Cooling Holstein cows for 60 days prepartum in summer: effects on prepartum physiology, postpartum productivity, and calf growth.

Heat stress (HS) during the dry period of dairy cows in hot and dry conditions compromises the physiological status and mammary gland development of dairy cows, thereby negatively affecting milk component yield in the subsequent lactation. Our objective was to evaluate the effects of cooling Holstein cows under moderate or higher HS conditions (i.e., ambient temperature higher than 30 °C, with a temperature-humidity index of 78.2 units) during the dry period on prepartum physiological status, postpartum productivity, and calf growth. Twenty-four multiparous Holstein cows were divided into two groups: one with a cooling system based on spray and fans under a pen shade (CL, n = 12) and the other not-cooled (NC, n = 12). The cooling system operated 10 h/d (09:00-19:00 h) for 60 d prepartum. During the morning, rectal temperature and respiration frequency were lower in CL cows, but not in the afternoon, which was attributed to higher (P < 0.01) dry matter intake by CL cows. Total serum protein was higher (P < 0.01) in CL cows, but hemoglobin was higher in NC cows (P < 0.01), with no differences in other electrolytes, hormones, hematological components, and metabolites. Milk fat and fat and fat-protein corrected milk were higher (P < 0.05) in CL cows. Female and birth weight trended (P = 0.08) to be higher in CL cows. Cooling cows during the dry period had a limited effect on physiology prepartum but increased postpartum productivity of Holstein cows under hot and dry conditions.

Biomolecular Analysis of Canine Distemper Virus Strains in Two Domestic Ferrets (Mustela putorius furo).

Canine distemper is a contagious and severe systemic viral disease that affects domestic and wild carnivores worldwide. In this study, two adult female ferrets (Mustela putorius furo) were evaluated for cutaneous lesions. Scab, fur, and swab samples from the external auditory canal, cutaneous lesions, and scrapings were analyzed. Canine distemper virus (CDV)-positive samples underwent RT-PCR/RFLP with the restriction enzyme PsiI, and the hemagglutinin gene sequence was obtained. According to the restriction enzyme and sequence analyses, the viral strains were typed as CDV field strains that are included within the Europe lineage and distinct from those including vaccinal CDV strains. The sequence analysis showed the highest nucleotide identity rates in older Europe lineage CDV strains collected from dogs and a fox in Europe. This study is the first to report on CDV infection in ferrets in southern Italy and contributes to the current knowledge about natural CDV infection in this species. In conclusion, vaccination remains crucial for preventing the disease and counteracting cross-species infection. Molecular biology techniques can enable the monitoring of susceptible wild animals by ensuring the active surveillance of CDV spread.

A SARS-CoV-2 full genome sequence of the B.1.1 lineage sheds light on viral evolution in Sicily in late 2020.

To investigate the influence of geographic constrains to mobility on SARS-CoV-2 circulation before the advent of vaccination, we recently characterized the occurrence in Sicily of viral lineages in the second pandemic wave (September to December 2020). Our data revealed wide prevalence of the then widespread through Europe B.1.177 variant, although some viral samples could not be classified with the limited Sanger sequencing tools used. A particularly interesting sample could not be fitted to a major variant then circulating in Europe and has been subjected here to full genome sequencing in an attempt to clarify its origin, lineage and relations with the seven full genome sequences deposited for that period in Sicily, hoping to provide clues on viral evolution. The obtained genome is unique (not present in databases). It hosts 20 single-base substitutions relative to the original Wuhan-Hu-1 sequence, 8 of them synonymous and the other 12 encoding 11 amino acid substitutions, all of them already reported one by one. They include four highly prevalent substitutions, NSP12:P323L, S:D614G, and N:R203K/G204R; the much less prevalent S:G181V, ORF3a:G49V and N:R209I changes; and the very rare mutations NSP3:L761I, NSP6:S106F, NSP8:S41F and NSP14:Y447H. GISAID labeled this genome as B.1.1 lineage, a lineage that appeared early on in the pandemic. Phylogenetic analysis also confirmed this lineage diagnosis. Comparison with the seven genome sequences deposited in late 2020 from Sicily revealed branching leading to B.1.177 in one branch and to Alpha in the other branch, and suggested a local origin for the S:G118V mutation.

