Molecular Surveillance for Bocaparvoviruses and Bufaviruses in the European Hedgehog (Erinaceus europaeus).

The presence of bocaparvoviruses (BoVs) and bufaviruses (BuVs) in the European hedgehog (Erinaceus europaeus) was investigated by screening duodenal and liver samples collected from 183 carcasses, delivered to wildlife rescue centers located in northwestern Italy. BoV DNA was detected in 15 animals (8.2%), with prevalences of 7.1% (13/183) and 2.7% (5/183) in intestine and liver samples, respectively. Upon the sequence analyses of the NS1 gene, two highly divergent BoVs (65.5-67.8% nt identities) were identified. Fourteen strains showed the highest identity (98.3-99.4% nt) to the hedgehog BoV strains recently detected in China in Amur hedgehogs (Erinaceus amurensis), whilst four strains were genetically related (98.9-99.4% nt identities) to the porcine BoVs identified in pigs and classified in the species Bocaparvovirus ungulate 4, which included related viruses also found in rats, minks, shrews, and mice. BuV DNA was detected in the duodenal samples of two hedgehogs, with a prevalence rate of 1.1%. The nearly full-length genome of two BuV strains, Hedgehog/331DU-2022/ITA and Hedgehog/1278DU/2019/ITA, was reconstructed. Upon phylogenetic analysis based on the NS and VP aa sequences, the Italian hedgehog BuVs tightly clustered with the BuVs recently identified in the Chinese Amur hedgehogs, within a potential novel candidate species of the genus Protoparvovirus.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

Potential Impact of Environmental Pollution by Human Antivirals on Avian Influenza Virus Evolution.

Antiviral (AV) drugs are the main line of defense against pandemic influenza. However, different administration policies are applied in countries with different stocks of AV drugs. These policies lead to different occurrences of drug metabolites in the aquatic environment, altering animal behavior with evolutionary consequences on viruses. The aim of this study was to investigate the potential impact of environmental pollution by human antivirals, such as oseltamivir carboxylate (OC), on the evolutionary rate of avian influenza. We used NA, HA, NP, and MP viral segments from two groups of neighboring countries sharing migratory routes of wild birds and characterized by different AV stockpiles. BEAST analyses were performed using the uncorrelated lognormal clock evolutionary model and the Bayesian skyline tree prior model. The ratios between the rate of evolution of the NA gene and the HA, NP, and MP segments were considered. The two groups of countries were compared by analyzing the differences in the ratio distributions. Our analyses highlighted a possible different behavior in the evolution of H5N1 2.3 clade viral strains when OC environmental pollution is present. In conclusion, the widespread consumption of antivirals and their presence in wastewater could influence the selective pressure on viruses.

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.

Evaluation of Side Effects and Long-Term Protection of a Sustained-Release Injectable Moxidectin Formulation against Dirofilaria immitis Infection in Dogs: An Observational-In Field Multicentric Study.

The sustained-release moxidectin formulation Afilaria SR is a relatively new product and has been labelled to prevent Dirofilaria immitis infection in dogs for a six months-period. An observational, in field multicentric study was performed, aiming to evaluate the tolerability and the long-term prevention of Afilaria SR in Italy, a country where filariasis is endemic. The study was designed to include not less than 300 dogs, older than 6 months, of any breed. Side effects were recorded by veterinarians and antigenic tests were performed after 210, 365, 730, and 1095 days after the administration of the drug. A total of 583 dogs were recruited from 2018 to 2021 and all of them were negative with respect to antigenic tests at all time points, indicating that 100% of protection was achieved. Ranking of adverse reactions and correlation to patient features were analyzed using descriptive statistics and χ2 square test, respectively. Afilaria SR was well tolerated: 13% of dogs experienced mild reactions and only two dogs out of 583 (0.3%) demonstrated anaphylactoid/angioneurotic reactions, resolved administering corticosteroids. These data support that Afilaria SR prevented Dirofilaria immitis disease in all enrolled dogs and the low number and the low grade of side effects indicate the high safety profile of the product.

