Canine parvovirus--a review of epidemiological and diagnostic aspects, with emphasis on type 2c

Canine parvovirus type 2 (CPV-2) emerged in late 1970s causing severe epizootics in kennels and dog shelters worldwide. Soon after its emergence, CPV-2 underwent genetic evolution giving rise consecutively to two antigenic variants, CPV-2a and CPV-2b that replaced progressively the original type. In 2000, a new antigenic variant, CPV-2c, was detected in Italy and rapidly spread to several countries. In comparison to the original type CPV-2, the antigenic variants display increased pathogenicity in dogs and extended host range, being able to infect and cause disease in cats. Epidemiological survey indicate that the newest type CPV-2c is becoming prevalent in different geographic regions and is often associated to severe disease in adult dogs and also in dogs that have completed the vaccination protocols. However, the primary cause of failure of CPV vaccination is interference by maternally derived immunity. Diagnosis of CPV infection by traditional methods has been shown to be poorly sensitive, especially in the late stages of infections. New diagnostic approaches based on molecular methods have been developed for sensitive detection of CPV in clinical samples and rapid characterisation of the viral type. Continuous surveillance will help assess whether there is a real need to update currently available vaccines and diagnostic tests.     

First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain

Canine parvovirus type 2 (CPV-2), the aetiological agent of haemorrhagic enteritis in dogs, includes three antigenic variants, types 2a, 2b and 2c. CPV-2c has been detected initially in Italy and subsequently in Vietnam. We report the first identification of this novel antigenic variant in Spain, where it caused an outbreak of fatal enteritis in basset hound pups in association with canine coronavirus type I and type II. We suggest that this new antigenic variant of CPV-2 could spread throughout Europe and that there is a subsequent need to update current CPV vaccines.     

Chronological analysis of canine parvovirus type 2 isolates in Japan

Fifty-five canine parvovirus type 2 (CPV) samples, 12 fecal specimens and 43 cell culture isolates, were examined for their genetic characteristics of VP2 gene. They were collected from the diseased dogs at various districts of Japan during 27 years from 1980 to 2006. A fragment of VP2 gene was analyzed by restriction fragment length polymorphism assay and DNA sequencing. The original antigenic type 2 of CPV (CPV-2) was no longer found in the samples since 1984, and two antigenic variants CPV-2a and CPV-2b replaced CPV-2 as predominant types for about 5 years from 1982. A new genetic variant of prototype CPV-2a with non-synonymous substitution at the VP2 amino acid residue 297 from Ser to Ala was first detected in 1987. New CPV-2b with the same amino acid substitution at position 297 as new CPV-2a was also detected from the samples collected in 1997. Since then new CPV-2b has been the predominant CPV over the field of Japan. Several additional amino acid substitutions were detected in the VP2 gene of some recent CPV strains. Neither CPV-2c(a), CPV-2c(b), nor "Glu-426" of the antigenic variants previously found outside the country was detected in any samples tested. Reactivity of new CPV-2a and 2b variants against antibodies produced by the current vaccine products was determined by a cross hemagglutination-inhibition test. The recent field CPV isolates reacted more efficiently to the antibodies produced in dogs vaccinated with the new CPV-2b vaccine strain than the conventional CPV-2 vaccine strain.     

Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India

Canine parvovirus 2 (CPV-2) causes a highly contagious and often fatal disease in dogs. Since its sudden emergence in the early 1970s, CPV-2 has been evolving through the generation of novel genetic and antigenic variants (CPV-2a/b/c) that are unevenly distributed throughout the world. In the present study we have examined 36 clinical cases of dogs suspected of CPV collected during year 2006. A fragment of the VP2 gene of the virus was analyzed using polymerase chain reaction (PCR), restriction endonuclease (RE) and DNA sequence analysis. Out of the 36 samples analyzed, 16 were found positive for CPV-2a/2b by conventional PCR. DNA sequencing was done for 6 PCR positive samples, out of which three were characterized as CPV-2c, indicating that this CPV type 2c is currently circulating in India.     