Leptospira in Slaughtered Fattening Pigs in Southern Italy: Serological Survey and Molecular Typing.

Leptospirosis is a re-emerging zoonosis of worldwide significance; a wide spectrum of wild and domestic animal species act as natural or accidental hosts. Swine can act as maintenance or accidental hosts of pathogenic Leptospira spp. This study aimed at investigation of Leptospira spp. prevalence and diversity in slaughtered pigs in southern Italy (Sicily). In total, 55 samples of kidneys and blood were collected. Microscopic agglutination test and real-time PCR were performed to detect pathogenic and intermediately pathogenic Leptospira. Partial rpoB gene sequencing and multi-locus sequence typing (MLST) were performed to characterize Leptospira species. The analysis showed a seropositivity rate of 16.4%, with Australis representing the most frequently identified serogroup (63.64%); Pomona and Sejroe were detected with a prevalence of 27.27% and 9.09%, respectively. Pathogenic Leptospiral DNA was detected in 2 kidney samples (3.64%). Leptospira were identified through MLST as L. borgpetersenii serovar Tarassovi (serogroup Tarassovi). Obtained data confirmed the presence of Leptospira infection among pigs in southern Italy, suggesting that management of these animals may be considered an occupational risk for humans.

SARS-CoV-2 detection in wastewater as an early warning indicator for COVID-19 pandemic. Madrid region case study.

COVID-19 pandemic is ongoing for more than a year and has changed priorities and boosted some WBE studies. The aim of this work is to contributed to our knowledge sharing the methodology developed for SARS-CoV-2 detection in wastewater of Madrid region of over six million and a half inhabitants, where the sewer system is a combined system. At first, a pilot test in a small metropolitan area was carried out in order to define the criteria for the selection of the sampling points to be applied to the entire region. Methodologys for laboratory analysis and statistical analysis and interpretation of data are also presented. This work relies highly on fieldwork, so sewer network safe accessibility is paramount. A total of 289 sampling points were weekly characterised. Each sampling point represents a sewershed, some of them in a cascade distribution. Samples are tested for SARS-CoV-2 concentration (gc/L, genome copies per litre) and physicochemical parameters are also analysed to validate or discard what at first could be an unusual virus presence. Field results are correlated with health indicators such as incidence rates and hospitalisation data. This information is daily shared with regional health authorities, disaggregated by municipalities, or aggregated for the entire Madrid region. Results have proved to anticipate health indicators. The tool is used as an early warning indicator for COVID-19 pandemic. Further work is planned to apply the current scheme for a permanent epidemiological surveillance system of 87 sampling points to pinpoint infection hotspots and activate the linked sewersheds in the event of an outbreak.

First neuroinvasive human case of West Nile Disease in Southern Italy: Results of the 'One Health' approach.

BACKGROUND: West Nile Disease (WND) is a zoonotic mosquito-borne infection involving viral pathogens, human and animal hosts, vectors and environment. Cooperation among medical, veterinary and entomological fields has been promoted by the Italian Public Health Authorities, and an integrated West Nile Virus (WNV) Surveillance Plan has been in force in Italy since 2016 to prevent the transmission risk of WND to humans through an early detection of viral circulation by animal and entomological surveillance. This managing model is unique in Europe. OBJECTIVES: This survey aimed at presenting the 'One Health' approach applied in 2016 to the first autochthonous human case of West Nile Neuroinvasive Disease (WNND) in Sicily (Southern Italy). METHODS: Serological (anti-WNV IgM and IgG ELISA, anti-WNV neutralizing antibodies) and molecular tests were conducted on blood, liquor and urine of a 38-year-old man with encephalitis and meningitis. Overall, 2704 adult culicides from 160 mosquito catches were morphologically identified. Female mosquitoes were analysed in pools for WNV RNA detection. Serological (anti-WNV IgM and IgG ELISA) and molecular analyses for WNV were carried out in 11 horses, 271 chickens and two dogs sampled in farms around the man's residence. RESULTS AND CONCLUSIONS: WNND was confirmed by serological analysis on patient's liquor and serum. Collected mosquito species included Culex pipiens (93.56%, CI95% 92.64%-94.49%), Aedes albopictus (5.25%, CI95% 4.41%-6.09%), Culex hortensis (0.59%, CI95% 0.30%-0.88%), Culiseta longiareolata (0.55%, CI95% 0.27%-0.83%) and Anopheles maculipennis s.l. (0.04%, CI95% -0.04% to 0.11%). Mosquito pools were negative for WNV RNA. Two dogs (100%) and two horses (18.18%, CI95% -4.61 to 40.97%) resulted positive for anti-WNV specific antibodies. The 'One Health' approach allowed to report the first human neuroinvasive WND in Sicily and to confirm the local circulation of WNV in animals of the same area where the clinical case occurred, defining the autochthonous origin of the infection.