Pulmonary fibrosis in a dog as a sequela of infection with Severe Acute Respiratory Syndrome Coronavirus 2? A case report.

BACKGROUND: Interstitial lung disease is a heterogeneous group of conditions characterized by severe radiographic changes and clinicopathological findings. However, in the vast majority of cases, the cause remains unknown. CASE DESCRIPTION: In the present study, we reported the clinical case of a 3 years old female Bull Terrier presented in October 2020 to the Advanced Diagnostic Imaging Department of the Turin Veterinary Teaching Hospital with a progressive pulmonary illness characterized by dyspnea, exercise intolerance, and a diffuse and severe pulmonary interstitial pattern at imaging investigations. Considering the clinical findings, the dog was included in a serological survey for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in companion animals, showing positive results. Due to the further clinical worsening, the owners opted for euthanasia. At necroscopy, dog showed severe and chronic bronchopneumonia compatible with a Canine Idiopathic Pulmonary Fibrosis and with serological features linked to a SARS-CoV-2 infection. CONCLUSIONS: The comparison of these lesions with those reported in humans affected by Coronavirus Disease 2019 (COVID-19) supports the hypothesis that these findings may be attributable to the post-acute sequelae of SARS-CoV-2 infection in a dog with breed predisposition to Canine Idiopathic Pulmonary Fibrosis (CIPF), although direct evidence of SARS-CoV-2 by molecular or antigenic approaches remained unsolved.

The evolution of dam-litter microbial flora from birth to 60 days of age.

BACKGROUND: Early bacterial colonization in puppies is still a poorly understood phenomenon. Although the topic is of considerable interest, a big gap in knowledge still exists on the understanding of timing and features of neonatal gut colonization. Thence, the purpose of this study was to evaluate the relationship between dam and litter microbial flora, in vaginally delivered puppies, from birth to two months of age. Bacteria were identified using MALDI-TOF, an accurate and sensitive method, and cluster analysis of data provided a new insight on the investigated topic. METHODS: Six dam-litter units of two medium size breeds were enrolled in the study. Vaginal and colostrum/milk samples were collected from dams after delivery and 48h post-partum, while rectal samples were taken from dams and puppies after delivery and at day 2, 30 and 60 (T2, T30 and T60, respectively) post-partum. Bacterial isolation and identification were performed following standard techniques, then the data were analyzed using a new approach based on bacterial genus population composition obtained using a wide MALDI-TOF screening and cluster analysis. RESULTS: Forty-eight bacteriological samples were collected from the dams and 145 from their 42 puppies. Colostrum/milk samples (n = 12) showed a bacterial growth mainly limited to few colonies. Staphylococci, Enterococci, E. coli, Proteus spp. were most frequently isolated. All vaginal swabs (n = 12) resulted in bacteria isolation (medium to high growth). Streptococci, Enterococci, E. coli were the most frequently detected. E. coli, Proteus mirabilis, Enterococcus spp., Streptococcus spp. were often obtained from dams' and puppies' rectal swabs. Clostridia, not isolated in any other sampling site, were rarely found (n = 3) in meconium while they were more frequently isolated at later times (T2: n = 30; T30: n = 17; T60: n = 27). Analysis of the bacterial genus pattern over time showed a statistically significant reduction (P < 0.01) in the heterogeneity of microbial composition in all time points if compared to birth for each dam-litter unit. These results were confirmed with cluster analysis and two-dimensional scaling. CONCLUSION: This novel data analysis suggests a fundamental role of the individual dam in seeding and shaping the microbiome of the litter. Thus, modulating the dam's microbiota may positively impact the puppy microbiota and benefit their health.

Rapid SARS-CoV-2 Intra-Host and Within-Household Emergence of Novel Haplotypes.

