Genetic characterization of parvoviruses identified in stray cats in Nigeria.

Parvoviruses are a major cause of haemorrhagic gastroenteritis, leukopenia and high mortality in cats and dogs. In this study, the presence and genetic characteristics of parvoviruses circulating among cats in Nigeria are reported. Faecal samples of stray cats from live animal markets in southwestern (Oyo and Osun States) and north-central (Kwara State) Nigeria were screened for the presence of parvoviral DNA using a qPCR. Positive samples were further characterized using a qPCR based on minor groove binder probes. Overall, 85/102 (83.3 %) stray cats tested positive for feline panleukopenia virus (FPV) DNA and one cat was co-infected with canine parvovirus-2 type a. Sequence analysis of the complete capsid region of 15 Nigerian FPV strains revealed that they were up to 99.9 % similar to the American reference strain FPV-b at the nucleotide level, and three of them presented amino acid mutations in key capsid residues. This is the first report of identification and molecular characterization of FPV strains in cats in Nigeria. The high prevalence of the virus emphasizes the need for constant surveillance of the circulation of parvoviruses in Nigeria and underscores the need to deploy an effective vaccination strategy.

Investigating the genetic diversity of CRESS DNA viruses in cats identifies a novel feline circovirus and unveils exposure of cats to canine circovirus.

Circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA viruses include Circoviruses which have been found in several animal species and in human specimens. Circoviruses are associated with severe disease in pigs and birds and with respiratory and gastrointestinal disorders and systemic disease in dogs. In cats there are only a few anecdotical studies reporting CRESS DNA viruses. In this study, a total of 530 samples (361 sera, 131 stools, and 38 respiratory swabs) from cats, were screened for the presence of CRESS DNA viruses. Overall, 48 (9.0%) of 530 samples tested positive using a pan-Rep PCR. A total of 30 Rep sequences were obtained. Ten sequences of fecal origin were tightly related to each other (82.4-100% nt identity) and more distantly related to mongoose circoviruses (68.3 to 77.2% nt identity). At genome level these circoviruses displayed the highest nt identity (74.3-78.7%) to mongoose circoviruses thus representing a novel circovirus species. Circoviruses from different animal hosts (n = 12) and from humans (n = 8) were also identified. However, six Rep sequences were obtained from serum samples, including canine circoviruses, a human cyclovirus and human and fish-associated CRESS DNA viruses. The presence of these viruses in the sera would imply, to various extent, virus replication in the animal host, able to sustain viremia. Overall, these findings indicate a wide genetic diversity of CRESS DNA viruses in cats and warrant further investigations.

Genomic characterization and phylogenetic evolution of the canine parvoviruses in Iranian dogs, a nationwide study: CPV evolutionary analysis in Iran.

Canine Parvo Virus 2 (CPV-2) culminated in lots of fatalities in domestic dogs since its emergence in 1978. Mainly, it is responsible for severe hemorrhagic diarrhea, vomiting, and dehydration. CPV-2 has three main variants known as 2a, 2b, and 2c. Due to the necessity of monitoring the evolutionary parameters of the virus, and also the lack of comprehensive study of CPV2 in Iran, this study is done for the first time in this country not only to characterize Iranian CPV genomes but also to study the evolutionary parameters and phylodynamics of CPV. The phylogenetic trees were constructed using the Maximum Likelihood (ML) method. By the use of the Bayesian Monte Carlo Markov Chain (BMCMC) method, evolutionary analysis and phylodynamics of the virus were investigated. Phylogenetic results showed that all Iranian isolates were classified in the CPV-2a variant. The central part of Iran was suggested to be the origin of the virus, especially the Alborz province. Before its prevalence throughout the country, the virus circulated in the central part, in Thran, Karaj, and Qom. Mutational analysis showed a positive selection pressure of CPV-2a. Investigating the evolutionary parameters of the virus proposed 1970 to be the date of birth of the virus, with a 95% credible interval between 1953 and 1987. The effective number of infections increased dramatically from 2012 to 2015, then faced a slightly decreasing trend from 2015 to 2019. A considerable up warding pattern was witnessed from the middle of 2019, which can be taken as a concern about the risk of vaccination failure.

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.

Persistence of DNA from canine parvovirus modified-live virus in canine tissues.

