犬细小病毒和犬冠状病毒双重PCR方法的建立及应用

根据GenBank中犬细小病毒(CPV)和犬冠状病毒(CCV)基因序列各设计了一对引物,经试验条件的优化,建立了检测CPV的PCR方法和CCV的RT-PCR方法,扩增目的片段大小分别为337 bp和852 bp.在此基础上建立了检测CPV和CCV双重PCR方法.该双重PCR方法能特异的扩增CPV和CCV,且分别扩增出1...     

Molecular characterization of canine coronavirus strains circulating in Brazil

To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.     

The seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in New Zealand

Abstract

AIM: To determine whether canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV) are present in dogs in New Zealand.

METHODS: Serum samples from 251 dogs of varying age, breed and clinical histories were tested for the presence of antibodies to CRCoV and CIV, using indirect fluorescent antibody (IFA) analysis. The population sampled represented a wide geographic area but principally encompassed the central and lower North Island of New Zealand.

RESULTS: Seventy-three of the 251 samples (29%) were seropositive for CRCoV. Dogs <2 years old were less likely to be seropositive for CRCoV than older dogs. None was seropositive for CIV.

CONCLUSIONS: This study revealed the presence of antibodies to CRCoV in dogs in New Zealand. Young dogs are less likely to be seropositive than older dogs, probably due to increased opportunity for exposure to CRCoV over time. Serum antibodies to CIV were not detected in any of the dogs sampled, suggesting that this virus is unlikely to be present in dogs in New Zealand.

CLINICAL RELEVENCE: Canine respiratory coronavirus is present in New Zealand. Although the role of this virus in canine infectious tracheobronchitis has not been fully elucidated, evidence suggests that it may have a causal role in this disease. Veterinarians should consider CRCoV as a differential diagnosis in cases of respiratory disease in dogs in New Zealand. While CIV appears not to be currently present in New Zealand, veterinarians should consider infection with this virus as a differential diagnosis in dogs presenting with respiratory signs.     

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.     

犬场蚊子与犬细小病毒

２０００年４月，应用套式ＰＣＲ技术从犬场蚊子的血液样品检测出犬细小病毒。将分离到的犬细小病毒ＶＰ２－Ｙ３４基因片段克隆到ＰＭＤ１８－Ｔ载全，其病毒基因组ＣＰＶ－ＰＶ２－Ｙ３４序列测定结果显示与作者在ＧＥＮＥＢＡＮＫ发表的肺病变犬细小病毒ＣＰＶ－ＨＮ－１株有９９％同源性。     

A serological survey of canine respiratory coronavirus in New Zealand

ABSTRACT

Aims: To determine the seroprevalence of canine respiratory coronavirus (CRCoV) in New Zealand dogs, and to explore associations with age, sex, breed, month, and geographical region of sampling and reported presence of clinical signs suggestive of respiratory disease.

Methods: A total of 1,015 canine serum samples were randomly selected from submissions to a diagnostic laboratory between March and December 2014, and were analysed for CRCoV antibodies using a competitive ELISA. Logistic regression analysis was used to determine associations between seroprevalence of CRCoV and breed category, age, sex, sampling month, region, and reported health status of dogs.

Results: Overall, 538/1,015 (53.0%) samples were seropositive for CRCoV, with 492/921 (53.4%) positive dogs in the North Island and 46/94 (49%) in the South Island. Age of dog, sampling month, region, and presence of abnormal respiratory signs were included in the initial logistic regression model. Seroprevalence was higher in dogs aged ≥3 compared with ≤2 years (p?<?0.01). The lowest seroprevalence was observed in July (30/105; 28.5%) and August (32/100; 32%), and the highest in June (74/100; 74%). Seroprevalence in dogs from Auckland was higher than in dogs from the Hawkes Bay, Manawatu, Marlborough, and Waikato regions (p?<?0.05). Abnormal respiratory signs (coughing, nasal discharge, or sneezing) were reported for 28/1,015 (2.8%) dogs sampled. Seroprevalence for CRCoV tended to be higher among dogs with respiratory signs (67.9 (95% CI?=?47.6–83.4)%) than dogs with no reported respiratory signs (52.6 (95% CI?=?49.5–55.7)%).

Conclusions: Serological evidence of infection with CRCoV was present in more than half of the dogs tested from throughout New Zealand. Differences in CRCoV seroprevalence between regions and lack of seasonal pattern indicate that factors other than external temperatures may be important in the epidemiology of CRCoV in New Zealand.

Clinical relevance: Our data suggest that CRCoV should be included in investigations of cases of infectious canine tracheobronchitis, particularly if these occur among dogs vaccinated with current vaccines, which do not include CRCoV antigens.     

