Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases.

The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay's clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets.

A novel defective recombinant porcine enterovirus G virus carrying a porcine torovirus papain-like cysteine protease gene and a putative anti-apoptosis gene in place of viral structural protein genes.

Enterovirus G (EV-G) belongs to the family of Picornaviridae. Two types of recombinant porcine EV-Gs carrying papain-like cysteine protease (PLCP) gene of porcine torovirus, a virus in Coronaviridae, are reported. Type 1 recombinant EV-Gs are detected in pig feces in Japan, USA, and Belgium and carry the PLPC gene at the junction site of 2C/3A genes, while PLPC gene replaces the viral structural genes in type 2 recombinant EV-G detected in pig feces in a Chinese farm. We identified a novel type 2 recombinant EV-G carrying the PLCP gene with flanking sequences in place of the viral structural genes in pig feces in Japan. The ~0.3 kb-long upstream flanking sequence had no sequence homology with any proteins deposited in GenBank, while the downstream ~0.9 kb-long flanking sequence included a domain having high amino acid sequence homology with a baculoviral inhibitor of apoptosis repeat superfamily. The pig feces, where the novel type 2 recombinant EV-G was detected, also carried type 1 recombinant EV-G. The amount of type 1 and type 2 recombinant EV-G genomes was almost same in the pig feces. Although the phylogenetic analysis suggested that these two recombinant EV-Gs have independently evolved, type 1 recombinant EV-G might have served as a helper virus by providing viral structural proteins for dissemination of the type 2 recombinant EV-G.

Characteristics of Staphylococcal Enterotoxin A Production and Growth of Staphylococcus aureus in Shaking and Stationary Cultures.

Staphylococcal food poisoning, which is still a serious health problem worldwide, is caused by staphylococcal enterotoxin (SE) . Among many types of SE, staphylococcal enterotoxin type A (SEA) is known to be the most responsible for staphylococcal food poisoning. Production by SEA-producing strains in shaking culture has been studied by many investigators, whereas the kinetical differences between shaking and stationary cultures have not been studied intensively. Therefore, this difference was studied at various temperatures from 14℃ to 46℃ in the present study. Consequently the maximum SEA concentration and populations of SEA producer in shaking culture was higher than those in stationary culture. Of interest, however, the productivity, which is the maximum SEA amount produced by one cell, in shaking cultures was lower than that in stationary culture. Kinetic analysis clarified that SEA gene expression in staphylococcal cells preceded toxin production at optimal temperature. Next, several SEA producers were studied for the maximum toxin production at various temperatures from 14℃ to 42℃ in shaking culture. Consequently all strains showed different patterns, suggesting that the characteristics of SEA production of these strains would be strain-specific. These results in this study would provide useful, basic information to prevent staphylococcal food poisoning outbreaks.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples.

Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.

Genetic diversity and intergenogroup recombination events of sapoviruses detected from feces of pigs in Japan.

Sapoviruses (SaV) are enteric viruses infecting humans and animals. SaVs are highly diverse and are divided into multiple genogroups based on structural protein (VP1) sequences. SaVs detected from pigs belong to eight genogroups (GIII, GV, GVI, GVII, GVIII, GIX, GX, and GXI), but little is known about the SaV genogroup distribution in the Japanese pig population. In the present study, 26 nearly complete genome (>6000 nucleotide: nt) and three partial sequences (2429nt, 4364nt, and 4419nt in length, including the entire VP1 coding region) of SaV were obtained from one diarrheic and 15 non-diarrheic porcine feces in Japan via a metagenomics approach. Phylogenetic analysis of the complete VP1 amino acid sequence (aa) revealed that 29 porcine SaVs were classified into seven genogroups; GIII (11 strains), GV (1 strain), GVI (3 strains), GVII (6 strains), GVIII (1 strain), GX (3 strains), and GXI (4 strains). This manuscript presents the first nearly complete genome sequences of GX and GXI, and demonstrates novel intergenogroup recombination events.

Discovery of fur seal feces-associated circular DNA virus in swine feces in Japan.

