Preventive Veterinary Medicine

Accuracy of qPCR and bacterial culture for the diagnosis of bovine intramammary infections and teat skin colonisation with Streptococcus agalactiae and Staphylococcus aureus using Bayesian analysis


Source: Line Svennesen a, Yasser S.Mahmmod ab1, Nanna K.Skjølstrup a, Louise R.Mathiasen a, JørgenKatholm c, Karl Pedersen d, Ilka C.Klaas a2, Søren S.Nielsen a.

a Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark
b Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig, Sharkia Province, Egypt
c DNA Diagnostic A/S, Voldbjergvej 14, 8240 Risskov, Denmark
d National Veterinary Institute, Technical University of Denmark, 2800, Kongens Lyngby, Denmark

ABSTRACT: Streptococcus agalactiae (Strep. agalactiae) and Staphylococcus aureus (Staph. aureus) are originally regarded as contagious mastitis pathogens, however, both pathogens have recently been isolated from extramammary and environmental sites, indicating that other sites than the udder might contribute to the spread of these pathogens potentially causing intramammary infections. Diagnostic tools to identify pathogens at extramammary sites are available but still needs to be validated. The objective of this cross-sectional field study was to estimate the diagnostic sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 qPCR assay and bacterial culture (BC) in identifying Strep. agalactiae and Staph. aureus from milk and teat skin samples. We randomly selected 3040 cows with high somatic cell counts from eight Danish Strep. agalactiae-positive dairy herds with automatic milking systems. Teat skin samples and aseptic milk samples were collected from right rear quarters (n?=?287) for BC and PCR analysis. Se and Sp were estimated in a Bayesian latent class analysis. For milk samples, the Se and Sp of qPCR for Strep. agalactiae were estimated to 0.97 and 0.99, respectively, whereas the Se and Sp of BC were 0.41 and 1.00, respectively. The Se and Sp of qPCR for Staph. aureus were estimated to 0.95 and 0.99, respectively, whereas the Se and Sp of BC were 0.54 and 0.77, respectively. For teat skin samples,

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Journal of Dairy Science

Elimination of selected mastitis pathogens during the dry period


SOURCE: Anri A. E. Timonen 1, Jørgen Katholm1, Anders Petersen 1,Toomas Orro2, Kerli Mõtus 2 and Piret Kalmus 1.

1 Chair of Clinical Veterinary Medicine, Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, 51014 Tartu, Estonia
2 DNA Diagnostic A/S, Voldbjergvej 16, 8240 Risskov, Denmark.

ABSTRACT: We aimed to evaluate the elimination of 4 different mastitis pathogens, Streptococcus agalactiae, Myco-plasma bovis, Staphylococcus aureus, and Streptococcus uberis, from infected udder quarters during the dry period using quantitative PCR. The second purpose of this study was to evaluate the association between milk haptoglobin (Hp) concentration and the presence of udder pathogens (Strep. agalactiae, Staph. aureus, M. bovis, and Strep. uberis) in udder quarter milk samples before and after dry period. Aseptic udder quarter milk samples (n = 1,001) were collected from 133 dairy cows at dry off and at the first milking after calving from 1 large dairy herd. Bacterial DNA of Strep. agalactiae, Staph. aureus, Strep. uberis, and M. bovis in the udder quarter milk samples was identified with commercial quantitative PCR analysis Mastitis 4B (DNA Diag-nostic A/S, Risskov, Denmark). Milk Hp concentration (mg/L) was measured from udder quarter milk samples. The elimination rates during the dry period for M. bo-vis, Staph. aureus, Strep. agalactiae, and Strep. uberis were 86.7, 93.6, 96.2, and 100.0%, respectively. The new IMI rate was 3.0% for M. bovis, 2.9% for Staph. aureus, 2.4% for Strep. agalactiae, and 3.1% for Strep. uberis. The milk Hp concentration was significantly higher in udder quarter milk samples with blood and in samples positive for Strep. agalactiae at dry off and for Staph. aureus postcalving.

