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

Cho, Yong Il; Kim, Won Il; Liu, Siyuan; Kinyon, Joann M.; Yoon, Kyoung‐Jin

doi:10.1177/104063871002200403

Cited by 81 publications

(81 citation statements)

References 29 publications

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“…These multiplex assays depend on pathogen differentiation based on the size of PCR amplicons, and therefore requires gel-electrophoresis following PCR amplification. Recently several multiplex real-time PCR assays have been developed to detect pathogens associated with BRDC and BED (Horwood and Mahony, 2011;Marley et al, 2008;Thonur et al, 2012;Cho et al, 2010). Alternatively, a real-time PCR based system was described that includes multiple reactions in one-run with similar thermal cycling conditions for detection of BRDC and BED pathogens (Kishimoto et al, 2017;Tsuchiaka et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Thanthrige-Don

Lung

Furukawa-Stoffer

et al. 2018

Journal of Virological Methods

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Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

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Section: Introductionmentioning

confidence: 99%

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Thanthrige-Don

Lung

Furukawa-Stoffer

et al. 2018

Journal of Virological Methods

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“…These procedures are reliable; however, they are time-consuming and require specialized knowledge. Recently, nucleic acid based tests, such as polymerase chain reaction (PCR) assays, have become popular for rapid and sensitive detection of infectious agents Cho et al, 2010). Multiplex real-time PCR panels have been proven to be a useful diagnostic tool for concurrent detection of several target enteric pathogens with high sensitivity and specificity Cho et al, 2010), which decreases bias in diagnostic outcome due to testing method.…”

Section: Introductionmentioning

confidence: 99%

Case–control study of microbiological etiology associated with calf diarrhea

Cho

Han

Wang

et al. 2013

Veterinary Microbiology

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132

128

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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.

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“…Diarrhea caused by a variety of bacteria has been recognized as one of the most public clinical problems for calves worldwide. Among these bacteria Eschirechia coli (E. coli) as "white scour", Salmonella typhyimurium (S. typhimurium), Clostridium perfringens (C. Perfringens) and Staphylococcus aureus (St. aureus) are believed to be the major microbial causes of diarrhea in calves [4][5][6].…”

Section: Introductionmentioning

confidence: 99%

Diagnostic Aspects and Novel Approach for Treatment of Antibiotic-Resistant Bacteria Isolated from Diarrheal Calves using Silver, Gold and Copper Nanoparticles

Behiry¹

2014

J Bacteriol Parasitol

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This study was performed to identify various bacteria from feces of calves suffering from diarrhea, and to determine in vitro antimicrobial activity of silver, gold and copper nanoparticles against the antibiotic-resistant bacteria isolated from fecal samples. Fecal samples were collected from 153 diarrheic calves and primarily tested for the presence of Escherichia coli, Salmonella spp. and Staphylococcus aureus using bacteriological examination, biochemical reactions and polymerase chain reaction. The minimum inhibitory concentrations of silver, gold and copper nanoparticles against identified bacterial isolates were determined by a broth dilution method. 84 bacterial isolates from the 153 fecal samples were identified using bacteriological, biochemical and genotypical methods. Escherichia coli was considered the most frequent bacterium isolated numbering 31 (36.90%) followed by Salmonella sp. as the second most prevalent 16 (19.04%). Further isolates such as Staphylococcus aureus 10 (11.90%) were isolated and identified. The MIC values of silver, gold and copper nanoparticles with a size of 10 nanometers (nm) against the three types of bacteria were ranged from 0.625 to 10 μg/ml, 2.5 to 20 μg/ml and 2.5 to 20 μg/ml, respectively. While these values with a size of 20 nm were 0.312 to 2.5 μg/ml, 1.25 to 10 μg/ml and 2.5 to 10 μg/ml, respectively. Additionally, the mean time of the antimicrobial action of silver, gold and copper nanoparticles at concentration 10 nm against all isolates was 5 min, 30 min and 15 min, respectively. Using of these nanoparticles in a concentration of 20 nm, the mean time was 1, 15 and 5 min, respectively. These in vitro results clearly indicate that the silver, gold and copper nanoparticles might have a superior activity and rapid onset of action against the Gram negative bacteria E. coli and Salmonella spp. and the Gram positive bacteria Staphylococcus aureus with diarrheal origin.

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

Cited by 81 publications

References 29 publications

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

Case–control study of microbiological etiology associated with calf diarrhea

Diagnostic Aspects and Novel Approach for Treatment of Antibiotic-Resistant Bacteria Isolated from Diarrheal Calves using Silver, Gold and Copper Nanoparticles

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