The Effect of a 7 Year-Long Cryopreservation on Stemness Features of Canine Adipose-Derived Mesenchymal Stem Cells (cAD-MSC).

Mesenchymal stem cells (MSCs) are used in therapy in animal models and veterinary medicine, due to their capacity of inducing tissue regeneration and immunomodulation. Their clinical application requires a ready off-the-shelf amount of viable therapeutics doses. For this purpose, it is useful to cryopreserve MSCs to gain a ready and controlled source of abundant autologous stem cells. We evaluated the effect of 7 years cryopreservation using 10% dimethyl sulfoxide (DMSO) with different fetal bovine serum (FBS) concentrations (from 10 to 90%) on different passages of MSCs isolated from canine adipose tissue (cAD-MSCs). The study aimed to evaluate the most adequate cell passage and FBS percentage for the long-term cryopreservation of cells by maintaining the stemness features. Phenotype morphology, cell viability, osteogenic and adipogenic differentiation potentials, proliferative potential and expression of pluripotency markers were analyzed in thawed cells and compared with fresh ones. We demonstrated that cells cryopreserved with at least 80% FBS maintain unaltered the stemness characteristics of the freshly isolated cells. In particular, cells of P0-P1 passages have to be expanded in vitro and subsequently cryopreserved and cells of P2-P4 passages should be considered in the studies on therapeutic application and in vitro study of cAD-MSCs.

Study on Bacteria Isolates and Antimicrobial Resistance in Wildlife in Sicily, Southern Italy.

Wild environments and wildlife can be reservoirs of pathogens and antibiotic resistance. Various studies have reported the presence of zoonotic bacteria, resistant strains, and genetic elements that determine antibiotic resistance in wild animals, especially near urban centers or agricultural and zootechnical activities. The purpose of this study was the analysis, by cultural and molecular methods, of bacteria isolated from wild animals in Sicily, Italy, regarding their susceptibility profile to antibiotics and the presence of antibiotic resistance genes. Bacteriological analyses were conducted on 368 wild animals, leading to the isolation of 222 bacterial strains identified by biochemical tests and 16S rRNA sequencing. The most isolated species was Escherichia coli, followed by Clostridium perfringens and Citrobacter freundii. Antibiograms and the determination of resistance genes showed a reduced spread of bacteria carrying antibiotic resistance among wild animals in Sicily. However, since several wild animals are becoming increasingly close to residential areas, it is important to monitor their health status and to perform microbiological analyses following a One Health approach.

Toxic mineral elements in Mytilus galloprovincialis from Sicilian coasts (Southern Italy).

We assessed the relationship between V, Cr, Mn, Hg, As, Cd, Sn, Sb and Pb concentrations in Mytilus galloprovincialis samples from the coasts of Sicily and the expression of metallothioneins. Toxic mineral elements assessment was carried out by A.A. Spectrometry and ICP-MS. The metallothioneins expression was performed by q-PCR method. Low metals' levels were found in the mussel samples examined, in comparison with what was reported in literature. The highest mean values of toxic mineral elements were found in Gela (Cr 0.178 ± 0.03 mg/Kg, Mn 4.325 ± 0.012 mg/Kg, As 3.706 ± 0.009 mg/Kg, Sn 0.148 ± 0.014 mg/Kg, Sb 0.009 ± 0.004 mg/Kg e Pb 0.364 ± 0.01 mg/Kg). Significant levels of Hg were found in samples from Catania (0.014 ± 0.005 mg/Kg). Only vanadium and lead concentrations showed significant differences between sampling areas (p < 0.05). Molecular analysis verified a basal expression of Mt1 and the absence of over-expression of Mt2, confirming the low mineral's concentrations found in the samples examined.

