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Different strategies have been proposed to overcome maternally derived antibody (MDA) interference with canine parvovirus type 2 (CPV-2) immunisation, including intranasal vaccination, which presents some practical limitations. In the present study, the results of the oral administration of a commercial CPV-2b modified live virus (MLV) vaccine in pups with MDA are reported. The CPV-2b vaccine was orally administered to 14 6-week-old pups with a bait. Blood samples and rectal swabs were collected at different days post-vaccination (dpv) to determine CPV-2 antibody titres and DNA loads. Thirteen pups were positive to serological and virological tests after the first vaccination and one pup became positive after the second vaccine administration. The findings of this study suggest that systemic immunity against CPV-2 may be achieved by the use of an MLV CPV-2b vaccine administered orally even in the presence of MDA titres that usually interfere with vaccination.

Canine parvovirus type 2 (CPV-2) causes a highly contagious and fatal disease, developing into acute hemorrhagic enteritis and myocarditis, in dogs. CPV-2 has evolved, generating antigenic variants CPV-2a/2b/2c that are globally distributed. However, investigating molecular characterization of CPV-2 among dog populations in Mongolia has been limited. Herein, 42 stool samples were collected from dogs with clinical signs of infection, and conventional PCR assays were employed to detect CPV-2 in 23. Our results indicated that during 2016-2018, the new CPV-2a and 2c subtypes were detected in 34.7% of the samples, and the new CPV-2b subtype was detected in 30.4% of samples. VP2 protein sequence analysis and next-generation sequencing of the complete viral genome confirmed these antigenic types. However, sequence analysis indicated new and unreported mutations, Pro580Thr, and Tyr584His in the CPV-2c subtype. From a PCR-positive sample, CPV-2c was successfully isolated, and we performed an immunofluorescence assay for antigen detection. Additionally, we performed genetic characterization and phylogenetic analysis to investigate genetic diversity among isolates from the region, resulting in high CPV-2 genetic diversity in the Mongolian dog population. Striking similarities were also observed between sequences of the strains isolated from Mongolia and China over a similar time span.

The variability and the intrinsically high mutation rate of canine parvovirus type 2(CPV-2) increased the diversity of CPV-2 in canine populations. Since the first occurrence of CPV-2, three antigenic variants (2a, 2b and 2c) were detected and distributed worldwide. CPV-2c infection has been detected and increasingly reported in China. Here, a CPV-2c strain CPV-SH1516 was isolated and its complete genome sequence was first characterized. Compared with other CPV-2c isolates, CPV-2c isolates from China continued to evolve into divergent CPV-2c variants with specific unique amino acid substitutions under purifying selection. Emergence of CPV-2c isolates from China was driven by the unique gradual point mutations in key sites of VP2 rather than introduction from outside China. Combining sequence comparison with phylogenetic analysis based on the amino acid sequences of VP2, the vast majority of CPV-2c isolates from China formed a monophyletic cluster and CPV-SH1516 was a representative isolate of CPV-2c circulating in China. Overall, our study provides valuable insight into the evolutionary mechanism of CPV-2c.

Feline panleukopenia (FPL) is caused by a Carnivore protoparvovirus infection. Feline parvovirus (FPV) causes most cases. When Canine parvovirus 2 (CPV-2) first emerged, it could not replicate in cats. All current CPV variants (CPV-2a-c) can infect cats to cause subclinical disease or FPL. Feline panleukopenia has re-emerged in Australia in shelter cats associated with failure to vaccinate. Parvoviruses can remain latent in mononuclear cells post-infection. Molecular methods such as polymerase chain reaction are used to determine the infecting strain. Current perspectives on causes, epidemiology, diagnosis, treatment, prognostic indicators, and management of outbreaks in shelters are reviewed.

Bufaviruses are small, nonenveloped, single-stranded DNA viruses belonging to the subfamily Parvovirinae. Human bufaviruses were first identified in 2012 in fecal samples from children with diarrhea. A new parvovirus of canines that was first detected in various samples from dogs with enteric and respiratory symptoms in Italy between 2014 and 2018 is closely related to the newly described human bufavirus. To explore the prevalence and genetic diversity of CBuV in Chinese dogs, 540 canine parvovirus (CPV)-positive serum and diarrhea samples were collected in Guangxi Province between 2016 and 2018. Among the samples, 6.25% (5/80) of rectal swabs and 2.5% (5/200) of CPV PCR-positive samples were positive for CBuV. However, the virus was not detected in CPV PCR-negative samples or nasal swabs. Two CBuV isolates were identified from CPV-positive fecal and serum samples by complete sequence analysis, with 99.8%–99.9% NS1 and VP2 protein identity to each another. Sequence analysis indicated that the CBuV GXNN01-2018 isolate VP2 protein shares 99.6% identity with the Italian CBuV ITA/2015/297 isolate and 62.3%–65.5% identity with human bufavirus. Phylogenetic analysis showed that CBuV was significantly distinct from other known bufaviruses and was most closely related to CBuV ITA/2015/297. This is the first report of the existence of CBuV in China, and our findings will strengthen the understanding of the epidemiology of bufaviruses in different animals.

A 4-month-old puppy died after showing intracranial signs a few days after a suspected viral enteritis. Grossly, the right cerebral hemisphere had a large irregular cavity external to the internal capsule. Histopathological examination revealed a cystic lesion in the right hemisphere and non-suppurative inflammation of the diencephalon and periaqueductal nervous tissue. Porencephaly associated with periventricular non-suppurative encephalitis was diagnosed. A nested polymerase chain reaction (PCR) identified the presence of parvovirus DNA in the brain and real-time PCR typed this as canine parvovirus (CPV) type 2a. Immunohistochemistry revealed the presence of CPV antigen in the cytoplasm of scattered cells in the subependymal layers and choroid plexus epithelium. The porencephaly was not associated with inflammatory lesions or CPV antigen and was considered to have preceded the neurological signs. In contrast, the detection of CPV antigen in the subependymal layers and choroid plexus epithelium supported the association of this virus with the periventricular encephalitis.