Canine parvovirus (CPV-2) modified-live virus vaccine strain can replicate in lymphoid tissues and intestinal mucosa after administration, being shed through canine faeces. Detection of vaccine strains has been reported in the bloodstream and faeces, potentially interfering with molecular diagnostic tests. The persistence of these strains in canine tissues has not yet been described. With this aim, canine tissues were tested during a molecular survey to screen for the presence of canine enteric viruses. Tissue samples from 165 dead dogs were tested by a conventional PCR assay. Positive samples and five commercial vaccines were subjected to sequence analysis. Vaccinal strains were detected and virus load was measured by using a set of real-time PCR assays using minor-groove binder (MGB) probes. Seventy-five dogs (45.4%) tested positive for CPV-2. Strains from 70 dogs were characterised as field variants. The presence of CPV sequences of vaccine origin was observed in the spleen, intestine, and mesenteric lymph nodes of five young dogs. Vaccinal strains were detected from 12 to 24 days after the last vaccine administration. Viral loads comprised between 6.3 × 102 and 9.95 × 104 DNA copies/10 µl of template. This study confirms that CPV vaccinal strains can be detected in canine tissues after vaccination, so post-mortem diagnosis of CPV infection needs further molecular analyses to assess the viral type (vaccine or field strains). The present study updates the current information on the persistence of CPV vaccine strains in canine tissues and their possible interference with molecular assays.

Feline Panleukopenia Virus in Dogs from Italy and Egypt.

Canine parvovirus and feline panleukopenia virus (FPV) are variants of Carnivore protoparvovirus 1. We identified and characterized FPV in dogs from Italy and Egypt using genomic sequencing and phylogenetic analyses. Cost-effective sequencing strategies should be used to monitor interspecies spread, evolution dynamics, and potential host jumping of FPV.

Molecular characterization of carnivore protoparvovirus 1 circulating in domestic carnivores in Egypt.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV), now included in the unique species Carnivore protoparvovirus 1 (CPPV1), have been circulating in dogs and cats for several decades and are considered the causes of clinically important diseases, especially in young animals. While genetic evidence of the circulation of parvoviruses in Egyptian domestic carnivores has been provided since 2016, to date, all available data are based on partial fragments of the VP2 gene. This study reports the molecular characterization of CPPV strains from Egypt based on the full VP2 gene. Overall, 196 blood samples were collected from dogs and cats presented at veterinary clinics for routine medical assessment in 2019 in Egypt. DNA extracts were screened and characterized by real-time PCR. Positive samples were amplified by conventional PCR and then were sequenced. Nucleotide and amino acid changes in the sequences were investigated and phylogeny was inferred. Carnivore protoparvovirus DNA was detected in 18 out of 96 dogs (18.8%) and 7 of 100 cats (7%). Phylogenetic analyses based on the full VP2 gene revealed that 9 sequenced strains clustered with different CPV clades (5 with 2c, 2 with 2a, 1 with 2b, and 1 with 2) and 1 strain with the FPV clade. All three CPV variants were detected in dog and cat populations with a predominance of CPV-2c strains (7 of 18, 38.9%) in dog samples, thus mirroring the circulation of this variant in African, European, and Asian countries. Deduced amino acid sequence alignment revealed the presence of the previously unreported unique mutations S542L, H543Q, Q549H, and N557T in the Egyptian CPV-2c strains.

Detection of canine circovirus in dogs infected with canine parvovirus.

Since the first detection of canine circovirus (CanineCV), several reports have been published over the last decade about the worldwide distribution of this emerging virus of dogs. In order to investigate the prevalence and genomic features of CanineCV in Iranian dogs, a total of 203 dog faecal samples was collected between February and November 2018 from five different geographical regions and screened by real-time PCR (qPCR). Thirteen dogs (6.4%) tested positive for CanineCV DNA, all being detected in co-infections with the highly virulent canine parvovirus (CPV). Three partial replicase nucleotide sequences of the detected CanineCV strains were obtained and compared with the reference sequences deposited in the GenBank database. The Iranian CanineCV sequences had a nucleotide identity of 96.4-98.2% each to other and of 88.3-98.2% with other sequences available on the GenBank. Phylogenetic analysis showed that the Iranian sequences are more closely related to Turkish strains than to strains reported from other countries. The present study provides new insights into the CanineCV molecular epidemiology and its possible role as a co-infectious pathogen.

Detection and Genetic Characterization of Canine Adenoviruses, Circoviruses, and Novel Cycloviruses From Wild Carnivores in Italy.