Fur seal feces-associated circular ssDNA virus (FSfaCV) was discovered in a pig for the first time in Japan using a next-generation sequencer with duplex-specific nuclease. Full genome of the virus showed approximately 92% similarity to FSfaCVs from New Zealand fur seals. Furthermore, we investigated the prevalence of the ssDNA virus in 85 piglets in Japan, and 65 piglets were positive (76%) for the virus.

Molecular characteristics and prevalence of small ruminant lentiviruses in goats in Japan.

Small ruminant lentiviruses (SRLVs), which comprise caprine arthritis-encephalitis virus (CAEV) and maedi-visna virus (MVV), are prevalent in goats and sheep worldwide, including in Japan. However, little is known about the molecular characteristics of goat lentiviruses in Japan. In this study, a molecular and phylogenetic analysis of the long gag region was performed. The phylogenic tree demonstrated that all samples belonged to SRLV subtype B1. Two clusters were identified, with one cluster distinct from previously reported strains of subtype B1. In addition, several alterations in the amino acid sequence were detected in immunodominant epitopes of the gag region. To gain a deeper understanding of the genetic diversity of SRLVs in Japan, it will be necessary to increase the sample size and conduct a broader survey. The present report is important for establishing baseline information on the prevalence of SRLV in Japan and providing data to develop a new, more sensitive diagnostic test for effective control of SRLV.

Complete genome analysis of porcine kobuviruses from the feces of pigs in Japan.

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

First isolation and characterization of pteropine orthoreoviruses in fruit bats in the Philippines.

Pteropine orthoreovirus (PRV) causes respiratory tract illness (RTI) in humans. PRVs were isolated from throat swabs collected from 9 of 91 wild bats captured on the Mindanao Islands, The Philippines, in 2013. The nucleic acid sequence of the whole genome of each of these isolates was determined. Phylogenetic analysis based on predicted amino acid sequences indicated that the isolated PRVs were novel strains in which re-assortment events had occurred in the viral genome. Serum specimens collected from 76 of 84 bats were positive for PRV-neutralizing antibodies suggesting a high prevalence of PRV in wild bats in the Philippines. The bat-borne PRVs isolated in the Philippines were characterized in comparison to an Indonesian PRV isolate, Miyazaki-Bali/2007 strain, recovered from a human patient, revealing that the Philippine bat-borne PRVs had similar characteristics in terms of antigenicity to those of the Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro). The impact of the Philippine bat-borne PRVs should be studied in human RTI cases in the Philippines.

Whole genome analysis of porcine astroviruses detected in Japanese pigs reveals genetic diversity and possible intra-genotypic recombination.

Porcine astroviruses (PoAstVs) are ubiquitous enteric virus of pigs that are distributed in several countries throughout the world. Since PoAstVs are detected in apparent healthy pigs, the clinical significance of infection is unknown. However, AstVs have recently been associated with a severe neurological disorder in animals, including humans, and zoonotic potential has been suggested. To date, little is known about the epidemiology of PoAstVs among the pig population in Japan. In this report, we present an analysis of nearly complete genomes of 36 PoAstVs detected by a metagenomics approach in the feces of Japanese pigs. Based on a phylogenetic analysis and pairwise sequence comparison, 10, 5, 15, and 6 sequences were classified as PoAstV2, PoAstV3, PoAstV4, and PoAstV5, respectively. Co-infection with two or three strains was found in individual fecal samples from eight pigs. The phylogenetic trees of ORF1a, ORF1b, and ORF2 of PoAstV2 and PoAstV4 showed differences in their topologies. The PoAstV3 and PoAstV5 strains shared high sequence identities within each genotype in all ORFs; however, one PoAstV3 strain and one PoAstV5 strain showed considerable sequence divergence from the other PoAstV3 and PoAstV5 strains, respectively, in ORF2. Recombination analysis using whole genomes revealed evidence of multiple possible intra-genotype recombination events in PoAstV2 and PoAstV4, suggesting that recombination might have contributed to the genetic diversity and played an important role in the evolution of Japanese PoAstVs.

