An Importance of Long-Term Clinical Analysis to Accurately Diagnose Calves Persistently and Acutely Infected by Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14-21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field.

Clinical Analysis for Long-Term Sporadic Bovine Viral Diarrhea Transmitted by Calves with an Acute Infection of Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) is a viral pathogen associated with serious problems in the cattle industry. Cattle persistently infected (PI) with BVDV are mild or asymptomatic; however, they become a source of BVDV transmission to other cattle. Hence, it is important to rapidly identify and remove the PI animals from cattle herds. Whereas cattle acutely infected (AI) with BVDV have various symptoms, yet they generally recover within 3 weeks. However, there is a paucity of information concerning clinical characteristics of AI cattle. Further accumulation of information would be required to accurately diagnose AI cattle with BVDV. Here, we attempted to obtain valuable information via various analyses using a case report of BVD outbreak that occurred for approximately four months in Iwate Prefecture in 2017. Using eight calves and multiple tests (real-time RT-PCR, virus isolation, enzyme-linked immunosorbent assay, and virus neutralization assay) over 6 weeks, we diagnosed the continuous BVD outbreak as an acute infection and not a persistent one. Additionally, we revealed that the sporadic case was caused by low pathogenic BVDV2 via BVDV genotyping and phylogenetic analysis. The data suggest that BVDV2 AI animals might also be a source of transmission to susceptible calves; hence, it might persist for a long period owing to multiple AI animals. These findings provide useful information to diagnose AI and PI cattle with BVDV in the field.

Detection of avian encephalomyelitis virus in chickens in Japan using RT-PCR.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for broadly detecting the avian encephalomyelitis virus (AEV). The new primers were based on conserved sequences of the 5'-untranslated region of AEV, because the virus was not detected using previous reported RT-PCR. By applying this method to the chicken samples with suspected AEV infection in Japan, we successfully obtained PCR products of the predicted size from all samples, and we confirmed the presence of AEV via sequence analysis.

Concurrent Fowlpox and Candidiasis Diseases in Backyard Chickens with Unusual Pox Lesions in the Bursa of Fabricius.

Concurrent fowlpox and candidiasis diseases occurred in a backyard chicken flock. Four deceased chickens (one Nagoya breed and three white silkie chickens) were examined for diagnosis. At necropsy, white curd-like plaques were observed in the crop. Fungal elements that stained positive for Candida albicans with immunohistochemistry were distributed throughout the tongue, choanal mucosa, esophagus, and crop. Typical fowlpox lesions, composed of proliferating epithelial cells with ballooning degeneration and viral intracytoplasmic inclusions, were observed in the conjunctiva, nasal mucosa, and skin around the cloaca. Interestingly, hyperplastic interfollicular epithelium with rare virus inclusions was observed in the bursa of Fabricius (BF). Some bursal follicles were replaced by proliferating epithelial cells. These proliferating cells immunohistochemically stained positive for cytokeratin. PCR and subsequent genetic sequencing detected the C. albicans gene in the crop, and fowlpox virus genes in the BF. These results indicate that this outbreak was a rare presentation of fowlpox in spontaneously infected chickens, with unusual pox lesions in the BF.

Detection and quantification of parapoxvirus DNA by use of a quantitative real-time polymerase chain reaction assay in calves without clinical signs of parapoxvirus infection.

OBJECTIVE: To investigate the presence of parapoxvirus (PPV) in cattle without clinical signs of infection and in farm environments of PPV-infected cattle. ANIMALS: 28 calves without clinical signs of PPV infection on 2 farms and 11 clinically affected calves on 6 farms. PROCEDURES: 164 oral swab samples were collected at regular intervals from 28 calves without clinical signs of PPV infection, and 11 swab samples were collected from 11 clinically affected calves. Viral DNA load was quantified by use of a PPV-specific quantitative real-time PCR (qRT-PCR) assay. RESULTS Of 28 calves without clinical signs of PPV infection, 12 had positive results for PPV DNA by use of the qRT-PCR assay. Viral DNA was detected continuously over a period of 2 to 5 months from 9 of these 12 calves, particularly from calves with dermatomycosis or respiratory tract disease. The PPV DNA loads in 32 oral swab samples from these 12 calves were significantly lower (median, 3.2 copies/mg) than those in samples collected from the 11 clinically affected calves (median, 3.2 × 10(4) copies/mg). Moreover, PPV DNA was detected in the residual feed and drinking water on both farms that housed the calves without clinical signs of PPV infection. CONCLUSIONS AND CLINICAL RELEVANCE: PPV in cattle without clinical signs of infection and in the environments of these cattle may represent sources of PPV transmission to susceptible cattle. IMPACT FOR HUMAN MEDICINE: Humans should wear gloves to prevent zoonotic disease transmission when handling cattle with or without clinical signs of PPV infection.

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans, animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis.

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson's diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.

Molecular analysis of parapoxvirus detected in eight calves in Japan.

Molecular analysis of parapoxvirus envelope genes was performed. Parapoxvirus DNA was detected in eight calves from eight farms in Iwate Prefecture, Japan, between April and September 2010. Seven of the detected viruses were identified as bovine papular stomatitis virus (BPSV) by sequencing, because their nucleotide identity was more than 96.8% similar compared with BPSV strain V660. Among them, two formed a subgroup, because their amplicons were digested with Xmn I (a marker for BPSV) and Hinc II and exhibited a T61C nucleotide substitution in the sequenced region. The remaining virus was pseudocowpox virus that had not been reported previously in Japan. Our results demonstrate the presence of a new BPSV variant in Japan with genetic variability in the envelope gene.

Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan.

The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.

Field application of the combination of neutralizing test and virus isolation, so-called spot test, to detect herds including cattle persistently infected with bovine viral diarrhea virus.

To detect herds including cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV), application of the combination of neutralizing antibody detection and virus isolation, so-called spot test, were performed on sera of 3 calves selected from each of 26 farms. Nine farms were judged as positive because 64 or more antibody titers were detected from 2 or more calves or BVDV was isolated from one or more calves. PI cattle were detected from 8 of the 9 farms. The positive judgment on one farm was obtained only when the indicator virus used on the neutralizing test was genotypically identical with the isolate from the farm. These results suggest that the spot test can be effective in detecting herds with PI cattle and that the accuracy may be influenced by the genotypes of the indicator viruses.

Identification of herds with cattle persistently infected with bovine viral diarrhea virus by virological evaluation of three calves.

For the identification of herds with cattle persistently infected (PI) with bovine viral diarrhea virus, 1,272 animals from 20 herds were subjected to serum neutralizing (SN) test using the Nose strain and virus isolation. Eighteen PI cattle were detected from 5 herds. On the phylogenetic tree based on the nucleotide sequences of the 5' untranslated region, the isolates from the PI cattle were classified into genotypes-1a or -1b. Of 3 unvaccinated calves aged 6 to 12 months selected from each herd, the probabilities of obtaining 2 or more non-PI cattle with SN antibody titers of 64 or more (P(SN)), one or more PI cattle (P(VI)), and either of the conditions (P(Total)) were calculated using the hypergeometric probability model. P(Total) for the 5 herds with PI cattle was 1.000. P(SN) for 3 herds with many PI cattle within the selected age group was as low as 0.500 or less, and P(VI) was as high as 0.886 or more. P(SN) in the 2 other herds with few PI cattle was 1.000, and P(VI) was as low as 0.375 or less. P(Total) in 13 of 15 herds without PI cattle was 0.000, and was 0.714 or 0.774 for the 2 other herds. These results suggest that herds with PI cattle can be predicted with high accuracy when both SN test and virus isolation are performed on only 3 unvaccinated calves aged 6 to 12 months selected from a herd.

Caprine enteritis associated with Yersinia pseudotuberculosis infection.

Yersiniosis was prevalent among a caprine herd during the late autumn of 2003 in Iwate Prefecture, Japan. The disease affected 29 of about 100 lactating goats, but not dried or nonparous goats, mature male goats or kids. Four animals died within an epidemic period of 20 days. Affected animals developed decreased milk production with subsequent watery diarrhea, neutrophilia with increased band forms and multiple microabscesses characteristic of yersiniosis in the intestinal mucosa from the jejunum to caecum as well as in the mesenteric lymph nodes. Y. pseudotuberculosis serotype III was isolated from intestinal contents and mesenteric lymph nodes. The organism was also cultured from clinically normal dried animals. The outbreak might have been precipitated by multiple stress factors, such as lactation, cold weather, Corynebacterium pseudotuberculosis infection resulting in abscess formation and tapeworm and coccidium parasitisms.

Genetic and antigenic analyses of bovine respiratory syncytial virus detected in Japan.

Genetic and antigenic analyses of bovine respiratory syncytial virus were conducted on 12 field strains from Tohoku and Hokuriku districts in Japan during from 2002 to 2004. On the phylogenetic tree of the nucleotide sequences of the glycoprotein region, the examined strains fell in the same cluster as the strain isolated in Nebraska and were classified as the subgroup III. The examined strains were subdivided into 2 lineages (A, B). Isoleucine 200 of the epitope domain was replaced by threonine as a feature of the lineage B strains. The examined strains showed the nucleotide sequence homologies of 88.3-93.3% with the known Japanese strains classified as the subgroup II and of 86.1-96.6% with those in the subgroup III. No significant difference was found on the neutralization index between the examined strain and the 52-163-13 phylogenetically similar to the Japanese vaccine one. The results suggest that the subgroup III strains have existed in Japan and that epidemics of the strains could be protected due to the present vaccination.

Occurrence of equine coital exanthema in pastured draft horses and isolation of equine herpesvirus 3 from progenital lesions.

During the period from 2001 to the following year, progenital diseases had been epidemic among the draft stallions and mares pastured together in Iwate Prefecture, the northeastern district of Japan. A stallion and 8 of 31 mares were affected in 2001, and 1 of 2 stallions and 10 of 36 mares in 2002. The clinical symptoms consisted of the formation of papules, pustules, ulcers and scabs on the progenital skin and mucosa in stallions and mares. In 2002, Equine herpesvirus 3 (EHV3) was isolated from 2 mares and the glycoprotein G gene of the virus detected from a stallion and 4 mares by polymerase chain reaction. Serum neutralizing tests showed that 12 of 38 horses, 10 clinically and 2 subclinically affected, changed to be positive for the EHV3 antibody. The results suggest that the horses were affected with equine coital exanthema (ECE) through coitus. Five mares with the antibody at the pre-pastured period may have been the possible origins of EHV3 infection in 2002, although the exact origin in 2001 remains unknown. The artificial insemination was performed for the prevention of ECE spreading through coitus on the pasture in 2003. There was no epidemic of the disease in 31 mares, although 3 mares with the antibody at the pre-pastured period showed the significant increase in the titers during the pastured period.