In February 2020, the municipality of Vo', a small town near Padua (Italy) was quarantined due to the first coronavirus disease 19 (COVID-19)-related death detected in Italy. To investigate the viral prevalence and clinical features, the entire population was swab tested in two sequential surveys. Here we report the analysis of 87 viral genomes, which revealed that the unique ancestor haplotype introduced in Vo' belongs to lineage B, carrying the mutations G11083T and G26144T. The viral sequences allowed us to investigate the viral evolution while being transmitted within and across households and the effectiveness of the non-pharmaceutical interventions implemented in Vo'. We report, for the first time, evidence that novel viral haplotypes can naturally arise intra-host within an interval as short as two weeks, in approximately 30% of the infected individuals, regardless of symptom severity or immune system deficiencies. Moreover, both phylogenetic and minimum spanning network analyses converge on the hypothesis that the viral sequences evolved from a unique common ancestor haplotype that was carried by an index case. The lockdown extinguished both the viral spread and the emergence of new variants.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

The Genetic Diversity of Rickettsiella Symbionts in Ixodes ricinus Throughout Europe.

Rickettsiella species are bacterial symbionts that are present in a great variety of arthropod species, including ixodid ticks. However, little is known about their genetic diversity and distribution in Ixodes ricinus, as well as their relationship with other tick-associated bacteria. In this study, we investigated the occurrence and the genetic diversity of Rickettsiella spp. in I. ricinus throughout Europe and evaluated any preferential and antagonistic associations with Candidatus Midichloria mitochondrii and the pathogens Borrelia burgdorferi sensu lato and Borrelia miyamotoi. Rickettsiella spp. were detected in most I. ricinus populations investigated, encompassing a wide array of climate types and environments. The infection prevalence significantly differed between geographic locations and was significantly higher in adults than in immature life stages. Phylogenetic investigations and protein characterization disclosed four Rickettsiella clades (I-IV). Close phylogenetic relations were observed between Rickettsiella strains of I. ricinus and other arthropod species. Isolation patterns were detected for Clades II and IV, which were restricted to specific geographic areas. Lastly, although coinfections occurred, we did not detect significant associations between Rickettsiella spp. and the other tick-associated bacteria investigated. Our results suggest that Rickettsiella spp. are a genetically and biologically diverse facultative symbiont of I. ricinus and that their distribution among tick populations could be influenced by environmental components.

Caprine Arthritis Encephalitis Virus Disease Modelling Review.

Mathematical modelling is used in disease studies to assess the economical impacts of diseases, as well as to better understand the epidemiological dynamics of the biological and environmental factors that are associated with disease spreading. For an incurable disease such as Caprine Arthritis Encephalitis (CAE), this knowledge is extremely valuable. However, the application of modelling techniques to CAE disease studies has not been significantly explored in the literature. The purpose of the present work was to review the published studies, highlighting their scope, strengths and limitations, as well to provide ideas for future modelling approaches for studying CAE disease. The reviewed studies were divided into the following two major themes: Mathematical epidemiological modelling and statistical modelling. Regarding the epidemiological modelling studies, two groups of models have been addressed in the literature: With and without the sexual transmission component. Regarding the statistical modelling studies, the reviewed articles varied on modelling assumptions and goals. These studies modelled the dairy production, the CAE risk factors and the hypothesis of CAE being a risk factor for other diseases. Finally, the present work concludes with further suggestions for modelling studies on CAE.

Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system.

The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.

Cross-Sectional Serosurvey of Companion Animals Housed with SARS-CoV-2-Infected Owners, Italy.

We conducted a serologic survey among dogs and cats in Italy to detect antibodies against severe acute respiratory syndrome virus 2 (SARS-CoV-2). We found that SARS-CoV-2 seroprevalence was higher among cats (16.2%) than dogs (2.3%). In addition, seroprevalence was higher among animals living in close contact with SARS-CoV-2-positive owners.