Canine parvovirus (CPV) is one of the most important cause of mortality in young dogs and no specific treatment exists. Since prolonged leukopenia greatly increases the risk of death in infected pups, strategies to counteract this decline were investigated. The outcomes of CPV naturally infected pups treated with the recombinant canine granulocyte-colony stimulating factor (rcG-CSF), in combination with the routine therapy, were compared with similarly-managed infected pups not treated with rcG-CSF. A non-randomized prospective clinical trial was performed on 62 CPV infected pups with WBC counts

We have isolated 4 naturally-occurring strains of CPV in mainland China and have identified them as CPV-2, 2a, 2b and 2c genotypes according to their VP2 sequences which also revealed substitutions within their right terminal regions. To determine if these substitutions affected the growth characteristics of the 4 strains, we constructed plasmids based on their genomic sequences minus their right terminal sequences, with the latter replaced by a single right terminal region. Analysis of rescued recombinants showed that the substitutions within their natural right termini had no significant effect on their growth characteristics.

Domestic dogs share habitats with human, a fact that makes them a potential source of zoonotic viruses. Moreover, knowledge regarding possible bloodborne pathogens is important due to the increasing application of blood transfusion in dogs. In the present study, we evaluated the serum virome of 520 dogs using throughput sequencing (HTS). The serum samples were pooled and sequenced using an Illumina MiSeq platform. Our unbiased method identified prevalent canine pathogens as canine protoparvovirus 1 (canine parvovirus 2), undersearched agents as canine bocaparvovirus 1 (minute virus of canines) and canine circovirus, circular viruses closely related to viruses recently found in human samples, and new parvovirus and anelloviruses. The dog virome described in the present work furthers the knowledge concerning the viral population in domestic animals. The present data includes information regarding viral agents that are potentially transmitted through blood transfusion among dogs.

Canine chaphamaparvovirus (CaChPV) is a newly recognised parvovirus discovered by metagenomic analysis during an outbreak of diarrhoea in dogs in Colorado, USA, in 2017 and more recently detected in diarrhoeic dogs in China. Whether the virus plays a role as canine pathogen and whether it is distributed elsewhere, in other geographical areas, is not known. We performed a case-control study to investigate the possible association of CaChPV with enteritis in dogs. CaChPV DNA was detected both in the stools of diarrhoeic dogs (1.9 %, 3/155) and of healthy animals (1.6 %, 2/120). All the CaChPV-infected dogs with diarrhea were mixed infected with other enteric viruses such as canine parvovirus (formerly CPV-2), canine bufavirus (CBuV) and canine coronavirus (CCoV), whilst none of the asymptomatic CaChPV positive animals resulted co-infected. The nearly full-length genome and the partial capsid protein (VP) gene of three canine strains, Te/36OVUD/19/ITA, Te/37OVUD/19/ITA and Te/70OVUD/19/ITA, were reconstructed. Upon phylogenetic analyses based on the NS1 and VP aa sequences, the Italian CaChPV strains tightly clustered with the American reference viruses. Distinctive residues could be mapped to the deduced variable regions of the VP of canine and feline chaphamaparvoviruses, considered as important markers of host range and pathogenicity for parvoviruses.

This study assessed the frequency and timing of feline panleukopenia virus (FPV) shedding in feces following administration of a modified live FPV vaccine. Feces were collected from 37 shelter cats that did not meet clinical criteria for panleukopenia on the day of vaccination or on days 3, 7, 14, and 21 post-vaccination (NCL group). A commercial quantitative PCR (qPCR) fecal pathogen panel and a canine parvovirus point-of-care antigen test were performed. FPV DNA copy numbers from a concurrent study of 39 cats with panleukopenia (CL group) were compared with the NCL group. Of the 165 samples from the NCL group, one had a weak positive antigen test result on day 7, while nine samples (5.5%) from eight cats (21.6%) produced positive FPV qPCR test results, one on day 3 and eight on day 7. There were no 21-day positive qPCR results in the 11 cats that were revaccinated on day 14. There was no association between the number of additional fecal pathogens identified and a positive FPV qPCR result. Of the cats with positive results, FPV DNA copy numbers differed between NCL group and CL group (median 1.13 × 107 and 5.01 × 10⁸ copies/g feces, respectively; P

As the outbreaks of COVID-19 in worldwide, coronavirus has once again caught the attention of people. Canine coronavirus is widespread among dog population, and sometimes causes even fatal cases. Here, to characterize the prevalence and evolution of current circulating canine coronavirus (CCoV) strains in China, we collected 213 fecal samples from diarrheic pet dogs between 2018 and 2019. Of the 213 samples, we found 51 (23.94%) were positive for CCoV. Co-infection with canine parvovirus (CPV), canine astrovirus (CaAstV), canine kobuvirus (CaKV), Torque teno canis virus (TTCaV) were ubiquitous existed. Mixed infection of different CCoV subtypes exists extensively. Considering the limited sequences data in recent years, we sequenced 7 nearly complete genomes and 10 complete spike gene. Phylogenetic analysis of spike gene revealed a new subtype CCoV-II Variant and CCoV-IIa was the most prevalent subtype currently circulating. Moreover, we identified strain B906_ZJ_2019 shared 93.24% nucleotide identifies with previous strain A76, and both of them clustered with CCoV-II Variant, which were not well clustered with the known subtypes. Recombination analysis of B906_ZJ_2019 indicated that strain B906_ZJ_2019 may a recombinant variant between CCoV-I and CCoV-II, which is consistent with strain A76. Furthermore, amino acid variations widely existed among current CCoV-IIa strains circulating in China and the classic CCoV-IIa strains, in spite of the unknown functions. In a word, we report a useful information as to the etiology and evolution of canine coronavirus in China based on the available sequences, which is urgent for the devise of future effective disease prevention and control strategies.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

Canine parvovirus (CPV) is a contagious disease in dogs that has high morbidity and mortality. In cases of infection, the pups tend to have a higher mortality and more severe clinical symptoms than the adult dogs because the dehydration is difficult for pups to bear. Following the natural infection, there is a rapid antibody response neutralizing the extracellular virus. As a result, virus titers in tissue and feces become markedly reduced. Hence, it is important to have an effective symptomatic therapy of supporting animals to survive in the early stages of CPV infection. Furthermore, the co-infection with bacteria could increase the severity of lesions and clinical signs as well. In this paper, we obtained the bacterial diversity in feces of CPV infected dogs with the enrichment of five bacteria genera (Shigella, Peptoclostridium, Peptostreptococcus, Streptococcus, Fusobacterium). These microorganisms may partly result in the intestinal pathology of the infection. In summary, the discussion of the bacterial biodiversity in feces of CPV infected dogs provides further insights into the pathology of CPV disease and the targets of developing more effective treatment strategies.

