%) is one of the highest reported without an associated outbreak. All marine sectors along the Italian coastlines, except for the northern Adriatic coast, reported a positive molecular diagnosis of CeMV. In one-third of the CeMV-positive cetaceans submitted to a histological evaluation, a chronic form of the infection (detectable viral antigen, the absence of associated lesions, and concomitant coinfections) was suspected. Tissues from 24 animals were used to characterize the strain, obtaining 57 sequences from phosphoprotein, nucleocapsid, and fusion protein genes, which were submitted to GenBank. Our sequences showed the highest identity with NE-Atlantic strain sequences, and in the phylogenetic study, they clustered together with them. Regarding age and species, most of these individuals were adults (17/24, 70.83%) and striped dolphins (19/24, 79.16%). This study improves our understanding on the NE-Atlantic CeMV strain in the Italian waters, supporting the hypothesis of an endemic circulation of the virus in this area; however, additional studies are necessary to deeply comprehend the epidemiology of this strain in the Mediterranean Sea.

The Gut Microbiota of Farmed and Wild Brook Trout (Salvelinus fontinalis): Evaluation of Feed-Related Differences Using 16S rRNA Gene Metabarcoding.

The gut microbiota has become a topic of increasing importance in various fields, including aquaculture. Several fish species have been the subject of investigations concerning the intestinal microbiota, which have compared different variables, including the intestinal portions, the environment, and diet. In this study, the microbiota of farmed and wild brook trout (Salvelinus fontinalis) were analyzed, in which the wall and content of the medial portion of the intestine were considered separately. A total of 66 fish (age class 2+) were sampled, of which 46 were wild and 20 were farmed brook trout, in two different years. Microbiota data were obtained using a 16S metabarcoding approach by analyzing the V3-V4 hypervariable regions of the corresponding 16S rRNA. The data showed that the core microbiota of these species consist of Proteobacteria (Alpha- and Gammaproteobacteria), Actinobacteria, Firmicutes (Bacilli and Clostridia), and, only for farmed animals, Fusobacteria. The latter taxon's presence is likely related to the fishmeal-based diet administered to farmed brook trout. Indeed, alpha and beta diversity analysis showed differences between wild and farmed fish. Finally, statistically significant differences in the microbiota composition were observed between the intestinal walls and contents of wild fish, while no differences were detected in reared animals. Our work represents the first study on the intestinal microbiota of brook trout with respect to both farmed and wild specimens. Future studies might focus on the comparison of our data with those pertaining to other fish species and on the study of other portions of the brook trout intestine.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

A Qualitative PCR Assay for the Discrimination of Bubaline Herpesvirus 1, Bovine Herpesvirus 1 and Bovine Herpesvirus 5.

Bubaline herpesvirus 1 (BuHV-1), Bovine herpesvirus 1 (BoHV-1) and Bovine herpesvirus 5 (BoHV-5) are classified in the genus Varicellovirus, subfamily Alphaherpesvirinae. BoHV-1 is the causative agent of infectious bovine rhinotracheitis, BoHV-5 induces moderate disease in adult cattle while BuHV-1 has instead been associated with a decline in livestock production of water buffaloes. The aim of this study was to develop a qualitative PCR assay that allows the discrimination of BuHV-1, BoHV-1 and BoHV-5. The alignment of homologous genes identified specific nucleotide sequences of BuHV- 1, BoHV-1 and BoHV-5. The design of the primers and the optimization of the PCR assay were focused on the target sequences located on the portions of gD, gE and gG genes. This assay involved the use of three different PCR end-points: the PCR of a portion of the gD gene identified only the presence of BoHV-1; the PCR of a portion of the gE gene confirmed the presence of both BoHV-5 and BuHV-1; the PCR of a portion of the gG gene discriminated between BoHV-5 and BuHV-1, as the amplification product was observed only for BoHV-5. This qualitative PCR assay allowed the differentiation of BoHV-1 and BoHV-5 infections both in cattle and water buffaloes and heterologous BuHV-1 infections in bovine.

First Report of Swinepox in a Wild Boar in Italy: Pathologic and Molecular Findings.

