Detection of Selected Canine Viruses in Nigerian Free-Ranging Dogs Traded for Meat Consumption.

Animal trade favors the spreading of emerging and re-emerging pathogens. Concerns have been previously expressed regarding the risks of dog trade in spreading zoonotic pathogens in Nigeria. However, the role of these dogs in disseminating highly pathogenic canine viruses has not yet been explored. The present study aimed to identify selected canine viruses in dogs traded for meat consumption in Nigeria. A total of 100 blood samples were screened for carnivore protoparvovirus-1 (CPPV-1), canine adenovirus 1/2 (CAdV-1/2), canine circovirus (CaCV), and canine distemper virus (CDV) by using real-time PCR and conventional PCR and/or sequencing. CPPV-1 DNA was identified in 83% of canine samples while CaCV DNA and CDV RNA were detected in 14% and 17% of the dog samples, respectively. None of the dogs tested positive for CAdV-1/2. The CaCVs identified in this study clustered along with other European, Asian, and American strains. Moreover, CDV strains identified in Nigeria clustered in a separate lineage with the closest genetic relatedness to the Europe-South America-1 clade. Further surveys prior to and after arrival of dogs at the slaughtering points are required to clarify the real virus burden in these animals.

Evaluation of antibody response to SARS-CoV-2 variants after 2 doses of mRNA COVID-19 vaccine in a correctional facility.

The SARS-CoV-2 pandemic has posed a challenge for correctional facilities worldwide. People in such settings are more vulnerable to severe forms of infection and it is impossible to completely isolate inmates from the outside world. This study aimed to assess the antibody-mediated immune response in terms of neutralizing antibodies against Alpha, Beta, Gamma and Omicron (sub-lineage BA.1) variants of concern after two doses of mRNA vaccine in correctional officers and inmates from an Italian correctional facility. Most of the correctional officers (56.5%) and inmates (52.3% and 63.6%) retained their neutralizing activity toward the Alpha and Gamma variants, respectively. By contrast, the most striking reduction in comparison with the ancestral virus was found in the antibody response toward the Beta and Omicron variants, in both correctional officers (91.2% and 93.9%) and inmates (85.1% and 92.8%). In addition, subjects who had undergone primary vaccination and had previously been naturally infected had higher neutralizing antibody titers toward the 4 variants than negative subjects. Overall, our findings indicate that primary mRNA vaccination is able to induce neutralizing antibodies toward the ancestral virus, while titers toward variants may vary, depending on the mutations harboring by the variants. Although the correctional setting is often considered distinct or isolated from the wider society and sanitary system, the health of correctional workers and prisoners is inexorably linked to the public health of the country as a whole and it is of paramount importance to monitor the antibody response in these settings.

The neutralizing response to SARS-CoV-2 Omicron variants BA.1 and BA.2 in COVID-19 patients and homologous and heterologous vaccinees.

The rapid replacement of Omicron BA.1 by BA.2 sublineage is very alarming, raising the question of whether BA.2 can escape the immunity acquired after BA.1 infection. We compared the neutralizing activity toward the Omicron BA.1 and BA.2 sub-lineages in five groups: COVID-19 patients; subjects who had received two doses of mRNA vaccine; subjects naturally infected with SARS-CoV-2 who had received two doses of mRNA; and subjects who had received three doses of homologous or heterologous vaccine. The results obtained highlight the importance of vaccine boosters in eliciting neutralizing antibody responses against Omicron sub-lineages, and suggest that the adenovirus vectored vaccine elicits a lower response against BA.1 than against BA.2 sub-lineage.

Antibody Avidity and Neutralizing Response against SARS-CoV-2 Omicron Variant after Infection or Vaccination.

Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in SARS-CoV-2 infected patients and vaccinees. Methods: Samples were collected from 42 SARS-CoV-2 infected patients during the first pandemic wave, 50 subjects who received 2 doses of mRNA vaccine before the Omicron wave, 44 subjects who received 3 doses of mRNA vaccine, and 35 subjects who received heterologous vaccination (2 doses of adenovirus-based vaccine plus mRNA vaccine) during the Omicron wave. Samples were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: In patients, RBD-binding IgG titers against the wild-type virus increased with time, but remained low. High neutralizing titers against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.

