Yong-Il Cho

Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Major Agents Causing Calf Diarrhea in Feces

Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99+, and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID50 (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colony-forming units for E. coli K99+ and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex realtime PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%–97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99+, Salmonella, and/or Cryptosporidium.

Case–control study of microbiological etiology associated with calf diarrhea

Abstract Calf diarrhea is a major economic burden for the US cattle industry. A variety of infectious agents are implicated in calf diarrhea and co-infection of multiple pathogens is not uncommon in diarrheic calves. A case–control study was conducted to assess infectious etiologies associated with calf diarrhea in Midwest cattle farms. A total of 199 and 245 fecal samples were obtained from diarrheic and healthy calves, respectively, from 165 cattle farms. Samples were tested by a panel of multiplex PCR assays for 11 enteric pathogens: bovine rotavirus group A (BRV-A), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine enterovirus (BEV), bovine norovirus (BNoV), Nebovirus, bovine torovirus (BToV) Salmonella spp. (Salmonella), Escherichia coli (E. coli) K99+, Clostridium perfringens with β toxin gene and Cryptosporidium parvum (C. parvum). The association between diarrhea and detection of each pathogen was analyzed using a multivariate logistic regression model. More than a half of the fecal samples from the diarrheic calves had multiple pathogens. Statistically, BRV-A, BCoV, BNoV, Nebovirus, Salmonella, E. coli K99+, and C. parvum were significantly associated with calf diarrhea (p <0.05). Among them, C. parvum and BRV-A were considered to be the most common enteric pathogens for calf diarrhea with high detection frequency (33.7% and 27.1%) and strong odds ratio (173 and 79.9). Unexpectedly BNoV (OR=2.0) and Nebovirus (OR=16.7) were identified with high frequency in diarrheic calves, suggesting these viruses may have a significant contribution to calf diarrhea.

Evaluation of a commercial rapid test kit for detecting bovine enteric pathogens in feces

Recently a commercial antigen-capture enzyme-linked immunosorbent assay kit in the form of a dipstick (Bovine Enterichek®, Biovet Inc.) was made available to bovine practitioners and producers for the rapid detection of Betacoronavirus 1 (BCV-1), Rotavirus A (RV-A), Escherichia coli K99+, and Cryptosporidium parvum in feces from diarrheic calves. The diagnostic performance of Bovine Enterichek was evaluated in comparison with a multiplex real-time polymerase chain reaction assay (mrtPCR). One hundred fecal samples were procured from diagnostic submissions to Iowa State University Veterinary Diagnostic Laboratory and were used for the assessment. The agreement quotient (kappa) in results for each pathogen between Bovine Enterichek and mrtPCR were 0.095 (BCV-1), 0.521 (RV-A), 0.823 (E. coli K99 + ), and 0.840 (C. parvum). In comparison to mrtPCR, the diagnostic sensitivity of Bovine Enterichek was 60.0%, 42.3%, 71.4%, and 81.5%, and the diagnostic specificity was 51.4%, 100%, 100%, and 98.6% for BCV-1, RV-A, E. coli K99 + , and C. parvum, respectively. The current study suggested that Bovine Enterichek can be a rapid test tool in the field for detection of RV-A, C. parvum, or E. coli K99+ in feces from calves at acute stage of clinical disease. However, test results for BCV-1 by the kit should be interpreted with caution due to low specificity and sensitivity of the kit.

Attempts to enhance cross-protection against porcine reproductive and respiratory syndrome viruses using chimeric viruses containing structural genes from two antigenically distinct strains.