Canine parvovirus (CPV) is the etiological agent of a severe viral disease of dogs. After its emergence in late 1970s, the CPV original type (CPV-2) was rapidly and totally replaced by three antigenic variants named CPV-2a, CPV-2b and CPV-2c. CPV has an evolutionary rate nearest to those of RNA viruses, with consequences on disease diagnosis and epidemiology. This paper reports the molecular characterization of eight CPV-2a strains collected from dogs in Italy in 2016–2017. Genetic analysis was conducted on a CPV genomic region encompassing both open reading frames (ORFs) encoding for nonstructural (NS1-NS2) and structural proteins (VP1-VP2). Sequence analysis indicates new and unreported sequence changes, mainly affecting the VP2 gene, which included the mutation Tyr324Leu. This study represents the first evidence of a new CPV-2a mutant (VP2 324Leu) and illustrates the importance of a continuous molecular survey in order to obtain more information on effective spread of new CPV mutants., Highlights • Canine parvovirus strains collected from dogs in southern Italy were analyzed. • Nearly complete genome sequences of the CPV strains were obtained and comparatively analyzed. • A novel CPV-2a mutant with unreported sequence changes has been characterized. • This study reports a novel CPV-2a mutant in genus Protoparvovirus. • Our data confirmed the importance of the continuous epidemiological survey.

Canine parvovirus (CPV) is an important pathogen in domestic dogs, and the original antigenic types CPV-2 and its variants, CPV-2a, 2b and 2c, are prevalent worldwide. A multiplex TaqMan real-time PCR method was developed for the detection and differentiation of four antigenic types of CPV. A set of primers and probes, CPV-305F/CPV-305R and CPV-2-305P (for CPV-2)/CPV-2a-305P (for CPV-2a, 2b and 2c), was able to differentiate CPV-2 and its variants (CPV-2a, 2b and 2c). Another set of primers and probes, CPV-426F/CPV-426R and CPV-2-426P (for CPV-2 and 2a)/CPV-2b-426P (for CPV-2b)/CPV-2c-426P (for CPV-2c), was able to differentiate CPV-2a (2), CPV-2b, and CPV-2c. With these primers and probes, the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2, 2a, 2b and 2c by two separate real-time PCRs. No cross reactivity was observed with canine distemper virus, canine adenovirus, and canine coronavirus. The detection limit of the assay is 101 genome copies/μL for CPV-2, CPV-2a, CPV-2b, and 102 copies/μL for CPV-2c. The multiplex real-time PCR has 100% agreement with DNA sequencing. We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome., Highlights • The Multiplex TaqMan real-time PCR can simultaneously detect and differentiate four antigenic types of CPV. • The method is suit for using in detection of CPVs in China. • The method showed a high specificity and sensitivity. • The method was also used for quantification of CPVs viral genome.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 μL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

In dogs, canine parvovirus (CPV) enteritis is associated with high morbidity and fatality rates requiring early diagnosis to facilitate treatment and reduce its spread. In recent times, various commercial immunochromatographic (IC) test kits are available for its rapid diagnosis, which require an assessment of their accuracy. Therefore, precision of a point-of-care IC combination test kit for canine coronavirus (CCoV)/CPV faecal antigen detection was evaluated in this study. Multicentred random faecal samples from 115 dogs with gastroenteritis were checked for the presence of CPV antigens using the SensPERT IC combination test kit and the result was compared with polymerase chain reaction (PCR) as a reference test. Parvovirus was detected in 105 (91.3%) and 108 (93.9%) faecal samples by the point-of-care test kit and PCR, respectively. The point-of-care IC test kit showed 95.4% relative sensitivity, 71.4% specificity, 98.1% positive predictive value, 50.0% negative predictive value, and 93.9% accuracy comparable to conventional PCR in the samples tested. This point-of-care test kit also demonstrated a fair positive likelihood ratio (3.34), a very low negative likelihood ratio (0.07) and a moderate agreement (Kappa = 0.6) compared with conventional PCR. This test kit has shown to be very useful in the screening of dogs for CPV infection, and is a reliable alternative for diagnosing CPV both in conventional laboratories and remote areas without laboratories. Negative results in the IC testing with high suspicion of CPV infection should be further confirmed using superior test such as PCR.

Parvovirus is a common element of the feline virus group and usually causes gastroenteritis and leukopenia in cats. In this study, we identified a novel protoparvovirus from the Chinese domestic cats, which is genetically similar to canine bufavirus (98.0%-99.8%), but sharing low amino acid identities in the viral structural proteins 2 (VP2) (36.1-37.2%) to the well-known canine parvovirus type 2 and feline panleukopenia virus. This virus was provisionally designated as feline bufavirus (FBuV). Screening of fecal samples revealed a prevalence of 7.4% (19/257) in domestic cats. Diarrhea was present in 52.6% (10/19) of cats positive for FBuV. However, statistical analysis showed no association between FBuV and clinical signs. VP2 gene of the 19 field FBuV was sequenced and phylogenetic analysis demonstrated that FBuV determined from China had a genetic diversity. This study will strengthen the understanding of the epidemiology and genetic diversity of bufavirus and provide a foundation for further studies.

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.

A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10-1, 10-1, and 10-1 TCID50/mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV.