Immune response to SARS-CoV-2 Omicron variant in patients and vaccinees following homologous and heterologous vaccinations.

The SARS-CoV-2 Omicron variant has rapidly replaced the Delta variant of concern. This new variant harbors worrisome mutations on the spike protein, which are able to escape the immunity elicited by vaccination and/or natural infection. To evaluate the impact and susceptibility of different serum samples to the Omicron variant BA.1, samples from COVID-19 patients and vaccinated individuals were tested for their ability to bind and neutralize the original SARS-CoV-2 virus and the Omicron variant BA.1. COVID-19 patients show the most drastic reduction in Omicron-specific antibody response in comparison with the response to the wild-type virus. Antibodies elicited by a triple homologous/heterologous vaccination regimen or following natural SARS-CoV-2 infection combined with a two-dose vaccine course, result in highest neutralization capacity against the Omicron variant BA.1. Overall, these findings confirm that vaccination of COVID-19 survivors and booster dose to vaccinees with mRNA vaccines is the correct strategy to enhance the antibody cross-protection against Omicron variant BA.1.

COVID-19 Outbreak and BNT162b2 mRNA Vaccination Coverage in a Correctional Facility during Circulation of the SARS-CoV-2 Omicron BA.1 Variant in Italy.

Background. The recent spread of the highly mutated SARS-CoV-2 Omicron variant (B.1.1.529) has raised concerns about protection against COVID-19 in congregate settings such as prisons, characterized by a high risk of transmission and possible difficulties in obtaining adequate vaccination coverage. The present study aims to investigate the spread of an outbreak of COVID-19 in an Italian correctional facility during the dominant circulation of the Omicron BA.1 variant, and also considers BNT162b2 mRNA vaccination coverage among inmates. A COVID-19 screening campaign by RT-PCR was performed on 515 detainees from 4−30 January 2022, in response to an outbreak that began in the correctional facility. Furthermore, 101 serum samples collected from healthy inmates 21 days after having received the second dose of the BNT162b2 vaccine were tested for neutralizing antibodies against both the wild-type SARS-CoV-2 strain and the Omicron BA.1 variant. The global attack rate during the study period was 43.6% (RR 0.8), progressively reducing from unvaccinated inmates (62.7%, RR 1.8) to those who had one dose (52.3%, RR 1.5), two doses (full cycle) (45.0%, RR 1.3), and the third dose (booster) vaccinated group (31.4%, RR 0.7). The percentage of SARS-CoV-2 positive subjects among unvaccinated inmates was significantly higher than in the other groups (p < 0.001), while no significant difference was observed between inmates with one or two vaccine doses. Only two of the positive inmates were hospitalized for COVID-19. The geometric mean titer of neutralizing antibodies in the tested sub-group after two doses of vaccine was lower than in previous studies against the wild-type virus, and showed a complete lack of neutralization against the Omicron variant in 92.1% of individuals. The findings support the need to prioritize vaccination in correctional facilities, as a public health measure to increase the protection of inmates and consequently of prison workers and the community against COVID-19, in coordination with the other prevention strategies.

Long-term persistence of neutralizing SARS-CoV-2 antibodies in pets.

We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS-CoV-2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID-19 human patients.

Absence of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in stray cats.