Prevalence and genotyping of Cryptosporidium in three cattle husbandry systems in Zambia

To determine the prevalence of Giardia genotypes in 12–24 month old dairy heifers, fecal specimens were collected from two farms each in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Specimens, cleaned of fecal debris and concentrated using CsCl density gradient centrifugation, were subjected to PCR and DNA sequence analysis. Prevalence of Giardia infection, ranged from 11% to 75% on 14 farms with an average prevalence of 36% (204 positive cattle out of 571 examined). DNA sequence analysis of the 16S rRNA gene revealed 91% of the 204 Giardia isolates were Assemblage E, and 9% were Assemblage A. The prevalence of these genotypes varied greatly from farm to farm, with four farms having exclusively Assemblage E Giardia. Overall, Assemblage E was present in 33% of all animals tested and Assemblage A was present in 3% of the animals. Thus, while many of the heifers were infected with a genotype that is not known to be infectious for humans, 1–2 year old heifers on 10 of 14 farms did harbor zoonotic Assemblage A Giardia. Therefore, heifers cannot be overlooked as a potential source of human infectious cysts in the environment, with some farms representing a much higher risk than others.     

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.     

RT-PCR检测犬粪便中的冠状病毒

根据犬冠状病毒(caninecoronavirus, CCV) 的S基因序列, 用计算机设计并合成了1对引物P1、P2, 以此引物及以从美国进口的CCV疫苗的反转录产物为模板, 初步建立了检测CCV的RT PCR。将纯化的PCR产物成功地克隆到pGEM T easy载体中, 鉴定、测序并进行同源性分析。结果表明, 与1 71和UCD 1株同源性分别为91 5%和94 3%。该RT PCR能对疫苗中CCV及CCV参考株USCV B1的S基因进行特异性地扩增,长度为575bp, 对犬的其他病毒及CRFK细胞未能扩增出任何片段, 该RT PCR能检测出0 5μLCCV培养液/2g粪便。用建立的RT PCR在上海对50条犬粪便进行检测, 结果表明CCV阳性6例。本研究建立了检测犬粪便中的CCV的RT PCR, 能用于犬冠状病毒流行病学调查。     

Monitoring of canine parvovirus (CPV) strains detected in vaccinated puppies in Brazil

The objective of this study was to investigate, by partial sequencing of VP2 protein, the variability of CPV detected in 37 fecal samples collected from vaccinated puppies with enteritis. Laboratorial diagnosis of CPV was confirmed by HA/HI and PCR and, for sequencing analyses, two different regions of the VP2 gene were amplified by PCR. From 1995 to 2004, all strains were characterized as CPV-2a. After that, both CPV-2a and CPV-2b were detected. All CPV-2a showed a non-synonymous mutation in the residue 297 (Ser → Ala). A synonymous substitution at the AA 574 was also observed in 15/37 samples. Our findings indicate that the cases of vaccine failure are most likely not associated to the mutations detected in the sequenced regions. However, the monitoring of genotyping mutations that led to new CPV strains is essential to determinate if current vaccines will keep providing protection against all new future variants.     

Current status and risk factors of canine parvovirus type 2 in North Central Nigeria

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.     

Prevalence of canine coronavirus (CCoV) in dog in Japan: detection of CCoV RNA and retrospective serological analysis

We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ²=8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies.     

复方苦芩注射液对人工感染犬细小病毒病的防治

目的:探讨复方苦芩注射液防治犬细小病毒病效果和作用原理。方法:人工感染犬细小病毒,其中预防试验组30只,治疗试验组40只。结果:复方苦芩注射液能显著提高犬的免疫功能,对犬细小病毒病的预防保护率达90%,治疗有效率为70%。结论:复方苦芩注射液能提高犬的免疫功能,对犬细小病毒病有良好的预防效果,且有一定的治疗作用。     

Outbreak of equine coronavirus infection among riding horses in Tokyo,Japan

In 2020, an outbreak of equine coronavirus (ECoV) infection occurred among 41 horses at a riding stable in Tokyo, Japan. This stable had 16 Thoroughbreds and 25 horses of other breeds, including Andalusians, ponies and miniature horses. Fifteen horses (37 %) showed mild clinical signs such as fever, lethargy, anorexia and diarrhoea, and they recovered within 3 days of onset. A virus neutralization test showed that all 41 horses were infected with ECoV, signifying that 26 horses (63 %) were subclinical. The results suggest that subclinical horses played an important role as spreaders. A genome sequence analysis revealed that the lengths from genes p4.7 to p12.7 or NS2 in ECoV differed from those of ECoVs detected previously, suggesting that this outbreak was caused by a virus different from those that caused previous outbreaks among draughthorses in Japan. Among 30 horses that tested positive by real-time RT-PCR, ECoV shedding periods of non-Thoroughbreds were significantly longer than those of Thoroughbreds. The difference in shedding periods may indicate that some breeds excrete ECoV longer than other breeds and can contribute to the spread of ECoV.     