Due to significant antigenic variations between field isolates of porcine reproductive and respiratory syndrome virus (PRRSV), suboptimal cross-protection between different viruses impedes the effective control of PRRS via vaccination. Our previous study showed that chimeric viruses containing mixed structural genes from two distinct strains (VR2332 and JA142) of PRRSV were highly susceptible to the viral neutralizing activity of antisera generated against both parental strains. In this study, three chimeric viruses (JAP5, JAP56 and JAP2-6) were constructed by replacing ORF5, ORFs 5 and 6, and ORFs 2-6 of VR2332 with the corresponding genes of JA142, respectively, and their ability to confer cross-protection against challenge with the VR2332 and JA142 strains was evaluated in vivo. A total of 114 pigs were divided into 6 groups, and each group was intramuscularly injected with one of the 3 chimeric viruses (n=16 pigs per group), VR2332 (n=24), JA142 (n=24), or sham inoculum (n=18). At 44days post-inoculation (dpi), these pigs were further divided into 15 groups (n=6 or 8 pigs per group) and intranasally challenged with VR2332, JA142, or sham inoculum. All pigs inoculated with one of the chimeric viruses prior to challenge had lower viremia levels than the challenge control pigs. Prior inoculation with JAP56 markedly decreased viremia to nearly undetectable levels in pigs challenged with either VR2332 or JA142. These results suggest that chimeric viruses harboring mixed structural genes from two distinct PRRSV strains can provide protection against both donor viruses.

Use of blood collected onto and dried on filter paper for diagnosing pregnancy in cattle.

The measurement of serum or plasma pregnancy-associated glycoproteins (PAGs) is increasingly used to diagnose pregnancy in cattle. This study evaluated whether a dried blood spot (DBS) collected on filter paper could be used as an alternative to serum or plasma for such tests. A total of 37 serum, 68 plasma and 68 DBS samples were collected from cows of known pregnancy status and tested using a commercial ELISA. None of the plasma or serum samples resulted in false positives or false negatives. No false positives (sample-negative (S-N) values >0.3 in non-pregnant cows) were observed with DBS samples, but false negatives were observed (S-N values <0.3 in pregnant cattle). The data suggested that PAGs in DBS samples were diluted during processing as samples from pregnant cattle had lower S-N values (0.111-0.494) than the corresponding serum (1.123-2.665) and/or plasma (0.764-2.042) samples. ROC analysis showed that lowering the cut-off S-N value from 0.3 to 0.1 for DBS samples prevented false negatives without increasing false positives. Modifications to the test protocol significantly increased mean S-N values of DBS samples from pregnant cows while those from non-pregnant cows were not affected. In conclusion, lowering the cut-off and modifying the protocol allowed DBS samples to be used for blood-based pregnancy testing.

Occurrence of Clostridium perfringens according to Raising Periods in Broilers

Department of Infectious Diseases & Avian Diseases, College Veterinary Medicine and Korea Zoonosis Research Institute,Jeonbuk National University, Jeonju 561-756, KoreaABSTRACT The objective of this study was to investigate occurrence patterns of Clostridium perfringens on different raising periods in broilers. In different raising periods, we investigated the change in the gross lesion and microscopic histological findings of the mucose of the small intestine, colony forming unit (CFU) and the types C. perfringens with PCR assay. According to the gross lesions on the mucose of small intestine with 10-days-old broilers, the non-antibiotic group showed a higher value (0.6) than the antibiotic group (0.0). Whereas 20-days-old broilers with, the antibiotic treatment had a slightly lower value (1.0) than the non-antibiotic group (1.3). In the histological examination on the villi of the small intestine, there was no damage of the villi of the small intestine with 1-day-old broilers in both groups; however, the non-antibiotic group showed a higher value (0.4) than the antibiotic group (0.0) with 10-days-old broilers. In the non-antibiotic group, the CFU of C. perfringens of the fecal samples from the small intestine increased from 10 days of raising broilers and rapidly increase after 20 and 30 days of raising broilers. There was no detection of C. perfringens types with PCR assy in 1-day-old broilers, but we found C. perfringens type A in 10-, 20- and 30-days-old broilers. Although it is possible to raise healthy broilers by using antibiotics, the addition of antibiotics to concentrate feed is prohibited for public health. The results of this study would contribute to proper feeding management through the careful use of antibiotics.(Key words : necrotic enteritis, Clostridium perfringens, broiler, antibiotics)