SARS-CoV-2 positive or seropositive owned cats have been reported worldwide. The detection of seropositive stray cats in the proximity of farms of infected minks, coupled with the demonstration of cat-to-cat transmission in experimental settings, raise the question whether stray cats may have an epidemiological role in the COVID-19 pandemic and may act as sentinel for the circulation of SARS-CoV-2. The aim of this study was to evaluate the presence of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in free roaming cats belonging to colonies located in an area highly affected by the COVID-19 pandemic and to correlate the results with the positivity rate in people sharing the same area. Interdigital, cutaneous, oropharyngeal, nasal and rectal swabs, as well as blood samples, were collected from 99 cats living in colonies and admitted to our hospital for neutering. This caseload corresponds to the 24.2% of the feline population living in the 25 sampled colonies and to the 5.6% of all the free-roaming registered cats. The presence of SARS-CoV-2 RNA in swabs was assessed using real time RT-PCR. Anti-SARS-CoV-2 serum antibodies were assessed using commercially available ELISA kits and confirmed by serum virus neutralization. In people, the SARS-CoV-2 positivity rate ranged from 3.0% to 5.1% (mean rate: 4.1%) and the seropositive rate from 12.1% to 16.3% (mean rate: 14.2%). Most of the colonies were in urban areas and resident cats had frequent contacts with external cats or people. A COVID-19 positive caretaker was found, whereas all the cats were negative for SARS-CoV-2 RNA and seronegative. Although the negative results cannot exclude previous infections followed by decrease of antibodies, this study suggests that colony cats do not have an important epidemiological role in SARS-CoV-2 transmission dynamics. Further studies on larger caseloads are warranted, also in the light of the emerging new viral variants, on a One Health perspective.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Identification of a Novel α-herpesvirus Associated with Ulcerative Stomatitis in Donkeys.

An outbreak of ulcerative stomatitis was observed in a donkey (Equus asinus) dairy herd. Similar lesions were also observed on the dams' udders and, sporadically, in genital areas. The lesions typically resolved in 1-3 weeks. An α-herpesvirus, Varicellovirus, genetically related to equid herpesvirus type 3, was identified.

Prevalence and risk factors for Felis catus gammaherpesvirus 1 detection in domestic cats in Italy.

Felis catus gammaherpesvirus 1 (FcaGHV1), a novel gammaherpesvirus of domestic cats identified in 2014, has been detected in different countries demonstrating a worldwide distribution. The aim of this study was to establish the prevalence of FcaGHV1 in Italy using a molecular epidemiological approach. FcaGHV1 DNA was detected with virus-specific real-time PCR in ≃1% of 2659 feline blood samples tested. Analysis of risk factors showed that being male and coinfection with feline immunodeficiency virus (FIV) increase the likelihood of FcaGHV1 detection. One-third of FcaGHV1-positive cats also tested positive for FIV provirus, whereas coinfections with feline panleukopenia virus were not demonstrated. Further studies are necessary to confirm the risk factors for FcaGHV1 detection and the pathobiology of the virus.

A nationwide survey of Leishmania infantum infection in cats and associated risk factors in Italy.

Though scantly investigated, Leishmania infantum infection and clinical cases of leishmaniasis in cats have been recently reported in several countries of the Mediterranean basin, with large variability in prevalence data. A major limitation in the comparability of the data available is attributed to the differences in diagnostic techniques employed and cat populations sampled. The aim of this study was to assess the prevalence of L. infantum infection in owned cats across Italy by serological and molecular tests and the identification of potential risk factors. Blood samples from 2,659 cats from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy were tested for antibodies against L. infantum, by an immunofluorescence antibody test and for the parasites' DNA, by real-time PCR. Samples were additionally screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. An overall cumulative L. infantum prevalence of 3.9% was recorded by serology (3.3%) and/or qPCR (0.8%), with a higher rate (10.5%) in southern Italy. The risk of L. infantum infection in cats was significantly associated to the geographical areas (South vs North and Centre; p<0.0001), age class (from 19 months to 6 years old vs ≤18 months old, p = 0.0003), neutering status (not neutered vs neutered, p = 0.0028) and FIV infection (p = 0.0051).Though the role of cats in the epidemiology of L. infantum is still debated, our findings indicate that cats are exposed to and/or infected by this protozoan, mainly in endemic regions of Italy. Hence, a standardization of procedures for a prompt diagnosis of L. infantum infection in cats and for screening cat population is crucial for a better understanding of the epidemiology of feline leishmaniasis, and of the potential role of cats in the transmission cycle of zoonotic visceral leishmaniasis.