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JIA 2018, 06

Evaluation of a new qPCR test to identify the organisms causing high total bacterial count in bulk tank milk



SOURCE: Jørgen Katholm1, Lene Trier Olesen2, Anders Petersen1, Snorri Sigurdsson3

1 DNA Diagnostic A/S, Risskov 8240, Denmark
2 ARLA Foods amba, Viby 8260, Denmark
3 SEGES, Aarhus N 8200, Denmark
ABSTRACT: Milk quality in bulk tank milk (BTM) is measured by flow cytometry technology as total bacterial count (TBC) and somatic cell count (SCC). To investigate SCC problems, culture or PCR can be used to identify mastitis causing bacteria, e.g., Mastit 4, a commercially available qPCR test. TBC in BTM can be investigated further using culture-based methods such as standard plate count, laboratory pasteurization count, coliform count, and spore counts. To our knowledge, no qPCR addressing the bacteria involved in TBC has been commercially introduced. The aim of this study is to evaluate a recently introduced 3-h qPCR test, TBC 4. The TBC 4 qPCR detects four target groups, Pseudomonas, Streptococci, Enterobacteriacea/ Enterococcus, and Bacillus/Clostridia. These target groups relate to problems on the farm such as cooling, mastitis, environment, and silage. We will continue with new research to compare the TBC 4 qPCR test with traditional culture. For this study, BTM samples from different TBC intervals were selected based on BactoCount results found at routine payment investigation at Eurofins laboratory (Vejen, Denmark). These samples were analyzed using TBC 4 qPCR assay within 24 h. In total, 346 BTM samples were divided into six different intervals of colony forming units (CFU).

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Preventive Veterinary Medicine

Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples



SOURCE: Ingrid H. Holmøy, Nils Toft, Hannah J. Jørgensen, Tormod Mørk, Liv Sølverød,. and Ane Nødtvedt


ABSTRACT: Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional acteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR.

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Journal of Dairy Science

Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples



SOURCE: J. B. Soltau, E. Einax, K. Klengel, J. Katholm, K. Failing, A. Wehrend, and K. Donat
ABSTRACT: The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis.

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Journal of Dairy Science

Within-herd prevalence of intramammary infection caused by Mycoplasma bovis and associations between cow udder health, milk yield, and composition

Published online: June 7, 2017

SOURCE: Anri A.E. Timonen, Jørgen Katholm, Anders Petersen, Kerli Mõtus, Piret Kalmus
ABSTRACT: Subclinical mastitis is one of the major health problems in dairy herds due to decreased milk production and reduced milk quality. The aim of this study was to examine the within-herd prevalence of subclinical intramammary infection caused by Mycoplasma bovis and to evaluate associations between M. bovis and cow daily milk yield, udder health, and milk composition. Individual cow composite milk samples (n = 522) were collected from all lactating dairy cows in 1 Estonian dairy farm in November 2014. Daily milk yield, days in milk, and parity were recorded. Collected milk samples were analyzed for somatic cell count, milk protein, fat, and urea content.

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ICAR The Global Standard For Livestock Data

Proceedings of the 40th ICAR Biennial Session held in Puerto Varas, Chile, 24-28 October 2016


SOURCE: J. Katholm and A. Pedersen, DNA Diagnostic
The use of Real-time PCR tests to identify mastitis pathogenes are growing, as they are faster and more sensitive than conventional bacteriological culturing, especially for Mycoplasma detection. Since 2014 a quantitative real-time PCR test kit (Mastit 4, DNA diagnostic) has been commercially available. the objective of this study was to investigate the correlation between Ct values of the Mastit 4 PCR test kit for and bacterial colony forming units (CFU) in fresh milk samples for the 11 bacteria that can be detected by the Mastit 4 BDF, CFU, PCR test kit.

