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.     

The use of enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

Complex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-, CCV- and rotavirus antigens in fecal samples were also developed. Both the serological and antigen-detection ELISAs were used to screen samples from dogs in The Netherlands, with or without a history of acute diarrhea. It was shown that the results of the respective serological ELISAs correlated well and that CPV was the major cause of virus-induced acute diarrhea in dogs in The Netherlands.     

广州地区犬细小病毒VP2基因的序列分析

犬细小病毒病是一种高度接触性传染病,临床上以急性出血性肠炎和心肌炎为特征。为研究广州地区犬细小病毒病的流行亚型,本试验采集2011—2012年间疑似病犬粪便,进行犬细小病毒（canine parvovirus,CPV）分离鉴定,提取病毒基因组进行VP2基因扩增和序列分析,与GenBank中登录的参考毒株进行比对,结果发现,分离的8株CPV中SCAU-5为CPV-2b 亚型,其余为CPV-2a亚型。结果表明,2011—2012年间广州地区的流行毒株以CPV-2a亚型为主。     

Genomic Typing of Canine Parvovirus Circulating in the State of Rio de Janeiro,Brazil from 1995 to 2001 Using Polymerase Chain Reaction Assay

In this study, the genomic types of canine parvovirus (CPV) circulating in the State of Rio de Janeiro, Brazil, from 1995 to 2001, were investigated using the polymerase chain reaction assay (PCR). A total of 78 faecal samples from gastroenteritic puppies, confirmed as positive for canine parvovirus by haemagglutination/haemagglutination inhibition tests or virus isolation in cell culture (MDCK), were examined. The viral DNA was extracted from faecal samples using a combination of phenol– chloroform and silica–guanidine thiocyanate methods. PCR was carried out with differential pairs of primers to distinguish the old (CPV-2) and new types of virus (CPv-2a or CPV-2b). Specific amplicons were observed for all samples using the primer pair P2ab, which detects CPV-2a and CPV-2b. Seventy-six from a total of 78 samples (97%) were considered as CPV-2b because of their reaction with the primer pair P2b. Thirty samples (30/78) were from previously vaccinated puppies and in 15 of them the enteritis symptoms began from 1 to 12 days after vaccination. PCR confirmed the infection by wild virus (CPV-2b) in 5 of these 15 puppies who had received old-type vaccines. Our results show that CPV-2b was the prevalent type circulating in the State of Rio de Janeiro from 1995 to 2001.     

ELISA法检测犬腹泻粪样中的犬冠状病毒

用ＦＥ细胞增殖犬冠状病毒（ＣＣＶ）参考株，分别免疫家兔和ＢＡＬＢ／ｃ小鼠制备ＣＣＶ多抗和单抗，建立了夹心ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ诊断方法。在检测的８４例犬腹泻粪样中，多抗、单抗夹心法显示ＣＣＶ阳性１６例，Ｄｏｔ－ＥＬＩＳＡ阳性１３例，后１３例包括在前１６例中。从８４例腹泻犬粪样中随机取３８例作ＣＣＶ、犬细小病毒（ＣＰＶ）双项检测，ＣＣＶ阳性１６例，ＣＰＶ阳性６例，ＣＣＶ、ＣＰＶ混合感染４例。结果显示，在南京地区流行的犬腹泻中，ＣＣＶ感染比例有超过ＣＰＶ的趋势。     

Recent epidemiological status of canine viral enteric infections and Giardia infection in Japan.

Epidemiology of canine enteric infections was studied. Rectal swabs collected from 95 dogs presented at animal hospitals during a period from January to June of 2000 were examined for enteric pathogens, including viruses and Giardia lamblia (G. lamblia). Most frequently detected in both diarrheal and normal feces were canine coronavirus (55.4%) and G. lamblia (48.2%). Canine parvovirus type 2 (CPV-2) was specifically associated with diarrheal cases and CPV-2b was the predominant antigenic type. Although canine rotavirus, canine adenovirus, and canine distemper virus were also detected in a small number of diarrheal cases, no evidence for calicivirus infection was obtained.     