Artemisinin and its derivatives; ancient tradition inspiring the latest therapeutic approaches against malaria.

Artemisinin (ART) is an endoperoxide sesquiterpene lactone, commonly used in the treatment of malaria. Although it was isolated from Artemisia annuaL., a plant widely applied in Chinese Traditional Medicine, its mechanism of action remains uncertain and its clinical use is still limited due to its low solubility, its poor bioavailability and short in vivo half-life. Over time, several studies have been aimed towards the discovery of potent ART derivatives that could overcome clinical drawbacks. In this review, we focus on the multifaced aspects of ART and on the efforts spent to improve its pharmacological profile that so far culminated in the discovery of more effective drugs. Lastly, we outline the new perspectives in the ART-derivatives scenario.

Discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis.

Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres≥1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with CT values>30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP.

Development of a serum neutralization assay to detect Pteropine Orthoreovirus Indonesia/2010 neutralizing antibodies.

Pteropine Orthoreoviruses (PRVs) are fusogenic bat-borne orthoreoviruses that cause flu-like upper respiratory tract infections in humans. The presence of this group of viruses in bats and humans has been well documented in areas where their biological reservoirs - fruit bats (family Pteropodidae) - live densely. In the present study, a serum neutralization (SN) assay to detect neutralizing antibodies against PRV Indonesia/2010 isolate was set up and used to assess the seroprevalence of this virus in Italian domestic animals. The new developed assay was able of detecting PRV neutralizing antibodies in the hyper-immune polyclonal serum produced in rabbits (titer of 1:160). The negative serum was negative at all tested dilutions. No cross-reactions have been evidenced neither against reference MRVs nor against their respective hyper-immune sera. Eight hundred and fifty-three serum samples collected from 524 bovines, 271 small ruminants, and 58 horses (all used as sentinel animals in the Bluetongue and West Nile disease National surveillance program) were also tested with the new developed SN assay. According to the results of this survey, neither PRV nor PRV cross- reacting viruses antibodies have been demonstrated in Italian domestic animals. However, the new developed SN assay could be a very valuable diagnostic tool to detect infection in animals and humans.

High mortality in foals associated with Salmonella enterica subsp. enterica Abortusequi infection in Italy.

Salmonella enterica subsp. enterica serovar Abortusequi is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries, but only sporadically in Europe and the United States. We report an outbreak of S. Abortusequi in foals in Italy, characterized by high mortality. In a herd of Murgese horses, 10 of 34 newborns died at birth and a further 7 died, after developing severe clinical signs, during the first 10 d of life. Tissue specimens from different organs of 2 dead foals, synovial fluids from 4 sick foals, and vaginal and rectal swabs from their dams were cultured. A total of 16 isolates, all as pure cultures, were obtained and identified as Salmonella. The isolates exhibited the same antimicrobial resistance pattern and the same sequence type, ST251, a type that has been associated with S. Abortusequi. Six of 16 isolates were serotyped and found to be S. Abortusequi 4,12:-:e,n,x. Equine practitioners should be aware of S. Abortusequi infection as a cause of neonatal mortality in foals.

Identification and genetic characterization of equine hepaciviruses in Italy.

Viruses similar to human hepatitis C virus, hepaciviruses, have been identified in various animal species. Equine hepacivirus (EqHV) is the closest relative of human hepaciviruses. Although detected worldwide, information on EqHV epidemiology, genetic diversity and pathogenicity is still limited. In this study we investigated the prevalence and genetic diversity of EqHV in Italian equids. The RNA of EqHV was detected in 91/1932 sera (4.7%) whilst it was not detectable in 134 donkey sera screened by a TaqMan-based quantitative assay. Upon sequencing and phylogenetic analysis of genomic portions located in the NS5B, 5'UTR and NS3 genes, the Italian EqHV strains segregated into two distinct clades that are also co-circulating globally, without apparent geographic restrictions.

Seroprevalence for norovirus genogroup II, IV and VI in dogs.