Feline panleukopenia virus (FPV) and canine parvovirus 2 (CPV-2) belong to the species Carnivore protoparvovirus 1 . While FPV has been endemic in cats for many years, CPV-2 emerged at the end of the 1970s and 3 antigenic variants (CPV-2a, b and c) are currently circulating in domestic and wild animals. Although raccoons played a fundamental role in the emergence of CPV-2a in dogs, knowledge about protoparvoviruses in these animals is still limited. We investigated 72 raccoons found dead or injured in southern British Columbia (BC), Canada, between 2009 and 2017. Among the 49 protoparvovirus-positive (66%) animals, 39 (80%) exhibited enteritis. Approximately 21% were FPV-positive and these were from Vancouver Island, while 79% were CPV-positive and found on the BC mainland, suggesting geographic separation of strains. Notably, one CPV-positive individual was FPV-vaccinated. The co-circulation of multiple FPV strains was observed, while the CPV–2 strain were all classified as CPV-2a, located in one unique clade, and likely originated from a single introduction. All BC CPV–2 sequences possessed a VP2-305His and therefore could represent one of the intermediate viruses that facilitated the emergence of CPV-2a in dogs. Like other raccoon viruses, all BC CPV–2 sequences possessed a VP2-300Asp but, differently, possessed a VP2-301Ala. Finally, two unique amino acid substitutions were identified in the NS1 of FPV (His554Gln and Gly573Cys). To our knowledge, this is the first molecular characterization of protoparvoviruses in wildlife from this region of North America and more studies are needed to elucidate the distribution of these viruses in wildlife outside the USA.

Canine parvovirus type 2 (CPV-2) infection is associated with severe gastroenteritis in puppies. Quantification of CPV-2 specific antibodies before vaccination can reveal the presence of interfering maternal-derived immunity and facilitate timing of effective immunisation. Inhibition of haemagglutination (HI) is commonly used to measure CPV-2-specific antibody levels in serum. However, the presence of nonspecific agglutinins in canine serum and artefactual precipitation of red blood cells (RBC) are both limitations of the assay. In this study, we compared the standard HI protocol with a refined HI protocol, in which canine serum was pre-incubated with porcine RBC for 12 h to remove nonspecific agglutinins and a lower concentration (0.1% vs. 0.8%) of porcine RBC suspensions was used to limit artefactual precipitation of RBC. A panel of canine sera, collected from 80 dogs of different ages and with different neutralising antibody titres, was analysed. Nonspecific agglutinins were identified in most (97%) serum samples from puppies4 months of age and in only 7% dogs 6 months old. Pre-treatment of serum samples was effective in removing nonspecific agglutinins from all samples and artefactual precipitation of RBCs was not noted when 0.1% RBC suspensions were used. Refinement of the HI protocol has increased the accuracy of interpretation and reduced the interference of nonspecific agglutinins, primarily seen in puppies. This reduces the likelihood of incorrect assessment of passive or active immunity in puppies when deciding whether to administer or defer vaccination, which could potentially leave them susceptible to CPV-2 infection.

Based on virus dependence from host cell machinery, their codon usage is expected to show a strong relation with the host one. Even if this association has been stated, especially for bacteria viruses, the linkage is considered to be less consistent for more complex organisms and a codon bias adaptation after host jump has never been proven. Canine parvovirus type 2 (CPV-2) was selected as a model because it represents a well characterized case of host jump, originating from Feline panleukopenia virus (FPV). The current study demonstrates that the adaptation to specific tissue and host codon bias affected CPV-2 evolution. Remarkably, FPV and CPV-2 showed a higher closeness toward the codon bias of the tissues they display the higher tropism for. Moreover, after the host jump, a clear and significant trend was evidenced toward a reduction in the distance between CPV-2 and the dog codon bias over time. This evidence was not confirmed for FPV, suggesting that an equilibrium has been reached during the prolonged virus-host co-evolution. Additionally, the presence of an intermediate pattern displayed by some strains infecting wild species suggests that these could have facilitated the host switch also by acting on codon bias.

Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive. To cut costs of screening peptides for a new serological assay, the concept was investigated of using cheap non-purified (crude) peptides instead of purified peptides. Peptides were selected that represent three well-described linear epitopes of viral proteins: VP2 of canine parvovirus (CPV), gp41 of human immunodeficiency virus (HIV) and E2 of classical swine fever virus (CSFV). Crude and purified biotinylated peptides with either a short or long spacer between the biotin and the epitope were used to test their capability to bind antibodies in a bead-based suspension array. The results show that, in a bead-based suspension array, crude peptides can function as antigen for specific monoclonal antibodies, and that the acquired signals are less than with purified peptides. CSFV-derived crude peptides were also able to detect specific antibodies in swine serum, indicating the applicability of crude peptides for pre-screening large numbers of different peptides during the development of serological peptide-based assays.

Type I interferons (IFNs) such as IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω represent cytokines, which are deeply involved in the regulation and activation of innate and adaptive immune responses. They possess strong antiviral, antiproliferative, and immunomodulatory activities allowing their use in the therapy of different viral diseases, neoplasms, and immune-mediated disorders, respectively. Initially, treatment strategies were based on nonspecific inducers of type I IFNs, which were soon replaced by different recombinant proteins. Drugs with type I IFNs as active agents are currently used in the treatment of hepatitis B and C virus infection, lymphoma, myeloid leukemia, renal carcinoma, malignant melanoma, and multiple sclerosis in humans. In addition, recombinant feline IFN-ω has been approved for the treatment of canine parvovirus, feline leukemia virus, and feline immunodeficiency virus infections. However, the role of type I IFNs in the pathogenesis of canine diseases remains largely undetermined so far, even though some share pathogenic mechanisms and clinical features with their human counterparts. This review summarizes the present knowledge of type I IFNs and down-stream targets such as Mx and 2',5'-oligoadenylate synthetase proteins in the pathogenesis of infectious and immune-mediated canine diseases. Moreover, studies investigating the potential use of type I IFNs in the treatment of canine lymphomas, melanomas, sarcomas, and carcinomas, canine distemper virus, parvovirus, and papillomavirus infections as well as immune-mediated keratoconjunctivitis sicca and atopic dermatitis are presented. A separate chapter is dedicated to the therapeutic potential of IFN-λ, a type III IFN, in canine diseases. However, further future studies are still needed to unravel the exact functions of the different subtypes of type I IFNs and their target genes in healthy and diseased dogs and the full potential action of type I IFNs as treatment strategy.