犬细小病毒四川株的分离与鉴定

为更好地了解我国细小病毒流行情况,笔者从四川农大动物医院采集5份患出血性肠炎犬的粪便,通过猫肾细胞(F81)传代培养后,成功分离到两株犬细小病毒。对两株病毒进行病毒形态学、理化学、血凝试验、PCR鉴定与分子生物学系统鉴定后,证实分离株是犬细小病毒,通过VP2结构蛋白测序分析表明,新分离到的两株细小病毒抗原型分别属于CPV-2a和CPV-2b。     

一株新分离犬细小病毒灭活疫苗的制备及免疫效果研究

JL0911株犬细小病毒是由军事兽医研究所流行病与病毒病防控技术实验室分离得到的一株新型犬细小病毒,其主要抗原位点上的氨基酸序列较国内先前分离到的其他株有较大差异,不能被归入目前已有的CPV-2a、CPV-2b、CPV-2c三个类型。本研究旨在利用JL0911株犬细小病毒研制灭活疫苗,并评价其免疫原性。试验采用10^5.5 TCID₅₀/mL的犬细小病毒JL0911,以甲醛灭活后,加入1/4体积的纳米佐剂,制备了犬细小病毒灭活疫苗。取1.5 mL上述疫苗,通过肌肉注射免疫普通家犬,免疫前后不同时间均采集血清,在F81细胞系上测定犬细小病毒的中和抗体。结果显示,免疫后14 d,试验犬血中细小病毒中和抗体效价较免疫前有显著提高,最高可由0提高至2⁹,表明本试验分离的JL0911株犬细小病毒具有生产犬细小病毒灭活疫苗的潜在价值。     

犬细小病毒四川株的分离与鉴定

采集5份患出血性肠炎犬的粪便,通过猫肾细胞(F81)传代培养后,成功分离到2株病毒。对2株病毒进行病毒形态学、理化学、血凝试验、PCR鉴定与分子生物学系统鉴定后,证实分离株是犬细小病毒,通过VP2结构蛋白测序分析表明,新分离到的2株细小病毒抗原型分别属于CPV-2a和CPV-2b。     

犬细小病毒病防控技术研究进展

感染犬细小病毒会导致典型的急性胃肠炎症状,包括呕吐、发热、白细胞显著减少和出血性腹泻等,常与犬瘟热、犬冠状病毒等混合感染。尽管相关疫苗进行了广泛接种,但其仍然是导致犬死亡的主要原因,幼犬死亡率常大于70 %。本文将介绍目前诊断CPV-2的主要方法及近年来取得的进展,并提供最新关于CPV-2疫苗类型和接种方案的信息,同时分析免疫失败的原因,对其防控技术进展进行综合描述。     

Validation of a fecal scoring scale in puppies during the weaning period

In puppies weaning is a high risk period. Fecal changes are frequent and can be signs of infection by digestive pathogens (bacteria, viruses, parasites) and indicators of nutritional and environmental stress. The aim of this study was to define a pathological fecal score for weaning puppies, and to study the impact on that score of two intestinal viruses (canine parvovirus type 2 and canine coronavirus). For this, the quality of stools was evaluated on 154 puppies between 4 and 8weeks of age (100 from small breeds and 54 from large breeds). The scoring was performed immediately after a spontaneous defecation based on a 13-point scale (from 1; liquid to 13; dry and hard feces). Fecal samples were frozen for further viral analysis. Each puppy was weighed once a week during the study period. The fecal score regarded as pathological was the highest score associated with a significant reduction in average daily gain (ADG). Fecal samples were checked by semi-quantitative PCR or RT-PCR for canine parvovirus type 2 and canine coronavirus identification, respectively. The quality of feces was affected by both age and breed size. In small breeds, the ADG was significantly reduced under a fecal score of 6 and 7 for puppies at 4-5 and 6-8weeks of age, respectively. In large breeds, the ADG was significantly reduced under a fecal score of 5 whatever the age of the puppy. Whereas a high viral load of canine parvovirus type 2 significantly impacted feces quality, no effect was recorded for canine coronavirus. This study provides an objective threshold for evaluation of fecal quality in weaning puppies. It also emphasizes the importance to be given to age and breed size in that evaluation.     

Do canine parvoviruses affect canine neurons? An immunohistochemical study

In cats (most of which died from panleukopenia), cerebral neurons have recently been shown to be susceptible to canine parvovirus infection. In addition to positive immunostaining and distinct in situ hybridization signals, signs of neurodegeneration were identified by histopathology, mainly in the diencephalic area. Similar histological lesions of the diencephalic regions in dogs have also attracted attention; therefore, an immunohistochemical study was initiated to determine the possible infection of canine neurons with canine parvoviruses. The study was carried out on formalin-fixed and paraffin-embedded brain tissue, with and without signs of neurodegeneration, from 40 dogs, most of them dying from parvovirus enteritis. Immunohistochemistry, using polyclonal antiserum against canine parvoviruses, was negative in all 40 cases, suggesting that, unlike cats, canine parvoviruses do not seem capable of infecting canine neurons.     