Swinepox virus (SWPV) is responsible for sporadic acute poxvirus infections in swine worldwide, causing a pathognomonic eruptive proliferative dermatitis. Beside direct and congenital transmission, the pig louse Haematopinus suis acts as a mechanical vector and favors virus infection through skin lesions. Infections are generally described in domestic pigs, while only a few cases have been reported in wild boars, in Austria and Germany. In September 2022, SWPV infection was suspected at post-mortem examination of a wild boar piglet with characteristic lesions in Liguria, Northwest Italy. The piglet was heavily parasitized by swine lice (H. suis). SWPV was then confirmed by histological and molecular analyses. Possible viral co-infections were also investigated (African swine fever virus, classical swine fever virus, parvovirus, circovirus, Aujeszky's disease virus and hepatitis E virus). This article describes gross and histopathologic features of SWPV infection, differential diagnosis, and potential vector-borne transmission to domestic pigs, presenting a brief review of the literature on the topic. SWPV infection is reported in wild boars in Italy for the first time. The finding of SWPV in a wild boar in an area with a very limited pig population may suggest the existence of a "wildlife cycle" in the area. Further investigations are needed to understand the real risk of transmission of SWPV to domestic pigs as well as the role of other arthropod vectors.

Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy.

BACKGROUND: Hepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits. RESULTS: Twenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM. CONCLUSIONS: The faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

Analysis of the bacterial communities associated with pupae and winged or wingless adults of Lipoptena fortisetosa collected from cervids in Italy.

The hippoboscid Lipoptena fortisetosa Maa, 1965 is a hematophagous ectoparasite of cervids that can bite humans. This fly is expanding its geographical range and is of concern for animal and human health since it can potentially harbour harmful microorganisms. This study was aimed at characterizing the bacterial communities of L. fortisetosa in its different life-cycle stages. Pupae and wingless adults were collected from cervids hunted in Tuscan-Emilian Apennines (central Italy) and pooled into groups of 10 by life stage (30 individual pupae; 1420 individual wingless adults). Winged flies were caught by sweep netting and separated into five pools of 10 insects. After DNA extraction, the bacterial content of each pool was analysed using 16 S metabarcoding. Results revealed that the composition and relative abundance of different taxa greatly differed in the three analysed groups. Wingless adults showed a high abundance of Bartonella (33.07%), which is almost absent in winged flies and pupae. Among the detected pathogens, four genera of concern for human health were found: Bartonella, Moraxella, Mycobacterium and Rickettsia. Interestingly reads similar to Bartonella bovis, Moraxella osloensis and Arsenophonus lipopteni Operational Taxonomic Unit (OTUs) were detected. These findings suggest the possible role of L. fortisetosa as a reservoir of pathogenic microorganisms, confirming the need for further investigation to ascertain its vectorial capacity.

First record of Aedes japonicus in Liguria region, Northwest of Italy.

Aedes japonicus is an invasive Asian mosquito species, and to date it is widespread in many European countries. In Italy, it was first recorded in 2015 at the Austrian border and it then spread throughout the Northeast of the country. In 2019, it was also identified in Piedmont region, near the Swiss border. In the framework of the Italian program for prevention, surveillance, and response to Arboviruses, from June to November 2021, biweekly entomological surveillance was performed in the Liguria region (Northwest Italy). The collected mosquitoes were morphologically and genetically identified and molecularly analysed for the detection of West Nile and Usutu viruses. Six female mosquitoes, trapped on the 6th of July 2021 using a gravid trap in Albenga (Savona province), were morphologically identified as Ae. japonicus and the identification was genetically confirmed. The pool tested was negative for the presence of West Nile and Usutu viruses. The detection of Ae. japonicus was performed in a coastal area characterized by the presence of many floriculture activities. Considering the distance from the established Ae. japonicus mosquito populations in Italy and other European countries, this could represent an independent introduction in this country.

Prokaryotic Diversity of Ruminal Content and Its Relationship with Methane Emissions in Cattle from Kazakhstan.

In this study, we analyzed the microbial composition of the rumen contents of cattle from Kazakhstan. Specifically, samples of the liquid and solid fractions of the rumen were collected to determine the quantitative and qualitative composition of methanogenic archaea. Cattle were six steers receiving hay-concentrate feeding. Methane emission was determined by repeated measurements for each animal. Rumen samples were then taken from fistulas and analyzed using 16S metabarcoding via Next-Generation Sequencing (NGS). The difference between the rumen fractions was investigated, resulting in differential distribution of the families Streptococccaceae, Lactobacillaceae, Desulfobulbaceae, and Succinivibrionaceae, which were more abundant in the liquid fraction, while Thalassospiraceae showed a higher presence in the solid fraction. These differences can be explained by the fact that fibrolytic bacteria are associated with the solid fraction compared to the liquid. A relationship between methane emission and methanogenic microbiota was also observed. Steers producing more methane showed microbiota richer in methanogens; specifically, most Mathanobacteriaceae resided in the liquid fraction and solid fraction of animals 1 and 6, respectively. The same animals carried most of the Methanobrevibacter and Methanosphaera genera. On the contrary, animals 2, 3, and 5 hosted a lower amount of methanogens, which also agreed with the data on methane emissions. In conclusion, this study demonstrated a relationship between methane emission and the content of methanogenic archaea in different rumen fractions collected from cattle in Kazakhstan. As a result of the studies, it was found that the solid fraction of the rumen contained more genera of methanogens than the liquid fraction of the rumen. These results prove that taking rumen contents through a fistula is more useful than taking it through a probe. The presented data may be of interest to scientists from all over the world engaged in similar research in a comparative aspect.