Highlights • Detection of CPV (24%) and CCoV (4.8%) in dogs on Caribbean island of St. Kitts. • New CPV-2a (CPV-2a-297A) was the predominant CPV variant in St. Kitts. • The CCoV strains from St. Kitts were assigned to CCoV-I genotype. • First report on genetic diversity of CPV and CCoV from the Caribbean region., Although canine parvovirus (CPV) and canine enteric coronavirus (CCoV) are important enteric pathogens of dogs and have been studied extensively in different parts of the world, there are no reports on these viruses from the Caribbean region. During 2015–2016, a total of 104 diarrheic fecal samples were collected from puppies and adult dogs, with or without hemorrhagic gastroenteritis, on the Caribbean island of St. Kitts (KNA). By PCR, 25 (24%, n = 104) samples tested positive for CPV. Based on analysis of the complete deduced VP2 amino acid sequences, 20 of the KNA CPV strains were assigned to new CPV-2a (also designated as CPV-2a-297A). On the other hand, the VP2 genes of the remaining 5 strains were partially characterized, or could not be sequenced. New CPV-2a was the predominant CPV variant in St. Kitts, contrasting the molecular epidemiology of CPV variants reported in most studies from nearby North and South American countries. By RT-PCR, CCoVs were detected in 5 samples (4.8%, n = 104). Based on analysis of partial M-protein gene, the KNA CCoV strains were assigned to CCoV-I genotype, and were closely related to CCoV-I strains from Brazil. To our knowledge, this is the first report on detection and genetic diversity of CPV and CCoV in dogs from the Caribbean region, and underscores the importance of similar studies in the other Caribbean islands.

The Torque teno canis virus (TTCaV) is a small virus with circular single-stranded DNA that has been reported to cause infections in dogs. The present study aimed to identify the presence of TTCaV in blood samples obtained from domestic dogs, and examine its diversity and evolution of the genomes. Five strains of TTCaV were detected, and the overall prevalence was found to be 7% (28/400). Phylogenetic analysis showed that the five genomes were closely clustered with the previously known Cf-TTV10 and LDL strains and formed a Thetatorque virus. Homology analysis of the whole genome showed a sequence identity of 94.6%–96.8% among the five genomes. The percent sequence similarity among the five complete genomes ranged from 95.3% to 97.4% and from 95.1% to 97% compared to the Cf-TTV10 and LDL strains respectively. The ORF1-encoded amino acid sequences showed 94.4%–97.2% identity among the five isolates. Our findings suggest that the TTCaV has a large genetic diversity and showed that TTCaV and canine parvovirus (CPV) co-infection exists in China. Further studies on the pathogenicity of TTCaV are required.

Highlights • A molecular survey for selected viral enteropathogens was conducted in dogs with and without acute gastroenteritis. • Canine parvovirus and coronavirus were strongly associated with occurrence of enteric disease. • Canine circovirus infections correlated with acute gastroenteritis only when associated with other enteric viruses., 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

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation., Highlights • We constructed a chimeric parvovirus virus-like particles displaying the MERS-CoV receptor-binding domain on the surface. • We found that the chimeric virus-like particles induced RBD-specific, neutralizing antibody responses in mice. • Splenocytes from immunized mice had considerably higher secretion of both Th1- and Th2-type cytokines. • Our results reveal that chimeric virus-like particles induce both specific humoral and cell-mediated immunity.

Since little is known about the persistence and faecal shedding of canine parvovirus (CPV) in dogs after modified-live vaccination, diagnostic tests for CPV can be difficult to interpret in the post-vaccination period. The primary aim of this study was to determine the incidence, duration and extent of CPV vaccine virus shedding in adult dogs and to investigate related factors, including the presence of protective antibodies, increase in anti-CPV antibody titres and development of any gastrointestinal side-effects. A secondary objective was to assess prevalence of CPV field virus shedding in clinically healthy dogs due to subclinical infections. One hundred adult, healthy privately owned dogs were vaccinated with a commercial CPV-2 modified-live vaccine (MLV). Faeces were tested for the presence of CPV DNA on days 0 (prior to vaccination), 3, 7, 14, 21 and 28 by quantitative real-time PCR. Pre- and post-vaccination serum titres were determined by haemagglutination inhibition on days 0, 7 and 28. Transient excretion of CPV DNA was detected in 2.0% of dogs before vaccination. About one quarter of dogs (23.0%) shed CPV DNA during the post-vaccination period, but field and vaccine virus differentiation by VP2 gene sequencing was only successful in few samples. Faecal CPV excretion occurred despite protective serum antibody titres. Post-vaccination CPV shedding was not related to adequate antibody response after vaccination or to the occurrence of gastrointestinal side-effects. Despite individual differences, CPV DNA was detectable for up to 28 days after vaccination, although the faecal CPV DNA load in these clinically healthy dogs was very low.

Cross-species transmission can often lead to deleterious effects in incidental hosts. Parvoviruses have a wide host range and primarily infect members of the order Carnivora. Here we describe juvenile common palm civet cats (Paradoxurus musangus) that were brought to the Singapore zoo and fell ill while quarantined. The tissues of two individual civets that died tested PCR-positive for parvovirus infection. Phylogenetic analysis revealed this parvovirus strain falls in a basal position to a clade of CPV that have infected dogs in China and Uruguay, suggesting cross-species transmission from domestic to wild animals. Our analysis further identified these viruses as genotype CPV-2a that is enzootic in carnivores. The ubiquity of virus infection in multiple tissues suggests this virus is pathogenic to civet cats. Here we document the cross-species transmission from domestic dogs and cats to wild civet populations, highlighting the vulnerability of wildlife to infectious agents in companion animals.

Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

Highlights • CPV, CCoV and β-hemolytic E. coli are associated with (severe) acute diarrhoea in puppies. • Puppies from high-volume breeders are more likely to be infected with CPV, CCoV and Cystoisospora spp. • CPV and CCoV are more often detected in winter/spring. • Multiple enteric infections are common in puppies with and without diarrhoea. • Screening of CPV, CCoV and β-hemolytic E. coli is advised in diarrhoeic puppies., Laboratory confirmation of the causative agent(s) of diarrhoea in puppies may allow for appropriate treatment. The presence of potential pathogens however, does not prove a causal relationship with diarrhoea. The aim of this study was to identify specific enteropathogens in ≤12 month old puppies with and without acute diarrhoea and to assess their associations with clinical signs, putative risk factors and pathogen co-occurrence. Faecal samples from puppies with (n = 113) and without (n = 56) acute diarrhoea were collected and screened for Canine Parvovirus (CPV), Canine Coronavirus (CCoV), Salmonella spp., Campylobacter spp., Clostridium perfringens, Clostridium difficile, β-hemolytic Eschericha coli (hEC), Giardia spp., Toxocara spp., Cystoisospora spp., and Cyniclomyces guttulatus. One or more pathogens were detected in 86.5% of diarrhoeic puppies and in 77.8% of asymptomatic puppies. Significant positive associations were found between CPV and CCoV, CPV and Cystoisospora spp., Toxocara spp. and hEC, Giardia spp. and C. guttulatus. Only CPV and CCoV were significantly associated with diarrhoea, hEC with a subset of puppies that had diarrhoea and severe clinical signs. CPV was more prevalent in puppies under 3 months of age. Puppies from high-volume dog breeders were significantly at increased risk for CPV (OR 4.20), CCoV (OR 4.50) and Cystoisospora spp. (OR 3.60). CCoV occurred significantly more often in winter (OR 3.35), and CPV in winter (OR 3.78) and spring (OR 4.72) as compared to summer. We conclude that routine screening for CPV, CCoV and hEC is recommended in puppies with acute diarrhoea, especially if they are under 3 months of age and originate from high-volume dog breeders. Routine screening for other pathogens may lead to less conclusive results.

Canine parvovirus type 2 (CPV-2) causes a highly contagious gastroenteritis disease of dogs and wild canids. To investigate the CPV-2 prevalence in Dakahlia Governorate, Egypt, a total of 50 fecal swabs were collected from suspected diseased dogs during 2016-2017. Out of 50 collected samples, 35 samples (70 %) presented positive results for CPV-2 using immuno-chromatography (IC) as a rapid test. CPV-2DNA was detected in 42 samples (84 %) by using polymerase chain reaction (PCR). The frequencies of CPV-2 were significantly higher in German shepherd breed (46 %; 23/50) and in age groups less than 6 months (76%; 38/50). We evaluated the breed, age, sex, rapid test results and clinical signs as predictors for classification of animal status into infected and not infected. The best predictors for classification process were rapid test result and clinical signs. Both CPV-2b and CPV-2c subtypes were detected by CPV2-VP2 gene sequences analysis. Deduced amino acid sequences alignment showed substitutions at 3 sites (Arg453Pro, Ala574Glu and Gln457Leu). Further investigations are needed to reveal the genetic and antigenic relation between field and vaccinal strains of CPV-2 in Egypt.

Since its emergence in Nigeria, canine parvovirus type 2 (CPV-2) infection has posed problems to dog breeding and requires constant awareness and monitoring. In this study, the status, the assessment of extrinsic risk factors of parvoviral infection in dog kennels in North Central Nigeria, and isolation of the CPV-2 were carried out. Potential risk factors were considered during sampling: age, breed, sex, location, vaccination and health status, using well-structured questionnaires on dog owners with experience of CPV-2 infection. There was high prevalence which depended on age, breed, location, clinical status of the dog while vaccination status of the dogs did not influence the prevalence. CPV-2 vaccination compliance by the breeders and management system of the kennels were also observed as risk factors. Isolation of CPV-2a and -2c strains from Nigeria for further study has been reported. The spread of CPV-2 in Nigeria is increasing, hence needs for continual epidemiological monitoring and review.

Since it first emergence in the mid-1970's, canine parvovirus 2 (CPV-2) has evolved giving rise to new antigenic variants termed CPV-2a, CPV-2b and CPV-2c, which have completely replaced the original strain and had been variously distributed worldwide. In Africa limited data are available on epidemiological prevalence of these new types. Hence, the aim of the present study was to determine circulating variants in Morocco. Through TaqMan-based real-time PCR assay, 91 samples, collected from symptomatic dogs originating from various cities between 2011 and 2015, were diagnosed. Positive specimens were characterised by means of minor groove binder (MGB) probe PCR. The results showed that all samples but one (98.9%) were CPV positive, of which 1 (1.1%) was characterised as CPV-2a, 43 (47.7%) as CPV-2b and 39 (43.3%) as CPV-2c. Interestingly, a co-infection with CPV-2b and CPV-2c was detected in 4 (4.4%) samples and 3 (3.3%) samples were not characterised. Sequencing of the full VP2 gene revealed these 3 uncharacterised strains as CPV-2c, displaying a change G4068A responsible for the replacement of aspartic acid with asparagine at residue 427, impacting the MGB probe binding. In this work we provide a better understanding of the current status of prevailing CPV strains in northern Africa.

Circoviruses are relatively novel pathogens with increased importance in canids. In this study, we first screened the presence of dog circovirus (DogCV) by molecular methods from a total number of 389 internal organ samples originating from 277 individuals of domestic dogs and wild animals including wolves, foxes and badgers. All the animals originated from Central-Southern Italy, specifically from Abruzzi and Molise regions, areas hosting several natural parks. DogCV was detected in 9/34 wolves (P=26.4%; IC 95%: 14.6–43.1%), 8/209 dogs (P=3.8%; IC 95%: 1.9–7.3%), 0/24 foxes (P=0%; IC 95%: 0–13.8%), 1/10 badgers (P=10%; IC 95%: 1.79–40.4%). However, all DogCV positive animals were shown to be infected at least by an additional key pathogen, including canine distemper virus (CDV) and canine parvovirus type 2. All wolves, but one, presenting DogCV in the internal tissues suffered from CDV infection. The DNA purified from 17 DogCV infected organs was used for whole genome sequencing and phylogenetic analysis.

Severe gastroenteritis caused by canine parvovirus type 2 (CPV2) is a serious life-threatening disease in puppies less than 4-months of age. The emergence of new variants has provoked some concern about the cross-protection elicited by licensed canine parvovirus modified-live type 2 (CPV2) and type 2b (CPV2b) vaccines against the most recent subtype CPV2c. A systematic review was carried out to assess the efficacy of commercial vaccines. We conducted a literature search of Pub Med/MEDLINE from January 1990 to May 2014. This was supplemented by hand-searching of related citations and searches in Google/Google Scholar. Controlled clinical trials in which vaccinated puppies were challenged with CPV2c virus were evaluated. Reporting of outcome measures and results for vaccine efficacy were critically appraised through a variety of clinical signs, serological tests, virus shedding and the ability to overcome maternally derived antibodies (MDA) titres. Six controlled clinical trials were included in the review. In most cases, the results of the selected studies reported benefits in terms of clinical signs, serological tests and virus shedding. However, MDA interference was not considered or evaluated in 5 of the selected trials. No accurate definitions of baseline healthy status and/or clinical outcomes were provided. Methods of randomization, allocation concealment and blinding were usually poorly reported. As a result of the limited number of included studies matching the inclusion criteria, the small sample sizes, short follow-up and the methodological limitations observed, it was not possible to reach a final conclusion regarding the cross-protection of licensed CPV2 and CPV2b vaccines against the subtype 2c in puppies. Further and specifically designed trials are required in order to elucidate whether cross-protection is acquired from licensed CPV vaccines.