Molecular and serological data suggest that noroviruses (NoVs) might be transmitted between humans and domestic carnivores. In this study we screened an age-stratified collection of canine sera (n=516) by using an ELISA assay based on virus-like particles (VLPs) of human NoVs GII.4 and GIV.1 and carnivore NoVs GIV.2 and GVI.2. Antibodies against GII.4 and GIV.1 human NoVs and GIV.2 and GVI.2 NoVs from carnivores were identified in dog sera (13.0%, 67/516) suggesting their exposure to homologous and heterologous NoVs. Analysis of the trends of age-class prevalence showed a gradual increase in the positive rate from 9.0% and 7.0%, in young dogs <1year of age to 15.0% in dogs older than 12 years, for GII.4 and GVI.2 NoVs, respectively. A significant difference in the IgG distribution by age classes was observed for GIV.1 NoVs, with the highest rate of antibodies (7.0%) in the age group <1year and the lowest (1.0%) in the age-classes 7-9 (P=0.049). High correlation between the reactivity to GII.4 and GVI.2 NoVs was observed, likely due to conserved epitopes in the capsid structure.

A molecular survey for selected viral enteropathogens revealed a limited role of Canine circovirus in the development of canine acute gastroenteritis.

Canine circovirus (CanineCV) is a canine virus, whose pathogenetic role is still uncertain. Based on recent data suggesting its role as entheropathogen, a case-control study was conducted between 2013 and 2016 to investigate the association of CanineCV with gastroenteritis in dogs, alone or in combination with other viral pathogens, including canine parvovirus (CPV), canine coronavirus (CCoV) and canine distemper virus (CDV). A total of 219 dogs suffering from acute gastroenteritis disorders and 67 controls randomly recruited among healthy dogs or patients presenting without enteric signs were screened by a panel of real-time (RT-)PCR assays for CanineCV, CPV, CCoV and CDV. A high prevalence of viral infections was detected in dogs with gastroenteritis (77.16%), with CPV representing the most frequently detected enteropathogen, followed by CanineCV and CCoV. While CPV and CCoV infections displayed a strong association with occurrence of acute gastroenteritis (p<0.00001), detection of CanineCV in control dogs (28.35%) occurred with prevalence comparable to that of clinical cases (32.42%), so that its correlation with gastrointestinal disease was not statistically supported (p=0.530988). Different from the clinical cases, where co-infections were frequently observed, all positive samples from the control group contained single infections. Noteworthy, a significant association was calculated between co-infections with CanineCV and occurrence of acute gastroenteritis (p<0.00001). This study supports the role of CanineCV as a co-pathogen in the development of gastrointestinal disease, mainly acting in synergism with other enteric viruses.

Identification of a novel canine norovirus.

By screening a collection of fecal samples from young dogs from different European countries, noroviruses (NoVs) were found in 13/294 (4.4%) animals with signs of enteritis whilst they were not detected in healthy dogs (0/42). An informative portion of the genome (3.4kb at the 3' end) was generated for four NoV strains. In the capsid protein VP1 region, strains 63.15/2015/ITA and FD53/2007/ITA were genetically related to the canine GVI.2 strain C33/Viseu/2007/PRT (97.4-98.6% nt and 90.3-98.6% aa). Strain FD210/2007/ITA displayed the highest identity to the GVI.1 canine strain Bari/91/2007/ITA (88.0% nt and 95.0% aa). Strain 5010/2009/ITA displayed only 66.6-67.6% nt and 75.5-81.6% aa identities to the GVI.1 canine strains FD210/2007/ITA and Bari/91/2007/ITA and the GVI feline strain M49-1/2012/JPN. Identity to the other canine/feline NoVs strains in the VP1 was lower than 67.6% nt and 62.7% aa. Based on the full-length VP1 amino acid sequence and the criteria proposed for distinction of NoV genotypes, the canine NoV 5010/2009/ITA could represent the prototype of a third GVI genotype, thus providing further evidence for the genetic heterogeneity of NoVs in carnivores.