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.     

Influence of chemotherapy for lymphoma in canine parvovirus DNA distribution and specific humoral immunity

In man, the combination of cancer and its treatment increases patients’ susceptibility to opportunistic infections, due to immune system impairment. In veterinary medicine little information is available concerning this issue. In order to evaluate if a similar dysfunction is induced in small animals undergoing chemotherapy, we assessed the complete blood count, leukocytic, plasma and fecal canine parvovirus (CPV) viral load, and anti-CPV protective antibody titers, in dogs with lymphoma treated with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) protocol, before and during chemotherapy.There was no evidence of decreased immune response, either at admission or after two chemotherapy cycles, indicating that the previously established immunity against CPV was not significantly impaired, supporting the idea that immunosuppression as a result of hematopoietic neoplasms and their treatment in dogs requires further investigation and conclusions cannot be extrapolated from human literature.     

Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs

The objectives of this study were to examine the potential roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme-linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media for C difficile, Salmonella spp., and Campylobacter spp. and selective media after heat shock for C perfringens. No significant difference was found in the isolation of C perfringens or C difficile among the 3 groups. A significant association was found between the presence of diarrhea and detection of C perfringens enterotoxin (CPE) or toxin A via ELISA for both C perfringens and C difficile, respectively. PCR performed on C difficile isolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene of C perfringens. Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence of C perfringens-associated diarrhea.     

Seroprevalence of antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in household dogs in Japan

We investigated the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among dogs in the Tokyo area via enzyme-linked immunosorbent assay (ELISA) using the spike protein as the target antigen. Plasma samples from 494 household dogs and blood-donor dogs were tested from July 2020 to January 2021. Of these samples, three showed optical densities that were higher than the mean plus two standard deviations of the mean of the negative-control optical densities (ODs). Of these three samples, only the sample with the highest OD by ELISA was confirmed positive by virus neutralization testing. The positive dog presented no SARS-CoV-2-related symptoms. The positivity rate of SARS-CoV-2 infections among dogs in the Tokyo area was approximately 0.2%.     

Canine coronavirus in Australian dogs

OBJECTIVE: To estimate the frequency of serum antibodies (IgG and IgM) to canine coronavirus (CCV) in the Australian dog population and evaluate the role of CCV as a causative agent of gastroenteritis. DESIGN: A serological survey of antibodies to CCV among different dog populations. PROCEDURE: The development and characterisation of an indirect ELISA for the detection of antibodies (IgG and IgM) to CCV was undertaken. Sera collected from both diarrhoeal and non-diarrhoeal dogs from various populations throughout Australia were tested for these antibodies to CCV. RESULTS: Serum samples (1396) collected from 1984 to 1998 were tested for the presence of IgG antibodies to CCV. Samples were divided into two categories on the basis of the number of dogs housed together. The groups were either an open population containing dogs housed as groups of three or less, or kennel populations. Sera from 15.8% of the open population and 40.8% of kennelled dogs were positive for CCV antibodies. The prevalence of antibodies varied from zero to 76% in kennelled dogs. About 23% of 128 dogs positive for IgG antibodies to CCV were also positive for IgM antibodies to CCV, indicating recent CCV infection. Of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting (n = 29), 85% were positive in the IgM ELISA and 85.7% in the IgG ELISA for antibodies to CCV. In comparison, for those dogs presented without any history of gastroenteritis only 15% were positive for IgM and 30% positive for IgG. CONCLUSION: Serological evidence indicates that infection with CCV in dogs is widespread throughout the Australian mainland. The prevalence of antibodies varies greatly among different populations, with an average of 40.8% positive in kennelled populations and 15.8% in the open population.     

犬细小+犬冠状+贾第鞭毛虫抗原三合一胶体金检测卡的制备与评价

为快速检测犬细小病毒(CPV)、犬冠状病毒(CCV)和贾第鞭毛虫(Giardia),研究利用胶体金免疫层析技术制备CPV+CCV+Giardia抗原三合一胶体金检测卡并评价其各项指标.结果显示,该三合一胶体金检测卡与CDV、CPV、CAV、CRV、CCV和Giardia(自身样本除外)无交叉反应;可检测浓度低至10.0...