Hepatitis E Virus (HEV): Identification of Subtypes 3b and 3m in Wild Boar Population in Liguria Region, Italy.

The wild boar is an important natural reservoir for the zoonotic transmission of the hepatitis E virus (HEV) around the world. In particular, HEV genotypes 3 and 4 are an emerging problem in industrialized countries, as the number of wild boars has increased, and their territory is ever closer to farms and populated areas. This study describes the HEV prevalence and geographic circulation among wild boar populations in the Ligurian region (Italy) during the period 2019-2022. Liver samples from 849 wild boars were analyzed for HEV RNA using real-time RT-PCR; positive samples were then subjected to sequencing and phylogenetic analysis. Overall, 6.7% of the wild boars were positive for HEV RNA; however, in the last two years, the percentage of positive animals almost doubled. Phylogenetic analysis showed that wild boar HEV sequences belonged to genotype 3 and clustered within subtypes 3a and 3c, and, for the first time in Italy, subtypes 3b and 3m were identified. Interestingly, 13 sequences could not be assigned to a recognized subtype. Furthermore, the results showed different circulations of identified subtypes across the territory. These findings increase the knowledge of HEV-3 heterogeneity in Italy and describe the role of wild boars in maintaining an active viral circulation in the environment.

Study of cattle microbiota in different regions of Kazakhstan using 16S metabarcoding analysis.

Methane (CH4) is an important greenhouse gas (GHG). Enteric methane emissions from farmed ruminant livestock account for approximately 15% of global GHG emissions, with approximately 44% of livestock emissions in the form of methane. The purpose of the research is to study the influence of feeding types and regional characteristics of Kazakhstan on the microbiota of feces and the number of methane-forming archaea of beef and meat-and-dairy cattle productivity. For this purpose, fecal samples were taken rectally from 37 cattle heads from four regions of Kazakhstan (Western, Southern, Northern and Southeast). The taxonomic composition of the community in all samples was determined by 16S metabarcoding; additionally alpha and beta diversities were calculated. The dominant phyla were: Firmicutes (57.30%), Bacteroidetes (17.00%), Verrucomicrobia (6.88%), Euryarchaeota (6.49%), Actinobacteria (4.77%) and Patescibacteria (3.38%). Significant differences with regard to methanogens bacteria were found: Euryarchaeota were less present in animals from Western Kazakhstan (2.40%), while Methanobacteriales and Methanobrevibacter were prevalent in Southeast, and less abundant in Western region. Western Kazakhstan differs from the other regions likely because animals are mainly grazed in the pasture. Thus, grazing animals has an impact on their microbiota thus leading to a decrease in methane emissions.

Equus caballus Papillomavirus Type-9 (EcPV9): First Detection in Asymptomatic Italian Horses.

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9-L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9-L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2-L1 was found in 7 out of the 11 EcPV9-L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

Neuropathological Characterization of Dolphin Morbillivirus Infection in Cetaceans Stranded in Italy.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Validation of suitable reference genes for quantitative expression analysis by qPCR in bovine terminal ileum and ileocecal valve.

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The aim of this study was to identify a set of reference genes suitable for gene expression analysis in the distal portion of small intestine and ileocecal valve in cattle. These sites of intestine are of interest in veterinary science as they are the main sites of inflammation caused by Mycobacterium avium subsp. paratuberculosis, agent of paratuberculosis. We employed ten PCR assays for commonly used reference genes belonging to various functional classes and then determined their expression stability. The most stable genes were RPL13A and HMBS, followed by TFRC and B-ACT. NormFinder analysis provided similar results with B-ACT as the best reference gene, followed by RLP13A and TFRC. This validated gene panel may be useful for studies on paratuberculosis aiming to identify genes differentially expressed by qRT-PCR.