A multi-target lateral flow immunoassay enabling the specific and sensitive detection of total antibodies to SARS COV-2.

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.

Sequelae of COVID-19 in Hospitalized Children: A 4-Months Follow-Up.

Little is known about the sequelae of SARS-CoV-2 infection in children. In a COVID-19 dedicated clinic, we followed-up for 4 months 25 children previously hospitalized for COVID-19, performing clinical, laboratory, and lung ultrasound evaluation. Mid-term sequelae were rarely observed in our COVID-19 children's cohort.

The evolution of African swine fever virus in Sardinia (1978-2014) as revealed by whole-genome sequencing and comparative analysis.

African swine fever (ASF) is a highly contagious and lethal viral disease of pigs and wild boars, which is enzootic in many African countries and on the Italian island of Sardinia, where it has been present since 1978. Previous genetic analyses of Sardinian ASF virus (ASFV) isolates have revealed that they all belong to p72 genotype I, with only minor sequence variations. However, these studies examined only a few selected genes. To distinguish between these closely related isolates and better investigate ASFV evolution in Sardinia, we sequenced the complete genomes of 12 Sardinian ASFV isolates collected between 1978 and 2012, and compared them with 47/Ss/2008 and 26544/OG10. Most of the observed changes occurred in a time-dependent manner; however, their biological significance remains unclear. As a whole, our results demonstrate the remarkable genetic stability of these strains, supporting a single-source introduction of the virus.

Compartmentalized evolution of Bovine Viral Diarrhoea Virus type 2 in an immunotolerant persistently infected cow.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV-1 is widely distributed in Italy, while BVDV-2 has been detected occasionally. BVDV can be classified in two biotypes, cytopathic (CP) or noncytopathic (NCP). The characteristic of the virus is linked with the infection of a pregnant dam with a NCP strain: due to viral establishment before maturation of the fetal immune system the calf remains persistently infected (PI) and immunotolerant to the infecting BVDV strain. Thanks to their immunotolerance, PI animals represent a unique model to study the viral distribution and compartmentalization in absence of immunoresponse in vivo. In the present study, NGS sequencing was used to characterize the BVDV2 viral strain infecting a PI calf and to describe the viral quasispecies in tissues. Even if the consensus sequences obtained by all the samples were highly similar, quasispecies was described evaluating the presence and the frequency of variants among all the sequencing reads in each tissue. The results suggest a high heterogeneity of the infecting viral strain suggesting viral compartmentalization. The quasispecies analysis highlights the complex dynamics of viral population structure and can increase the knowledge about viral evolution in BVDV-2 persistently infected animals.

A single nucleotide variant in the promoter region of the CCR5 gene increases susceptibility to arthritis encephalitis virus in goats.

BACKGROUND: The small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses that includes caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV). SRLVs affect the production and welfare of sheep and goats worldwide. There is currently no effective treatment. Their high mutation rate precludes vaccine development, making innovative control measures necessary. A variant of the chemokine (C-C motif) receptor 5 (CCR5) gene is reportedly involved in resistance to human immunodeficiency (HIV) infection in humans and to SRLV in sheep. The aim of this study was to analyse the genetic structure and variability of the CCR5 gene in goats and to carry out a cross-sectional study to investigate the role of CCR5 genetic variants in controlling susceptibility/resistance to CAEV. RESULTS: The variant g.1059 T located in the promoter region revealed an interesting association with high proviral loads (a 2.8-fold increased risk). A possible explanation could be an alteration of the transcriptional level. Overexpression of the CCR5 receptor on the cell surface may increase virus internalization and proviral load as a consequence. CONCLUSIONS: Our findings could be advantageously used to reduce the susceptibility of goat herds to CAEV by negatively selecting animals carrying the g.1059 T mutation. Eliminating animals predisposed to high proviral loads could also limit the development of clinical signs and the spread of the virus, since these animals are also highly efficient in shedding the virus.