Wild carnivores are known to play a role in the epidemiology of several canine viruses, including canine adenoviruses types 1 (CAdV-1) and 2 (CAdV-2), canine circovirus (CanineCV) and canine distemper virus (CDV). In the present study, we report an epidemiological survey for these viruses in free ranging carnivores from Italy. A total of 262 wild carnivores, including red foxes (Vulpes vulpes), wolves (Canis lupus) and Eurasian badgers (Meles meles) were sampled. Viral nucleic acid was extracted and screened by real-time PCR assays (qPCR) for the presence of CAdVs and CanineCV DNA, as well as for CDV RNA. CAdV-1 DNA was detected only in red foxes (4/232, 1.7%) whilst the wolves (0/8, 0%) and Eurasian badgers (0/22, 0%) tested negative. CanineCV DNA was detected in 4 (18%) Eurasian badgers, 4 (50%) wolves and 0 (0%) red foxes. None of the animals tested positive for CDV or CAdV-2. By sequence and phylogenetic analyses, CAdV-1 and CanineCV sequences from wild carnivores were closely related to reference sequences from domestic dogs and wild carnivores. Surprisingly, two sequences from wolf intestines were identified as cycloviruses with one sequence (145.20-5432) displaying 68.6% nucleotide identity to a cyclovirus detected in a domestic cat, while the other (145.201329) was more closely related (79.4% nucleotide identity) to a cyclovirus sequence from bats. A continuous surveillance in wild carnivores should be carried out in order to monitor the circulation in wildlife of viruses pathogenic for domestic carnivores and endangered wild species.

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.

Do Dogs and Cats Passively Carry SARS-CoV-2 on Hair and Pads?

The epidemiological role of domestic animals in the spread and transmission of SARS-CoV-2 to humans has been investigated in recent reports, but some aspects need to be further clarified. To date, only in rare cases have dogs and cats living with COVID-19 patients been found to harbour SARS-CoV-2, with no evidence of pet-to-human transmission. The aim of the present study was to verify whether dogs and cats act as passive mechanical carriers of SARS-CoV-2 when they live in close contact with COVID-19 patients. Cutaneous and interdigital swabs collected from 48 dogs and 15 cats owned by COVID-19 patients were tested for SARS-CoV-2 by qRT-PCR. The time elapsed between owner swab positivity and sample collection from pets ranged from 1 to 72 days, with a median time of 23 days for dogs and 39 days for cats. All samples tested negative, suggesting that pets do not passively carry SARS-CoV-2 on their hair and pads, and thus they likely do not play an important role in the virus transmission to humans. This data may contribute to confirming that the direct contact with the hair and pads of pets does not represent a route for the transmission of SARS-CoV-2.

Canine parvovirus is shed infrequently by cats without diarrhoea in multi-cat environments.

Whether subclinical shedding of canine parvovirus (CPV) by cats might contribute to the epidemiology of canine CPV infections, particularly in facilities housing both cats and dogs, requires clarification. Conflicting results are reported to date. Using conventional PCR (cPCR) to amplify the VP2 gene, shedding of the CPV variants (CPV-2a, 2b, 2c) by healthy cats in multi-cat environments was reportedly common in Europe but rare in Australia. The aim of this study was to determine whether low-level faecal CPV shedding occurs in multi-cat environments in Australia and Italy using a TaqMan real-time PCR to detect Carnivore protoparvovirus 1 (CPV and feline parvovirus, FPV) DNA, and minor-groove binder probe real-time PCR assay to differentiate FPV and CPV types and to characterize CPV variants. In total, 741 non-diarrhoeic faecal samples from shelters in Australia (n = 263) and from shelters or cat colonies in Italy (n = 478) were tested. Overall, Carnivore protoparvovirus 1 DNA was detected in 49 of 741 (6.61 %) samples. Differentiation was possible for 31 positive samples. FPV was most common among positive samples (28/31, 90.3 %). CPV was detected in 4/31 samples (12.9 %) including CPV-2a in one sample, CPV-2b in another and co-infections of FPV/CPV-2b and CPV-2a/CPV-2b in the remaining two samples. A high rate of subclinical FPV infection was detected in one shelter during an outbreak of feline panleukopenia, during which 21 of 22 asymptomatic cats (95.5 %) sampled were shedding FPV. Faecal shedding of CPV by cats in multi-cat environments is uncommon suggesting that domestic cats are not significant reservoirs of CPV.

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.