Recent spreading of a divergent canine parvovirus type 2a (CPV-2a) strain in a CPV-2c homogenous population

Canine parvovirus type 2 (CPV-2), which causes acute hemorrhagic enteritis in dogs, is comprised of three antigenic variants (2a, 2b, and 2c) that are distributed worldwide with different frequencies. Variant prevalence was analyzed in 150 CPV-2-positive samples collected from the Uruguayan dog population in 2007-2010. Samples were analyzed with polymerase chain reaction, restriction fragment length polymorphism, and sequencing of the coding region for the largest and most variable loop of the VP2 capsid protein. CPV-2c was the only strain detected from 2007 to 2009. Uruguayan CPV-2c showed high homogeneity in both nucleotide and amino acid sequences, indicating a low level of genetic variability. In 2010, an unexpected epidemiological change occurred in Uruguay as a consequence of the appearance of a novel CPV-2a strain. This variant rapidly spread through the Uruguayan dog population and was detected in 20 of the 52 cases (38%) analyzed in 2010. CPV-2a sequences were identical in all field viruses analyzed, and in addition to the characteristic 426Asn residue, the sequences showed amino acid substitutions (267Tyr, 324Ile, and 440Ala) not observed in the Uruguayan CPV-2c. These data and the first detection in April 2010 suggest that the CPV-2a variant recently emerged in Uruguay and underwent clonal expansion. This observation is the first case in which a CPV-2a variant increased its frequency in a dog population where CPV-2c was prevalent. Our results emphasize the dynamic changes in CPV variants and highlight the importance of ongoing surveillance programs to provide a better understanding of virus epidemiology.     

Vaccination with canine parvovirus type 2 (CPV-2) protects against challenge with virulent CPV-2b and CPV-2c

Mutations in canine parvovirus (CPV) field isolates have created concerns regarding the ability of vaccines containing CPV-2 to protect against infection with the newly identified antigenic types CPV-2b and CPV-2c. To address this concern, the efficacy of CPV-2 strain NL-35-D currently in use as a commercial vaccine was demonstrated against an oral challenge with CPV-2b and CPV-2c, respectively. Clinically healthy specific pathogen free Beagle dogs were either vaccinated or treated with water for injection first at 8-9 weeks of age and again at 11-12 weeks of age. All dogs were challenged either with CPV-2b or CPV-2c three weeks after the second vaccination. During the two week period following challenge, clinical signs, white blood cell counts, serology by haemagglutination inhibition (HI) and serum neutralisation tests, and virus shedding by haemagglutination test were assessed. All control dogs developed clinical signs of parvovirosis (including pyrexia and leucopenia) and shed virus. Vaccinated dogs seroconverted (HI titres > or =80), remained healthy throughout the study and shed more than 100 times less virus than controls. In conclusion, vaccination with the low passage, high titre CPV-2 strain NL-35-D cross-protects dogs against virulent challenges with CPV-2b or CPV-2c by preventing disease and substantially reducing viral shedding.     

First detection of canine parvovirus type 2c in South America

Since its sudden emergence in the early 1970s, canine parvovirus type-2 (CPV-2) has been evolving through the generation of novel genetic and antigenic variants (CPV-2a/b/c and a number of additional mutations) that are unevenly distributed throughout the world. In order to develop strategies to control the spread of these variants and to understand virus evolution is fundamental to genotype field isolates from different geographic locations. In the present paper we have examined 25 isolates of CPV from clinical samples of Uruguayan dogs collected during year 2006. A fragment of the VP2 gene of the virus was analyzed using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and DNA sequence analysis. Out of the 25 isolates analyzed, only one was characterized as CPV-2a and 24 were characterized as CPV-2c, indicating that this type is currently the prevalent field CPV circulating in Uruguay. This is the first report of CPV-2c in the American continent and it also represents the highest frequency of this type observed in a dog population so far. Its presence in South American supports the assumption that CPV-2c is reaching a worldwide distribution as occurred with 2a/2b antigenic types.     

Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay

Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.     

New approaches for the molecular characterization of canine parvovirus type 2 strains

Characterization of the canine parvovirus type 2 (CPV-2) is sometimes ambiguous, frequently requiring more than one technique for definitive prediction of the viral type. Taking into account the single-nucleotide polymorphisms encountered in the VP2-protein gene between types 2a and 2b and between type 2b and Glu-426 mutant (type 2c), two different minor groove binder (MGB) probe assays were developed for rapid identification of the CPV-2 variants. A total of 315 samples collected from dogs with diarrhoea were screened for CPV-2 by a real-time polymerase chain reaction (PCR) assay capable of detecting all CPV-2 types. In order to compare the type-specific assays with the traditional techniques [haemagglutination inhibition with monoclonal antibodies, PCR-restriction fragment-length polymorphism (RFLP), sequence analysis] for prediction of CPV-2 antigen specificity, the 203 samples tested CPV-2 positive were analysed using the different methods. The results showed a 100% concordance between the MGB probe assays and the combined conventional methods, with 116 samples characterized as type 2a, 32 as type 2b and 55 as type 2c. Therefore, the MGB probe assays represent a quick, reliable tool for prediction of CPV-2 antigen specificity, with regard to the more time-consuming assays currently used.     