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NMC Annual Meeting Proceedings (2017)

Providing Routine PCR Testing as Part of Official Milk Recordings Options


SOURCE: Angela Dawn Coburn, Agsource Cooperative Service, Verona, Wisconsin USA
Introduction: Obtaining Somatic Cell Count (SCC) value from routine milk sample analysis is, historically, a primary reason U.S. dairy producers participate in official milk recordings program. With improves management of environmental conditions and general cow health, SCC levels have shown a steady decline over the past 4 years. Among Agsource herds, weighted average SCC for Holstein have dropped from 263,000 in 2012 to 227,000 in 2016. The percent of cows with SCC over 200,000 SCC has dropped from 24,3 % to 20,4 %.

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Jena at IDF Nantes Mastitis Conference (2016)

Jena at IDF Nantes Mastitis conference


SOURCE: J. B. Soltau, E. einax, K. Klengel, K. Donat, Animal Health Service, Thuringian animal Diseases Fund, Jena, Thuringia Germany. J. Katholm, DNA diagnostic, Risskov Denmark. K. Failing, Unit for Biomathermatics and Data Processing, Justus-liebig University, Giessen, Hesse Germany. A. Wehrend, Clinic of Obstetrics, gynaecology and Andrology for small and large Animals, Justus-Liebig University, Giessen, Hesse Germany.
Introduction: The aim of the study was to analyse the value of a polymerase chain reaction (PCR) examination of bulk tank samples for bovine mastitis pathogens as a tool to estimate the within herd prevalenceof these pathogens.
A total of 6,335 quarter milk samples were collected from 1,615 cows farms in Germany. Additionally, two bulk tank milk samples were collected from each farm. The quarter milk samples were cultivated in the laboratory of Animal Disease Fund (Jena, Thuringia, Germany). The bulk tank milk samples were tested by the commercial PCR assay Mastit 4 (DNA Diagnostic, Denmark) that identified Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and Streptocuccus uberis.

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NMC Annual Meeting Proceedings (2016)

Test characteristics of the qPCR test Mastit 4 to identify Major Pathogenes in spiked and originally infected Milk samples.


SOURCE: Torben W. Bennedsgaard, Line Svennesen, Ilka C. Klaas Aarhus University, Foulum Denmak and University of Copenhagen Denmark
Introduction: Real-time PCR tests are increasingly used to identify mastitis pathogenes as they are faster and more sensitive than conventional bacteriogical culturing. Recently a new commercially availeble quantitative real-time PCR test kit (Mastit 4, DNA Diagnostic) was introduced in Denmak and is mainly used to test cows prior to dry -off for subclinical intramammary infections or in herd screenings for eradication of Streptocuccus agalactiae. The objective of our study was to investigate the association between ct values of the Mastit 4 PCR test kit for and colony forming units in fresh milk samples from quaters without clinical mastitis for Streptocuccus agalactiae, Staphylocuccus aureus, Streptocuccus uberis and Streptococcus dysgalactiae.

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NMC Annual Metting Proceedings (2015)

Bulk Tank milk Surveillance For Contagios Mastitis Pathogenes: Comparison Of Two Commercial Real-Time PCR Test Kits


SOURCE: Erik Rattenborg, Carl O. Paulrud, Søren K. S. Jensen and Jørgen Katholm
Knowledge center for agriculture, Cattle, Aarhus N, Denmak
Eurofins, Vejen, Denmark
DNA diagnostic, Risskov, Denmark
As part of the Danish Surveillance program for Streptococcus agalactia. bulk tank milk (BTM) samples from all dairy herds are tested annually. since 2009 the bulk tank samples has been analyzed using real-time PCR PathoProof Mastitis assay (Thermo fisher Scientific, Vantaa, Fiinland) In 2009 and 2010 the analyses were carried out with the complete 12 Kit and in 2011-2013 with the complete 16 kit. Indseptember 2014 the real-time PCR Mastit 4 B (DNA Diagnostic, risskov, Denmark) was made commercially availeble. the objective of this study was to compare the PathoProof Mastitis Complete 16 kit with the Mastit 4b kit regarding the detection of four species of bacteria tested by both test (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis and Mycoplasma bovis) the comparison reveals essential knowledge tha may be used in designing the future surveillance program.

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