Do two current canine parvovirus type 2 and 2b vaccines provide protection against the new type 2c variant?

Three groups (n=9 or 10) of 12-week-old canine parvovirus type 2 (CPV-2) antibody-negative puppies were vaccinated: one group with a product containing modified-live CPV-2b (Galaxy DA2PPv; Schering-Plough Animal Health), one group with a product containing modified-live CPV-2 (Continuum DAP, Intervet), and one group (controls) with sterile saline. All puppies receiving CPV-2 and CPV-2b vaccines developed antibody as determined by the hemagglutination inhibition assay. All groups of puppies were challenged with a combination of virulent CPV-2b and CPV-2c 5 weeks after vaccination. All puppies in the CPV-2 and CPV-2b vaccinated groups were protected from disease, whereas all control group puppies developed disease and 50% died or were euthanized. This study demonstrated that the CPV-2 and CPV-2b vaccine components of the Continuum DAP and Galaxy DA2PPv products, respectively, provided protection against the CPV-2b virus and also provided complete protection against the new CPV-2c variant.     

Detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis

A new monoclonal antibody (mAb), CCV2-2, was compared with the widely used FIPV3-70 mAb, both directed against canine coronavirus (CCoV), as a diagnostic and research tool. Western blot showed that both anti-CCoV mAbs only reacted with a protein of 50 kD, a weight consistent with the feline coronavirus (FCoV) viral nucleocapsid. A competitive inhibition enzyme-linked immunosorbent assay showed that the 2 recognized epitopes are distinct. Preincubation of CCV2-2 mAb with FCoV antigen suppressed the immunostaining. Formalin-fixed, paraffin-embedded sections from brains of 15 cats with the dry form of feline infectious peritonitis (FIP) were examined by immunohistochemistry. Immunohistochemistry was performed with both anti-CCoV mAbs, either on consecutive or on the same sections. A myeloid-histiocytic marker, MAC 387, was also used to identify FIP virus-infected cells. In all regions where MAC 387-positive cells were present, positive staining with the CCV2-2 mAb was systematically detected, except at some levels in 1 cat. In contrast, none or only a few cells were positive for the FIPV3-70 mAb. Double immunostaining showed macrophages that were immunopositive for either CCV2-2 alone or alternatively for CCV2-2 and FIPV3-70 mAbs. This reveals the coexistence of 2 cohorts of phagocytes whose FIP viral contents differed by the presence or absence of the FIPV3-70-recognized epitope. These findings provide evidence for antigenic heterogeneity in coronavirus nucleocapsid protein in FIP lesions, a result that is in line with molecular observations. In addition, we provide for the first time morphologic depiction of viral variants distribution in these lesions.     

Duodenal perforation in a puppy with canine parvovirus infection

A 2-month-old puppy was brought to a veterinary hospital with diarrhea, vomiting, and anorexia. The test for canine parvovirus was positive, and she was hospitalized for supportive care. Her gastrointestinal symptoms initially improved; however, vomiting and lethargy developed again in the second week of hospitalization. Abdominal ultrasonography results were suspicious of a duodenal perforation. Cytology of the abdominal effusion confirmed septic peritonitis; therefore, emergency exploratory laparotomy was performed. The surgery was successful, and the puppy recovered fully. When symptoms recur or deteriorate in patients with parvoviral infection, surgically curable complications may be disregarded if supportive therapy is continued without additional investigative examinations. This report highlights the usefulness of abdominal ultrasound in conjunction with fluid cytology to identify subsequent complications when the clinical signs of parvovirus deteriorate.Key clinical message:This case report demonstrates duodenal perforation as a complication of parvoviral infection. Abdominal ultrasonography and peritoneal fluid cytology can be crucial for the early recognition of intestinal complications requiring immediate successful perioperative treatment.     