Diversity in VP3, NSP3, and NSP4 of rotavirus B detected from Japanese cattle.

Bovine rotavirus B (RVB) is an etiological agent of diarrhea mostly in adult cattle. Currently, a few sequences of viral protein (VP)1, 2, 4, 6, and 7 and nonstructural protein (NSP)1, 2, and 5 of bovine RVB are available in the DDBJ/EMBL/GenBank databases, and none have been reported for VP3, NSP3, and NSP4. In order to fill this gap in the genetic characterization of bovine RVB strains, we used a metagenomics approach and sequenced and analyzed the complete coding sequences (CDS) of VP3, NSP3, and NSP4 genes, as well as the partial or complete CDS of other genes of RVBs detected from Japanese cattle. VP3, NSP3, and NSP4 of bovine RVBs shared low nucleotide sequence identities (63.3-64.9% for VP3, 65.9-68.2% for NSP3, and 52.6-56.2% for NSP4) with those of murine, human, and porcine RVBs, suggesting that bovine RVBs belong to a novel genotype. Furthermore, significantly low amino acid sequence identities were observed for NSP4 (36.1-39.3%) between bovine RVBs and the RVBs of other species. In contrast, hydrophobic plot analysis of NSP4 revealed profiles similar to those of RVBs of other species and rotavirus A (RVA) strains. Phylogenetic analyses of all gene segments revealed that bovine RVB strains formed a cluster that branched distantly from other RVBs. These results suggest that bovine RVBs have evolved independently from other RVBs but in a similar manner to other rotaviruses. These findings provide insights into the evolution and diversity of RVB strains.

Identification of a novel bovine enterovirus possessing highly divergent amino acid sequences in capsid protein.

BACKGROUND: Bovine enterovirus (BEV) belongs to the species Enterovirus E or F, genus Enterovirus and family Picornaviridae. Although numerous studies have identified BEVs in the feces of cattle with diarrhea, the pathogenicity of BEVs remains unclear. Previously, we reported the detection of novel kobu-like virus in calf feces, by metagenomics analysis. In the present study, we identified a novel BEV in diarrheal feces collected for that survey. Complete genome sequences were determined by deep sequencing in feces. Secondary RNA structure analysis of the 5' untranslated region (UTR), phylogenetic tree construction and pairwise identity analysis were conducted. RESULTS: The complete genome sequences of BEV were genetically distant from other EVs and the VP1 coding region contained novel and unique amino acid sequences. We named this strain as BEV AN12/Bos taurus/JPN/2014 (referred to as BEV-AN12). According to genome analysis, the genome length of this virus is 7414 nucleotides excluding the poly (A) tail and its genome consists of a 5'UTR, open reading frame encoding a single polyprotein, and 3'UTR. The results of secondary RNA structure analysis showed that in the 5'UTR, BEV-AN12 had an additional clover leaf structure and small stem loop structure, similarly to other BEVs. In pairwise identity analysis, BEV-AN12 showed high amino acid (aa) identities to Enterovirus F in the polyprotein, P2 and P3 regions (aa identity ≥82.4%). Therefore, BEV-AN12 is closely related to Enterovirus F. However, aa sequences in the capsid protein regions, particularly the VP1 encoding region, showed significantly low aa identity to other viruses in genus Enterovirus (VP1 aa identity ≤58.6%). In addition, BEV-AN12 branched separately from Enterovirus E and F in phylogenetic trees based on the aa sequences of P1 and VP1, although it clustered with Enterovirus F in trees based on sequences in the P2 and P3 genome region. CONCLUSIONS: We identified novel BEV possessing highly divergent aa sequences in the VP1 coding region in Japan. According to species definition, we proposed naming this strain as "Enterovirus K", which is a novel species within genus Enterovirus. Further genomic studies are needed to understand the pathogenicity of BEVs.

Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex.

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Identification of further diversity among posaviruses.