Occurrence of canine parvovirus type 2c in the United States.

Canine parvovirus (CPV) type 2 (CPV-2) emerged around 1978 as a major pathogen of dogs worldwide. In the mid-1980s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant of CPV (named CPV-2c) was detected in Italy and now cocirculates with types 2a and 2b in that country. The CPV-2c has also been reported from single outbreaks in Vietnam and Spain. This study was conducted to determine if CPV-2c occurs in the United States. Thirty-three fecal samples were collected from dogs in 16 states between April 2006 and April 2007 and were tested for CPV using real-time polymerase chain reaction (PCR). Positive samples were further tested using conventional PCR and minor-groove binding TaqMan PCR assays to determine the viral type and to differentiate vaccine strains from field strains. Twenty-seven samples were positive for CPV, 7 of which were CPV-2c from 5 states: Arizona, California, Georgia, Oklahoma, and Texas. Of the 7 isolates, 4 differed from European CPV-2c isolates by 2 additional single-nucleotide mutations at positions 4076 and 4104, the latter of which produces a ThrAla change at residue 440 located near a major antigenic site. The coast-to-coast geographic distribution of the states in which CPV-2c was detected strongly suggests that this new CPV variant is probably widespread in the United States. The continuous evolution of CPV requires that monoclonal antibody-based and nucleic acid-based diagnostic assays should be periodically checked for sensitivity on prevalent CPV strains.     

Canine parvovirus 2c infection in central Portugal

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.     

Genetic analysis of canine parvovirus from dogs in Australia

OBJECTIVE: To determine the genetic variants of canine parvovirus-2 (CPV) present in domestic dogs in Australia and to investigate 26 cases of apparent vaccine failure. DESIGN: Thirty-three samples of faeces or intestinal tissues and 16 cell culture virus isolates collected over a period from 1980 to 2005 from five Australian states were analysed. Procedure DNA was extracted from the samples and a 1975 bp fragment of the VP1/2 gene of CPV was amplified by polymerase chain reaction (PCR) and sequenced. Sequences were compared to published strains of CPV-2, CPV-2a, CPV-2b and CPV-2c. RESULTS: Forty-one of 43 PCR-positive samples contained CPV-2a viruses. One sample collected in 2002 from a pup in northern NSW contained a CPV-2b virus. One sample that had been included in the study as a CPV-antigen negative control sample contained a CPV-2 virus. CONCLUSION: CPV-2a remains the predominant genetic variant of CPV in dogs in Australia and has not been replaced by CPV-2b or CPV-2c as in many other countries. The vaccine failures investigated in the study were likely caused not by genetic variation of field viruses but by maternal antibody interference in the response of pups to vaccination.     

The pathogenicity of canine parvovirus type-2b, FP84 strain isolated from a domestic cat, in domestic cats

Canine parvovirus type-2a (CPV-2a) and type-2b (CPV-2b) have recently been isolated from domestic cats. The pathogenicity of CPV-2b in domestic cats is still unclear. In this study, we performed infection tests to examine the pathogenicity of CPV-2b, FP84 strain, isolated from a domestic cat. The results demonstrated that the CPV strain FP84 is able to infect and replicate well in domestic cats. Two of the 3 cats used in the test died. They showed loss of appetite, diarrhea, leukopenia and dehydration. Since FP84 was found to be virulent to domestic cats, it is necessary to examine the efficacy of inactivated feline panleukopenia virus vaccines against CPV infection in domestic cats.     

Antibodies to parvovirus, distemper virus and adenovirus conferred to household dogs using commercial combination vaccines containing Leptospira bacterin

To examine how the inclusion (+) or exclusion (-) of inactivated Leptospira antigens in a vaccine for canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type 2 (CAdV-2) affects antibody titres to CPV-2, CDV and CAdV-1 antigens, household dogs were vaccinated with commercially available vaccines from one of three manufacturers. CPV-2, CDV and CAdV-1 antibody titres were measured 11 to 13 months later and compared within three different age groups and three different bodyweight groups. There were significant differences between CPV-2 antibody titres in dogs vaccinated with (+) vaccine and those vaccinated with (-) vaccine for two products in the two-year-old group and for one product in the greater than seven-year-old group; no significant differences were seen that could be attributed to bodyweight. No differences in CDV antibody titres were observed within age groups, but a significant difference was seen in the 11 to 20 kg weight group for one product. Significant differences in CAdV-1 antibody titres were seen for one product in both the two-year-old group and the ≤10 kg weight group.     