Canine parvovirus (CPV) is the most important enteric virus infecting canids worldwide. The purpose of this study was to detect CPV in naturally infected dogs from several veterinary clinics distributed throughout Portugal between 2012 and 2014 and to identify risk factors associated with CPV infection. From 209 dogs suspected of being infected with CPV, historical data and clinical signs were collected. Fecal samples were screened for CPV by PCR assay and those positive were confirmed by sequencing. The data was analyzed using logistic regression to investigate associations between each of the predisposing factors and CPV status. Of the samples collected, 77.5% tested CPV-positive. Statistical analysis showed that animals in the three age categories (p

Highlights • CDV, CPV and CCoV nucleic acids were detected in samples from Portuguese wild carnivores. • Exposure to Canine parvovirus and Canine distemper virus was confirmed by serology. • Sample conservation status was significant for antibody detection. • Co-infections by multiple pathogens (two or three viruses) were recorded., Pathogen surveillance in free-ranging carnivores presents challenges due to their low densitie and secretive nature. We combined molecular and serological assays to investigate infections by viral pathogens (Canine parvovirus (CPV), Canine distemper virus (CDV) and Canine coronavirus (CCoV)) in Portuguese carnivores (Canis lupus, Vulpes vulpes, Lutra lutra, Martes foina, M. martes, Meles meles, and Genetta genetta) over a period of 16 years. Additionally we explored spatio-temporal patterns of virus occurrence in Canis lupus. Our study identified CPV DNA in all carnivore species with an overall prevalence of 91.9 %. CPV was detected in all sampled years and seasons in Canis lupus, supporting its enzootic nature. CDV RNA was mainly detected in the Canidae family, with viral nucleic acid recorded between 2005 and 2008 with a peak prevalence of 67 % among the wolf population, followed by a sharp decline, suggesting an epizootic behaviour of the virus. Antibodies show that mustelids and viverrids were often exposed to CDV. CCoV was first recorded by molecular methods in wolf samples in 2002, remaining in the wolf populations with marked fluctuations over time. The dual serological and molecular approach provided important epidemiological data on pathogens of wild carnivores in Portugal. These programmes should also include monitoring of other potential reservoir hosts such as domestic cats and dogs.

Canine Parvovirus (CPV) causes severe morbidity and mortality in dogs, particularly puppies, worldwide. Although vaccination is highly efficacious in preventing disease, cases continue to occur and vaccination failures are well documented. Maternally derived antibody interference is the leading cause of vaccination failure and age at vaccine administration is a significant risk factor for failure. However, no studies have been performed on practicing veterinarians' usage of and compliance with published vaccination guidelines and label recommendations. Likewise, there are no published studies of veterinarian perceptions on CPV occurrence and mortality and its influence on case outcome. We report a study in which all Australian small companion animal (canine and feline) veterinary hospitals were surveyed, yielding a response rate of 23.5% (534 unique veterinary hospitals). Respondents overall perceived national CPV occurrence ten-times lower (median 2000 cases) than the estimated national caseload (20,000 cases). Respondents from hospitals that did not diagnose CPV perceived national occurrence twenty-times lower (median 1000 cases) than the estimated rate (p 0.0001). Perceived disease mortality (50%) was 2.74 times higher than that reported (18.2%). In addition, 26.7% of veterinarians reported using serological titer testing to some degree, which some practitioners use in lieu of vaccination if a titer is perceived to reflect sufficient immunity. Based on this study veterinarians appear to be aware of the disease risk in their region but unaware of the burden of CPV disease nationally, and perceive mortality risk higher than it actually is. This might lead to an overestimation of cost to treat, and over-recommendation of euthanasia. Nearly half (48.7%) of respondents recommended final puppy vaccination earlier than guidelines recommend, while 2.8% of respondents recommended a puppy re-vaccination interval longer than supported by vaccine labels and guidelines. Both of these practices may put puppies at risk of CPV infection.

Canine parvovirus (CPV) has emerged as an acute pathogen of young canine causing haemorrhagic enteritis and myocarditis. It is widely distributed and underreported in India. Therefore the study was conducted to type the CPV circulating in western Maharashtra. The faecal samples (n = 150) from clinically ill dogs showing diarrhoea and vomition were collected and subjected to haemagglutination (HA) with porcine RBC's. The DNA was extracted from the samples showing HA titres above 64 and subjected for amplification of VP2 gene fragment by PCR. The amplicons were subjected for restriction fragment length polymorphism (RFLP), sequencing and BEAST phylogenetic analysis. The results revealed 6% positivity by PCR. The RFLP results indicated single cleavage site for ApaLI and HinfI with an exception of two sites for HinfI. The nucleotide sequences showed nonfunctional nucleotide changes at different locations. The sequence analysis indicated that the nucleotide divergence within isolates under study was 0.00–0.42%, while the nucleotide homology was 99.58–100%. The most recent common ancestor was determined by molecular clock analysis using Bayesian methods. The sequence and phylogenetic analysis suggested the isolates as CPV-2a and KATN1 (KU866391, 2014) isolate from Tamilnadu, India as time to most recent common ancestor (TMRCA). The results revealed the circulating CPV in canines from western India as CPV2a genotype.