Recently, there have been reports of new members of posavirus-like viruses in the order Picornavirales. In this study, using a metagenomics approach, 11 posavirus-like sequences (>7,000 nucleotides) were detected in 155 porcine fecal samples. Phylogenetic analysis revealed that the newly identified virus sequences, together with other posavirus-like viruses, form distinct clusters within the order Picornavirales, composed of eight genogroups and unassigned sequences based on amino acid sequences of the helicase and RNA-dependent RNA polymerase regions, with <40 % and <50 % sequence identity, respectively. We propose further classifications of highly diverse posavirus populations based on newly identified sequences from Japanese pig feces.

Development of one-step real-time reverse transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea.

Porcine diarrhea caused by viruses is a major problem of the pig farming industry and can result in substantial losses of revenue. Thus, diagnosing the infectious agents is important to prevent and control diseases in pigs. We developed novel one-step real-time quantitative RT-PCR (qPCR) assays that can detect four porcine diarrheal viruses simultaneously: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine group A rotavirus (PRVA). The qPCR analysis takes only 75 minutes to detect the presence of the four viruses. The limits of detection of our new assays for PEDV, TGEV, PDCoV, and PRVA were 100, 10, 10 and 10 copies per reaction, respectively. The sensitivity of qPCR was 1-1000 times higher than that of published gel-based RT-PCR. We used our qPCR method to successfully diagnose clinical samples from infected pigs, and no false positive results were obtained. In conclusion, qPCR can drastically reduce the diagnostic time to detect viruses compared to currently employed methods. We predict that the qPCR assays will become a useful tool for detecting viral infections that cause diarrhea and other complications in pigs.

Detection of Campylobacter jejuni in rectal swab samples from Rousettus amplexicaudatus in the Philippines.

Bats are the second diversity species of mammals and widely distributed in the world. They are thought to be reservoir and vectors of zoonotic pathogens. However, there is scarce report of the evidence of pathogenic bacteria kept in bats. The precise knowledge of the pathogenic bacteria in bat microbiota is important for zoonosis control. Thus, metagenomic analysis targeting the V3-V4 region of the 16S rRNA of the rectal microbiota in Rousettus amplexicaudatus was performed using high throughput sequencing. The results revealed that 103 genera of bacteria including Camplyobacter were detected. Campylobacter was second predominant genus, and Campylobacter coli and Campylobacter jejuni were identified in microbiome of R. amplexicaudatus. Campylobacteriosis is one of the serious bacterial diarrhea in human, and the most often implicated species as the causative agent of campylobacteriosis is C. jejuni. Therefore, we investigated the prevalence of C. jejuni in 91 wild bats with PCR. As a result of PCR assay targeted on 16S-23S intergenic spacer, partial genome of C. jejuni was detected only in five R. amplexicaudatus. This is the first report that C. jejuni was detected in bat rectal swab samples. C. jejuni is the most common cause of campylobacteriosis in humans, transmitted through water and contact with livestock animals. This result indicated that R. amplexicaudatus may be a carrier of C. jejuni.

Characterization and phylogenetic analysis of a novel picornavirus from swine feces in Japan.

During an investigation of porcine fecal viruses using a metagenomics approach, a novel picornavirus was identified from the feces of a healthy two-month-old pig. This virus, named porcine picornavirus Japan (PPVJ), had a standard picornavirus genome organization, including the L protein region. The 5' untranslated region harbored a type II internal ribosomal entry site. This virus was most closely related to lesavirus 1 (amino acid sequence identity: 38.2 %) in P1, equine rhinitis A virus (25.8 %) in P2, and lesavirus 2 (40.9 %) in P3. According to the genus demarcations for the family Picornaviridae (less than 40 %, 40 %, and 50 % amino acid sequence identity in P1, P2, and P3, respectively), PPVJ represents a new genus in the family Picornaviridae. PPVJ was detected in 23.3 % of the fecal samples (from 58.3 % of the farms across a wide area) from pigs less than four months old, by reverse transcription PCR, using specific primers designed from the 3D sequence, followed by sequencing. The host range and pathogenic potential of this virus in animals is yet to be determined.

Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.