兔抗犬细小病毒2a型多克隆抗体交叉保护研究

为探究犬细小病毒（canine parvovirus,CPV）血清型只有一种的条件下,常用疫苗CPV-2型和CPV-2a病毒免疫血清抗体对国内流行CPV-2a病毒的中和率,试验将藏獒源CPV-2a毒株（10⁵ TCID₅₀/100 μL）和CPV-2（10^4.5 TCID₅₀/100 μL）疫苗接于F81细胞增殖,PEG6000法浓缩制成免疫原,各免疫新西兰长白兔3只,间隔2周1次,共免疫4次。收集血清纯化制备多克隆抗体,通过血凝抑制试验（HI）和血清中和试验（SN）分别用2种抗体中和CPV-2、CPV-2a病毒,对两者保护率进行初步评价,并对本地感染CPV-2a的4只犬,每组2只进行治疗,每日跟踪白细胞消长规律。结果显示,CPV-2a多抗中和国内流行病毒CPV-2a的HI抗体、中和抗体水平都极显著高于CPV-2多抗（P<0.01）,而中和CPV-2病毒的HI抗体、中和抗体水平两者差异不显著（P>0.05）,CPV-2a多抗组治疗能较快恢复正常值,2组白细胞数有显著差异（P＜0.05）。结果表明,CPV-2a多抗与常用CPV-2型疫苗免疫抗体相比能高滴度的中和CPV-2a,更适用于中国犬细小病毒的防制,对研发新型生物制品有一定意义。     

Molecular investigation on the presence of canine parvovirus in Egypt

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.     

Passive protection of dogs against clinical disease due to Canine parvovirus-2 by specific antibody from chicken egg yolk

The protective effect of immunoglobulins derived from chicken egg yolk (IgY) against infection by Canine parvovirus 2 (CPV-2) was evaluated in 10 beagle dogs orally challenged with a strain of the virus. The 2-mo-old dogs were divided into 3 groups and treated with powders containing CPV-2 IgY or normal egg yolk for 7 d after the challenge. The 4 dogs receiving normal egg yolk (control group) demonstrated mild symptoms typical of CPV-2 infection, such as vomiting, diarrhea, and weight loss. No symptoms were observed by 16 d after challenge in the 3 dogs receiving 2 g of IgY powder. Of the 3 dogs receiving 0.5 g of IgY powder, 2 had clinical CPV-2 disease; however, the manifestations were less severe than in the control group. Furthermore, the IgY-treated groups had significantly greater weight gain and shorter duration of virus shedding than the control group. These results indicate that IgY is useful in protecting dogs from CPV-2-induced clinical disease.     

Three-year serologic immunity against canine parvovirus type 2 and canine adenovirus type 2 in dogs vaccinated with a canine combination vaccine

A group of client-owned dogs and a group of dogs at a commercial kennel were evaluated for duration of antibody responses against canine parvovirus type 2 (CPV-2) and canine adenovirus type 1 (CAV-1) after receiving a combination vaccine containing recombinant canarypox-vectored canine distemper virus (CDV) and modified-live CPV-2, CAV-2, and canine parainfluenza virus, with (C6) or without (C4) two serovars of Leptospira (Recombitek C4 or C6, Merial). Duration of antibody, which correlates with protective immunity, was found to be at least 36 months in both groups. Recombitek combination vaccines can confidently be given every 3 years with assurance of protection in immunocompetent dogs against CPV-2 and CAV-1 as well as CDV. This allows this combination vaccine, like other, similar modified- live virus combination products containing CDV, CAV-2, and CPV-2, to be administered in accordance with the recommendations of the American Animal Hospital Association Canine Vaccine Task Force.     

A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogs

We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2 (CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA quantitation over a range of eight orders of magnitude (from 10(2) to 10(9) copies of standard DNA). The reproducibility of the CPV-2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to detect low viral titers of CPV-2 in infected dogs.     

Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs

Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.