Spreading of canine parvovirus type 2c mutants of Asian origin in southern Italy.

Canine parvovirus type 2 (CPV-2) emerged as dog pathogen in the late 1970s, causing severe and often fatal epizootics of gastroenteritis in the canine population worldwide. Although to date CPV-2 is circulating in all continents, most of the current studies have analysed the amino acid changes accounted in the VP2 gene sequence, with limited information on virus introductions from other countries. The aim of this study was to analyse the genetic features of CPV-2c strains currently spreading in Italy. Swabs and tissue samples were collected from dogs suspected of CPV infection. The nearly complete genome sequence from the CPV-positive samples was obtained. The co-circulation of two different but related CPV-2c strains, with amino acid changes characteristic of CPV strains of Asian origin (NS1: 60V, 544F, 545F, 630P - NS2: 60V, 151N, 152V - VP2: 5A/G, 267Y, 297A, 324I, 370R), were observed. The phylogenetic analyses inferred from the NS1 and VP2 gene sequences confirmed the relationship with Asian CPV-2c strains. This study reports the spread of novel CPV-2c mutants in Italy and supports further studies to evaluate the coexistence of genetically divergent CPV strains in the same geographical environment.

Molecular Characterization and Evolutionary Analyses of Carnivore Protoparvovirus 1 NS1 Gene.

Carnivore protoparvovirus 1 is the etiological agent of a severe disease of terrestrial carnivores. This unique specie encompasses canine parvovirus type 2 (CPV-2) and feline panleukopenia virus (FPLV). Studies widely analyzed the main capsid protein (VP2), but limited information is available on the nonstructural genes (NS1/NS2). This paper analyzed the NS1 gene sequence of FPLV and CPV strains collected in Italy in 2009⁻2017, along with worldwide related sequences. Differently from VP2, only one NS1 amino-acid residue (248) clearly and constantly distinguished FPLV from CPV-2, while five possible convergent amino-acid changes were observed that may affect the functional domains of the NS1. Some synonymous mutation in NS1 were non-synonymous in NS2 and vice versa. No evidence for recombination between the two lineages was found, and the predominance of negative selection pressure on NS1 proteins was observed, with low and no overlap between the two lineages in negatively and positively selected codons, respectively. More sites were under selection in the CPV-2 lineage. NS1 phylogenetic analysis showed divergent evolution between FPLV and CPV, and strains were clustered mostly by country and year of detection. We highlight the importance of obtaining the NS1/NS2 coding sequence in molecular epidemiology investigations.

Molecular characterization of human enteric viruses in food, water samples, and surface swabs in Sicily.

OBJECTIVES: Enteric viruses are responsible for foodborne and waterborne infections affecting a large number of people. Data on food and water viral contamination in the south of Italy (Sicily) are scarce and fragmentary. The aim of this study was to evaluate the presence of viral contamination in food, water samples, and surface swabs collected in Sicily METHODS: The survey was conducted on 108 shellfish, 23 water samples (seawater, pipe water, and torrent water), 52 vegetables, one peach and 17 berries, 11 gastronomic preparations containing fish products and/or raw vegetables, and 28 surface swabs. Hepatitis A virus (HAV), genogroup GI, GII, and GIV norovirus (NoV), enterovirus (EV), rotavirus (RoV), hepatitis E virus (HEV), adenovirus (AdV), and bocavirus (BoV) were detected by nested (RT) PCR, real-time PCR, and sequence analysis. RESULTS: The most frequently detected viruses in shellfish were HAV (13%), NoV (18.5%), and EV (7.4%). Bocavirus was found in 3.7%, HEV in 0.9%, and AdV in 1.9% of the molluscs. Of the 23 water samples, 21.7% were positive for GII NoV and 4.3% for RoV and HEV genotype 3. Of the 70 vegetable samples, 2.9% were positive for NoV GI (GI.5 and GI.6), 2.9% for EV, and 1.4% for HEV. In the gastronomic preparations, only one EV (9%) was detected. No enteric viruses were detected in the berries, fruit, or swabs analyzed. CONCLUSIONS: Molecular surveillance of water and food samples clearly demonstrated that human pathogenic viruses are widely found in aquatic environments and on vegetables, and confirmed the role of vegetables and bivalve molluscs as the main reservoirs.