Discrepancy Between In-clinic and Haemagglutination-Inhibition Tests in Detecting Maternally-Derived Antibodies Against Canine Parvovirus in Puppies.

Canine parvovirus (CPV) is one of the most common causes of mortality in puppies worldwide. Protection against CPV infection is based on vaccination, but maternally-derived antibodies (MDA) can interfere with vaccination. The aim of this study was to evaluate the applicability of an in-clinic ELISA test to assess the CPV MDA in unvaccinated puppies and CPV antibodies in bitches, comparing the results with the gold standard haemagglutination inhibition (HI) test. Serum samples of 136 unvaccinated puppies were tested, along with sera of 16 vaccinated bitches. Five unvaccinated puppies were retested after vaccination. Both assays showed that the 16 vaccinated bitches had protective antibody levels against CPV. Conversely, significant discrepancies were observed for the MDA titers in unvaccinated puppies. Protective MDA titers were observed in 91.9% puppies using HI and in 40.4% by the in-clinic ELISA test, and only the latter one showed a decrease of MDA titers and percentages of protected puppies after the first weeks of age. Vaccination of five puppies with high HI and low in-clinic ELISA MDA titers resulted in seroconversion. Our results confirm the reliability of the in-clinic ELISA test in determining protective antibodies against CPV in adult dogs. Our findings also suggest that the in-clinic ELISA test kit may also be a useful tool to detect and quantify CPV MDA, thus allowing prediction of the best time to vaccinate puppies and reduction of the rate of vaccination failures due to interference by maternally-derived antibodies.

Circulation of diverse protoparvoviruses in wild carnivores, Italy.

Protoparvovirus is a monophyletic viral genus that includes the species Carnivore protoparvovirus-1 infecting domestic and wild carnivores. In this paper, the results of an epidemiological survey for Carnivore protoparvovirus-1 in wild carnivores in Italy are reported. Overall, 34 (11.4%) out of 297 tested animals were positive for Carnivore protoparvovirus-1, but the frequency of detection was much higher in intestine (54%) than in spleen samples (2.8%), thus suggesting that the intestine is the best sample to collect from wild animals for parvovirus detection. Feline panleukopenia virus (FPV) was detected in red foxes (Vulpes vulpes) (2.8%, 7/252) and Eurasian badgers (Meles meles) (10%, 1/10), whilst canine parvovirus (CPV) was found in wolves (54.3%, 19/35), Eurasian badgers (60%, 6/10) and one beech marten (Martes foina) (100%, 1/1), with more than one parvovirus type detected in some animals. Protoparvoviral DNA sequences from this study were found to be related to CPV/FPV strains detected in Asia and Europe, displaying some amino acid changes in the main capsid protein VP2 in comparison with other parvovirus strains from wildlife. In particular, the two most common mutations were Ile418Thr and Ala371Gly, which were observed in 6/12 (50%) and 5/12 (41.7%) of the CPV sequences from this study. Continuous surveillance for parvoviruses in wild carnivores and genetic analysis of the detected strains may help obtain new insight into the role of these animals in the evolution and epidemiology of carnivore parvoviruses.

Circulation of pantropic canine coronavirus in autochthonous and imported dogs, Italy.

Canine coronavirus (CCoV) strains with the ability to spread to internal organs, also known as pantropic CCoVs (pCCoVs), have been detected in domestic dogs and wild carnivores. Our study focused on the detection and molecular characterization of pCCoV strains circulating in Italy during the period 2014-2017 in autochthonous dogs, in dogs imported from eastern Europe or illegally imported from an unknown country. Samples from the gut and internal organs of 352 dogs were screened for CCoV; putative pCCoV strains, belonging to subtype CCoV-IIa, were identified in the internal organs of 35 of the examined dogs. Fifteen pCCoV strains were subjected to sequence and phylogenetic analyses, showing that three strains (98960-1/2016, 98960-3/2016, 98960-4/2016) did not cluster either with Italian or European CCoVs, being more closely related to alphacoronaviruses circulating in Asia with which they displayed a 94%-96% nucleotide identity in partial spike protein gene sequences. The pCCoV-positive samples were also tested for other canine viruses, showing co-infections mainly with canine parvovirus.

Feline Parvovirus Seroprevalence Is High in Domestic Cats from Disease Outbreak and Non-Outbreak Regions in Australia.

