Degree Type

Dissertation

Date of Award

2012

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Kyoungjin Yoon

Abstract

Calf diarrhea is a commonly reported disease in young animal and still a major cause of productivity and economic loss to cattle producers. In the report of 2007 National Animal Health Monitoring System for U.S. dairy, a half of the deaths in unweaned calves were reported to be attributed to diarrhea. Multiple pathogens are known or postulated to cause or contribute to calf diarrhea. However, their significance and interaction in the disease has not been clearly addressed, not to mention uncertainty on the role of emerging viral pathogens such as bovine caliciviruses in calf diarrhea. It was of interest how diagnostic testing influences such an assessment. The long term goal of our study was to better understand the epidemiology, ecology and pathogenesis of well-known and potential bovine enteric pathogens in the field and developing better intervention strategies. Specific aims of the current study were to: a) develop highly specific and sensitive diagnostic methods for simultaneous detection of major bovine enteric pathogens; b) determine the prevalence and molecular characteristics of bovine norovirus (BNoV) which has recently emerged as a potential enteric pathogen worldwide, in the US Midwest cattle farms; and c) characterize the epidemiology of historically well-known and emerging bovine enteric pathogens in calf diarrhea. A series of four studies were conducted to address these specific aims. In a separate study the prevalence of bovine enterovirus (BEV), bovine coronavirus (BCoV), bovine rotavirus group A (BRV-A) and coliform bacteria in pasture streams in Southern Iowa over a 3-year period was surveyed to assess their association with cattle grazing.

The objective of the first study was to develop a PCR panel (hereafter, bovine enteric panel) which can simultaneously detect multiple enteric pathogens with high sensitivity and specificity. The bovine enteric panel (BEP) was consisted of 2 multiplex real-time PCR assays for simultaneous detection of five major bovine enteric pathogens [i.e., BCoV (formally known as Betacoronavirus 1), BRV-A, Salmonella spp. (Salmonella), Escherichia coli (E. coli) K99+, and Cryptosporidium parvum (C. parvum)]. The analytic sensitivity of the panel was 0.1 TCID50 for BCoV and BRV-A, 5 and 0.5 CFU for E. coli K99+ and Salmonella respectively, and 50 oocysts for C. parvum per reaction. Diagnostically, the panel was able to detect all five target pathogens directly from fecal samples and was more rapid and sensitive than conventional diagnostic tests (i.e., antigen-capturing ELISA, bacterial culture, direct microscopy with acid-fast staining).

The second study evaluated the diagnostic performance of a commercial “rapid” antigen detection kit named “Bovine Enterichek® ” (hereafter, Enterichek) in comparison to BEP. The test agreement (κ value) between Enterichek and BEP were 0.095 (BCoV), 0.521 (BRV-A), 0.823 (E. coli K99+), and 0.840 (C. parvum). In comparison to BEP, the diagnostic sensitivity of Enterichek was 60.0%, 42.3%, 71.4% and 81.5%; and the diagnostic specificity was 51.4%, 100%, 100%, and 98.6% for BCoV, BRV-A, E. coli K99+, and C. parvum, respectively. While Enterichek can be an animal-side or at-clinic rapid test tool in the field for detection of C. parvum or E. coli K99+ in feces from calves at acute stage of clinical disease, BCoV positive and BRV-A negative results by the kit requires careful interpretation due to its relatively low specificity and sensitivity.

In the third study, the detection frequency and genetic relatedness of BNoV among bovine diarrhea cases in the US Midwest was assessed. Total 102 fecal samples were collected from clinically diarrheic animals originated in 82 different cattle farms in 8 states and tested by PCR-based assays. BNoV was detected in 53 samples (52%), suggesting endemic status in diarrheic bovine and emphasizing the need for further evaluation of its clinical significance. Among 38 BNoVs successfully sequenced for polymerase gene, 14 and 24 BNoVs were phylogenetically classified into GIII-1 and GIII-2, respectively. Interestingly GIII-1 BNoVs were identified at a much higher rate than expected based on previous reports in US. Clustering with ≥10% sequence divergence between clusters was observed within each genotype, justifying establishment of subtypes. Besides mutations, recombination among BNoVs appeared to occur frequently since the genotype of viruses was frequently switched when compared by capsid gene, raising the need for better classification criteria.

The fourth study was conducted in a case-control manner to: 1) survey the prevalence of 7 historically well-known and 4 emerging bovine enteric agents and 2) determine their association with calf diarrhea. Fecal samples were obtained from diarrheic (n=199) and healthy (n=249) calves and tested by multiple multiplex PCRs for the 11 enteric pathogens[BRV-A, BCoV, bovine viral diarrhea virus (BVDV), BEV, BNoV, Nebovirus, bovine torovirus (BToV), Salmonella, E. coli K99+, and Clostridium perfringens (C. perfringens) with β toxin gene, and C. parvum]. The association between the presence of pathogens individually or concurrently and diarrhea was analyzed by 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 calf diarrhea (p<0.05). Among them, BRV-A, Nebovirus, Salmonella, E. coli K99+, and C. parvum showed a stronger association with diarrhea, and BRV-A infection appeared to be responsible for ‘watery&rsquo diarrhea. To our surprise none of the samples tested was positive for C. perfringens type C. Overall, viral etiology or co-infection of virus and C. parvum were the major contributor to calf diarrhea.

In testing 1274 water samples collected from 13 pasture streams in Southern Iowa during 2007-2009 grazing seasons for viral bovine biomarkers and coliform bacteria, BEV, BCoV and BRV were detected in 3.91%, 1.12% and 0.48% of the samples, respectively. There was a trend of BEV incidence difference between up- and down-stream sites, implying a dilution effect and/or loading of contaminant from the pasture. Total coliform bacteria counts did not correlate with BEV incidence as well as cattle presence or stocking density, indicating that other sources of fecal contamination may contribute to bacterial loading of pasture streams. Although the study results suggest that multiple factors affect the quality of pasture streams rather than solely cattle-originated contaminant, appropriate cattle grazing or pasture management practices should be considered to minimize bovine fecal contamination of pasture stream.

In conclusion, multiple pathogens were involved in calf diarrhea and frequently infected animals concurrently; hence, PCR-based panel testing for simultaneous detection of multiple pathogens may be a better way to assess their epidemiology and clinical significance in calf diarrhea. Historically known major bovine enteric pathogens, such as BRV-A, BCoV, Salmonella, E. coli K99+ and C. parvum, were commonly and significantly associated with calf diarrhea, suggesting that lack of appropriate maternally derived herd immunity is still of concern for controlling calf diarrhea. Unexpectedly bovine caliciviruses (i.e., BNoV and Nebovirus) were identified as significant bovine enteric pathogens. In particular BNoV was found to be widely distributed among diarrheic bovine in the Midwest USA with considerable genetic diversity. These observations raise the need to pay attention to these emerging pathogens for better control of bovine enteric diseases. Transmission of enteric pathogens through pasture streams is unlikely although bovine feces can be a source for microbial contamination of stream water, which could be minimized by better grazing management.

Copyright Owner

Yong-il Cho

Language

en

File Format

application/pdf

File Size

218 pages

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