5种常见犬腹泻病毒基因芯片检测方法的建立

为建立一种简便、快速、高效的可同时区分犬腺病毒1型（canine adenovirus type 1,CAV-1）、犬腺病毒2型（canine adenovirus type 2,CAV-2）、犬冠状病毒（canine coronavirus,CCV）、犬瘟热病毒（canine distemper virus,CDV）、犬细小病毒（canine parvovirus,CPV） 5种常见犬腹泻病毒的基因芯片诊断方法,本研究以CAV-1、CAV-2、CCV、CDV、CPV 5种犬腹泻病毒为靶病毒,根据NCBI上收录的病毒基因序列在其保守区域内设计引物,在此基础上根据变异区域设计针对每种病毒的探针2~3条,优化检测体系的各反应条件,确定该检测方法的特异性与敏感性,建立可同时区分5种病毒的基因芯片检测方法。结果显示,建立的基因芯片检测方法可同时检测以上述5种犬腹泻病毒,其中PCR的退火温度为55℃、延伸时间为1 min 15 s;探针与PCR产物的杂交温度40℃、杂交时间2.5 h时,该方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒标准品的检测限分别为0.2 fg/μL、2 fg/μL、2 fg/μL、20 pg/μL和0.02 fg/μL,具有较高的灵敏性;同时对犬副流感病毒进行特异性试验,发现无阳性信号出现,具有较强的特异性;对14份临床腹泻样品检测结果显示,基因芯片方法对CAV-1、CAV-2、CCV、CDV和CPV 5种病毒的阳性检出率分别为28.57%、50.00%、64.28%、14.28%和85.71%,并且基因芯片检测方法的敏感性较PCR要高10~100倍。以上结果表明,本研究建立的基因芯片检测方法具有特异、敏感等特点,对临床中犬类混合感染病毒检测具有一定的诊断意义。     

Oxidative stress indices in gastroenteritis in dogs with canine parvoviral infection

Gastroenteritis of viral origin has emerged as a major cause of morbidity and mortality in dogs during the last two decades. Amongst the viral etiologies responsible for gastroenteritis in dogs, canine parvovirus (CPV) is considered as the most pathogenic. The disease is characterized by hemorrhagic enteritis, bloody diarrhoea and myocarditis in young pups. The present study was carried out to examine alterations in oxidative stress indices in the erythrocytes from dogs suffering from gastroenteritis with or without canine parvoviral infection as confirmed by CPV-DNA amplification from faeces using specific primers for CPV-2 as well as CPV-2a and CPV-2b variants by polymerase chain reaction (PCR). The present investigation utilized clinical cases of dogs with signs of acute diarrhea (n = 56), and 14 more apparently healthy dogs of similar age group. Erythrocytic oxidative stress indices such as lipid peroxides level and antioxidant enzymes like superoxide dismutase and catalase activity, and blood micro-mineral (iron, copper, cobalt and zinc) status were analyzed in each dog (n = 70). The acute cases of gastroenteritis in dogs were associated with altered erythrocytic lipid peroxidation as evident by estimation of malonaldehyde (MDA) concentration. The activities of antioxidant enzymes catalase and superoxide dismutase, the first line of antioxidant defense against damaging effects of free radicals, were also altered. The alterations in oxidative stress indices were more pronounced in cases with involvement of canine parvovirus as compared to parvo-negative cases. Our results also revealed decreased blood zinc level in diarrhoea in dogs irrespective of involvement of canine parvovirus.     

Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan

To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p < 0.0001 and p = 0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date.     

Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats

ABSTRACT: Parvoviruses of carnivores include three closely related autonomous parvoviruses: canine parvovirus (CPV), feline panleukopenia virus (FPV) and mink enteritis virus (MEV). These viruses cause a variety of serious diseases, especially in young patients, since they have a remarkable predilection for replication in rapidly dividing cells. FPV is not the only parvovirus species which infects cats; in addition to MEV, the new variants of canine parvovirus, CPV-2a, 2b and 2c have also penetrated the feline host-range, and they are able to infect and replicate in cats, causing diseases indistinguishable from feline panleukopenia. Furthermore, as cats are susceptible to both CPV-2 and FPV viruses, superinfection and co-infection with multiple parvovirus strains may occur, potentially facilitating recombination and high genetic heterogeneity. In the light of the importance of cats as a potential source of genetic diversity for parvoviruses and, since feline panleukopenia virus has re-emerged as a major cause of mortality in felines, the present study has explored the molecular characteristics of parvovirus strains circulating in cat populations. The most significant findings reported in this study were (a) the detection of mixed infection FPV/CPV with the presence of one parvovirus variant which is a true intermediate between FPV/CPV and (b) the quasispecies cloud size of one CPV sample variant 2c. In conclusion, this study provides new important results about the evolutionary dynamics of CPV infections in cats, showing that CPV has presumably started a new process of readaptation in feline hosts.