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infectionand analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPVpositive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belongedto the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since2008.It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas thismutation was absent in the four CPV2a strains reported herein.Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples,it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previouslocal CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reportedUruguayan CPV2a strains.The results obtained in this work, together with those reported previously in Uruguay strongly suggestthat, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathwaysin each country, resulting in the prevalence of different strains in related dog populations.Further extensive epidemiological studies are needed in order to improve the understanding of CPVevolution. Fil: Gallo Calderon, Marina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina Fil: Romanutti, Carina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina Fil: Wilda, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina Fil: D'antuono, Alejandra Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina Fil: Keller, Leticia. Fundación de Estudios en Virología Animal; Argentina Fil: Giacomodonato, Mónica Nancy. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Mattion, Nora Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina Fil: la Torre, Jose Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina

Canine parvovirus 2 (CPV-2) is a well-recognized cause of acute haemorrhagic enteritis in dogs worldwide. The aim of the current study was to identify which CPV-2 subtypes circulate among dogs in New Zealand, and to investigate the evolutionary patterns of contemporary CPV-2 viruses. Faecal samples were collected from 79 dogs with suspected CPV-2 infection over the period of 13 months, and tested for the presence of CPV-2 DNA by PCR. Of 70 positive samples, 69 were subtyped as CPV-2a and one as CPV-2. A majority of CPV-2 positive samples were collected from unvaccinated or not-fully vaccinated puppies ≤6 months of age. The haplotype network produced from New Zealand CPV-2 sequences showed no structure when assessed based on location, vaccination status or age of the animals sampled. International haplotype network indicated that, unlike CPV-2 from other countries, the population of CPV-2 in New Zealand appeared to be monophyletic.

Highlights • A novel RT-LAMP method was developed to detect canine distemper virus (CDV). • A set of four primers were designed to target the H gene for the specific detection of wild-type CDV variants. • The assay was 100-fold more sensitive than conventional RT-PCR. • The system showed a preference for wild-type CDV, and exhibited less sensitivity to CPV., Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers—two internal and two external—were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65 °C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10−1 TCID50 ml−1. The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

False negative faecal canine parvovirus (CPV) antigen ELISA results in dogs with CPV infection are common, but the factors that lead to these false negative results are still unknown. The aim of this study was to investigate whether dogs with a false negative faecal CPV antigen ELISA result have milder clinical signs and laboratory changes, a lower faecal virus load, higher faecal and serum CPV antibody titres and a faster recovery than dogs with a positive result. Eighty dogs with CPV infection, confirmed by the presence of clinical signs and a positive faecal CPV polymerase chain reaction (PCR), were assigned to two groups according to their faecal antigen ELISA result. Time until presentation, severity of symptoms, laboratory parameters, faecal virus load, faecal and serum antibody titres, and CPV sequencing data were compared between both groups. In 38/80 dogs that were hospitalised until recovery, the time to recovery, mortality, and the course of the disease were compared between dogs with positive and negative faecal antigen ELISA results. Of the 80 dogs included, 41 (51.3%) had a false negative faecal antigen ELISA result. ELISA-negative dogs had a significantly shorter time until presentation, lower frequency of defaecation, lower faecal virus load, and higher serum antibody concentrations than ELISA-positive dogs. Laboratory changes, CPV shedding, and outcomes were not associated with faecal antigen ELISA results. In conclusion, low faecal CPV load and antibodies binding to CPV antigen in faeces are likely to be important reasons for false negative faecal antigen ELISA results. Dogs with clinical signs of CPV infection should be retested by faecal PCR.

Highlights • We performed a search for canine coronavirus in fecal samples and organs from dogs. • Pantropic canine coronavirus was found in different organs. • Similar lesions to those found in canine parvovirus were observed. • This study reinforces the need for differential diagnosis between canine parvovirus and canine coronavirus., Characterization of canine coronavirus (CCoV) strains currently in circulation is essential for understanding viral evolution. The aim of this study was to determine the presence of pantropic CCoV type IIa in tissue samples from five puppies that died in Southern Brazil as a result of severe gastroenteritis. Reverse-transcriptase PCR was used to generate amplicons for sequence analysis. Phylogenetic analysis of the CCoV-IIa strains indicated that they were similar to those found in other countries, suggesting a common ancestor of these Brazilian isolates. This is the first report of pantropic CCoV-II in puppies from Latin America and our findings highlight that CCoV should be included as a differential diagnosis when dogs present with clinical signs and lesions typically seen with canine parvovirus infection.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been developed to detect and differentiate between canine parvovirus (CPV) and mink enteritis virus (MEV). Eight CPV and three MEV epidemic strains isolated from 28 pathological samples from dogs and minks suspected of being infected with parvovirus were amplified by PCR using a pair of specific primers designed based on the CPV-N strain (M19296). PCR amplified a fragment of 1016bp from the genomic DNA of both MEV and CPV. The MEV-derived fragment could be digested with the restriction enzyme BSP1407I into three fragments of 102bp, 312bp and 602bp, while the fragment amplified from the CPV genomic DNA was digested into only two fragments of 414bp and 602bp. The lowest DNA concentration of CPV and MEV that could be detected using this assay was 0.004μg/ml and 0.03μg/ml, respectively. The PCR-RFLP assay developed in the present study can, therefore, be used to detect and differentiate MEV from CPV with high specificity and sensitivity.

Canine parvovirus (CPV) infection is a common and severe disease particularly affecting young dogs. The paramunity inducer PIND-ORF is reported to stimulate the innate immune system and, if used as a supplementary medication, might lead to a more rapid improvement in clinical signs in dogs with CPV infection. The aim of this study was to evaluate the efficacy of PIND-ORF in dogs with CPV infection in a prospective, placebo-controlled, double-blinded trial using 38 dogs randomly assigned to two groups. Inclusion criteria were clinical signs consistent with CPV infection and a positive faecal CPV PCR. Dogs received either PIND-ORF ( n = 20) or placebo ( n = 18) and additional symptomatic treatment. Time to recovery and mortality rate were compared between the two groups. Clinical signs, complete blood counts (CBC), and serum protein and albumin concentrations were evaluated daily during hospitalisation and on day 14. Viral shedding and antibody titres were measured by faecal CPV PCR and serum neutralisation assay. There was no significant difference in time to recovery, clinical signs, blood parameters, duration of virus shedding, and antibody titres between the two groups. The only significant difference was an increase in lymphocyte counts and antibody titres observed in the PIND-ORF group only. Three dogs receiving placebo did not survive, but the mortality rate was not significantly different between groups ( P = 0.097). No significant effect of PIND-ORF on recovery and outcome could be demonstrated.