Multiple, epizootic outbreaks of feline panleukopenia (FPL) caused by feline parvovirus(FPV) occurred in eastern Australia between 2014 and 2018. Most affected cats were unvaccinated.We hypothesised that low population immunity was a major driver of re-emergent FPL. The aim ofthis study was to (i) determine the prevalence and predictors of seroprotective titres to FPV amongshelter-housed and owned cats, and (ii) compare the prevalence of seroprotection between a regionaffected and unaffected by FPL outbreaks. FPV antibodies were detected by haemagglutinationinhibition assay on sera from 523 cats and titres ≥1:40 were considered protective. Socioeconomicindices based on postcode and census data were included in the risk factor analysis. The prevalenceof protective FPV antibody titres was high overall (94.3%), even though only 42% of cats wereknown to be vaccinated, and was not significantly different between outbreak and non-outbreakregions. On multivariable logistic regression analysis vaccinated cats were 29.94 times more likelyto have protective FPV titres than cats not known to be vaccinated. Cats from postcodes of relativelyless socioeconomic disadvantage were 5.93 times more likely to have protective FPV titres. Thepredictors identified for FPV seroprotective titres indicate targeted vaccination strategies in regionsof socioeconomic disadvantage would be beneficial to increase population immunity. The criticallevel of vaccine coverage required to halt FPV transmission and prevent FPL outbreaks should bedetermined.

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.

General and type 2 parvovirus-specific passive immune transfer in puppies - Evaluation by early growth.

Failure of passive immune transfer put puppies at a higher risk of neonatal and weaning mortality due to low immune protection against infectious agents. The aim of this study was to investigate the evolution of the general via serum IgG concentration (IgG) and the specific via serum maternally derived canine parvovirus type 2-specific antibody titer (CPV2 MDA) passive immune transfer within the first 4 weeks of age. Furthermore, the relationship between general and specific immune transfer and the possibility of non-invasive evaluation was assessed. Puppies (169) were weighed systematically between birth and Day 28. IgG and CPV2 MDA were assayed in serum samples at 2 and at 28 days of age. At Day 2, there was a positive correlation between IgG and CPV2 MDA (ρ = 0.71; p < 0.001). At Day 2, 17.9% (27/151) of puppies presented a deficit of passive immune transfer according to IgG result (defined as IgG < 2.3 g/L) and 25.8% (39/151) of puppies were under the minimal protective CPV2 MDA titer (defined as <1:160). No correlation between IgG and CPV2 MDA was observed at Day 28 (ρ = 0.14; p = 0.11). Growth rate within the first 48 hours <-2.7% allowed to distinguish puppies at high risk of the general and specific passive immune failure (Youden's index = 0.79 and 0.75, respectively). The threshold value of early growth rate, although applicable only in puppies non-supplemented with milk replacer, allows identifying via non-invasive way individuals requiring a special care. Further investigation of the mechanism of passive immune transfer in dogs is necessary to understand the relationship between the general and specific immunoglobulin levels.

Detection of parvovirus and herpesvirus DNA in the blood of feline and canine blood donors.

With the increase of blood transfusion in veterinary medicine, the presence of endemic viral agents in the blood should be carefully investigated. For this reason, the blood of feline and canine blood donors was screened to detect the presence of herpesviruses, especially gammaherpesviruses and parvoviruses, and to characterize the viruses detected. A retrospective cross-sectional study was performed on 31 cats and 54 dogs, enrolled as voluntary blood donors. Nested PCR was carried out to detect herpesvirus and parvovirus DNA. Sequencing and real-time PCR were used to confirm and quantify positive samples. The feline and canine samples were negative for the presence of herpesviruses. Fourteen specimens of blood (45.16%, 95% confidence interval, CI: 27.78-63.70) from feline blood donors and two (3.7%, 95% CI: 0.64-13.84) from canine blood donors were positive for parvovirus DNA. The percentage positivity was significantly different in cats and dogs (P < 0.0001), giving an odds ratio of 21.41 (95% CI: 4.4-103.9). The lack of detection of herpesviral DNA confirms previous results obtained in dogs, but contrasts with the evidence of the worldwide distribution of gammaherpesviruses in cats. Selection of blood donors is a useful tool adopted to reduce the risk of transfusion-transmitted infections for the majority of known microorganisms. The results obtained for parvovirus, however, confirm the presence of this pathogen in the blood of healthy cats, with a significant difference from dogs. The implications of the detection of parvoviral DNA in the blood of donors must be clarified in order to exclude the risk of transmission.