This study demonstrated and evaluated practical applicability of the integrated VetMAX™ MAP Real-Time PCR kit on reconstituted infant milk samples, artificially spiked milk, and field milk samples obtained from dairy herds. The standardized protocol was provided by a DNA isolation method, possible cross contamination between samples was avoided with a semi-automatic system, resulting in a high throughput rate of tested samples. An IAC is critical for PCR systems because it allows monitoring of reactions for associated inhibition and amplification efficiency. The results of this study suggest that real-time PCR using ISMAP02 primers is useful in practice, being both fast and cost-effective, to identify MAP as a possible cause for chronic individual and herd health problems. Furthermore, the results supported the validity of the MAP real-time PCR kit as a powerful tool to detect MAP in milk samples. However, fecal samples appear to be more suitable for predicting an association between MAP infection and the health status.
SUMMARY
69 7 SUMMARY
Ahmad Alajmi
Occurence of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk and feces samples from Nothern Germany
MAP, the etiological agent of paratuberculosis, is economically important in dairy operations because it affects ruminants such as cattle as the causative agent of Johne's disease as well as perhaps the causative agent of CD. The many different commercially produced tests for detecting MAP in different matrices have varied advantages, disadvantages and applications.
In the present study, the first aim was to evaluate the VetMAX™ MAP Real-Time PCR Screening Kit (Thermo Fisher Scientific™) using milk samples since the commercial kit is certified only for the detecting of MAP in feces and environmental samples. The second aim was to evaluate the practicability of the applied new molecular technique using the previously mentioned real-time PCR kit and the MagVET™ Mycobacterium paratuberculosis Isolation Kit (Thermo Fisher Scientific™) to quickly detect MAP in feces, composite foremilk samples and bulk milk samples of chronically diseased milking cows on dairy farms in Northern Germany.
The integrated VetMAX™ MAP Real-Time PCR kit was evaluated, including a unique transposon sequence; (ISMAP02) that was targeted to provide sensitive-specific results. The analytical sensitivity of the assays was determined using MAP type strain ATCC 19698 (DSM 44133). The assay's specificity was validated by testing 15 isolates of MAP, 13 isolates of non-MAP Mycobacterium species and 8 isolates of other related bacterial non-Mycobacterium species. Six spiked experiments were performed using multiple 50 mL-1 samples of both raw milk and reconstituted infant milk (BEBA®) that were spiked with a tenfold serial dilution containing 100 to 105 MAP cells mL-1. However, 3 raw milk samples from a local farm and 3 reconstituted infant milk formula samples were tested independently.
The detection probability in raw milk for the samples containing 1.4 × 101 MAP cell 50 mL-1 amounted to 16.6% and the detection probability for reconstituted infant milk samples containing 1.7 × 101 MAP cell 50 mL-1 amounted to 91.6%.
Additionally, the diagnostic kit was applied on the field samples for MAP detection. A total of 928 fecal, 922 composite foremilk and 92 bulk milk samples obtained from 58 case and 35
70
control dairy herds were investigated. The real-time PCR showed MAP positive results for 11 (18.96%) and 6 (17.14%) of the case and control herds, respectively. The odds ratio for the association between MAP-positive PCR results from milk and fecal samples and the herd disease status were OR = 1.9 (95% CI = 0.75.4) and OR = 5.7 (95% CI = 2.016.2), respectively. All bulk milk samples were MAP negative. The result of fecal and milk samples were moderately correlated (kappa = 0.27).
Finally, the validity of the commercial MAP real-time PCR kit to detect MAP in milk was supported by the study results. The data results indicate that real-time PCR results have diagnostic value for diagnosing MAP positive animals, whereby fecal samples are more suitable than composite foremilk samples when the assay is applied on samples from diseased cows.
ZUSAMMENFASSUNG
71 8 ZUSAMMENFASSUNG
Ahmad Alajmi
Vorkommen von Mycobacterium avium subspecies paratuberculosis (MAP) in Kuhmilch und Facesproben aus Norddeutschland
Mycobacterium avium subspecies paratuberculosis (MAP), der Erreger der Paratuberkulose (auch als Johne’sche Krankheit bekannt), stellt aufgrund des chronisch-auszehrenden Verlaufes einen wichtigen Wirtschaftsfaktor für die Milchwirtschaft dar. Darüber hinaus besteht die Möglichkeit eines Zusammenhangs mit dem den Menschen betreffenden Morbus Crohn. Die vielen verschiedenen kommerziellen Tests zur Erkennung von MAP in unterschiedlichen Matrices haben eine Reihe von Vor- und Nachteilen. In der hier vorgelegten Studie ging es vorrangig darum, die Effizienz des VetMAX™ MAP Real-Time PCR Screening Kit (Thermo Fisher Scientific™) in Milchproben zu ermitteln, da sich die Zertifizierung dieses kommerziellen Testsystems eigentlich auf den Nachweis von MAP in Kot- und Umweltproben begrenzt. Das zweite Ziel war, die Praktikabilität der angewandten, neuen molekularbiologischen Technik unter Verwendung des oben genannten Real-Time-PCR-Kits und dem MagVET™ Mycobacterium paratuberculosis Isolationskits (Thermo Fisher Scientific™) zum schnellen Nachweis von MAP in Kot- und Gesamtviertelgemelksproben chronisch erkrankter Milchrinder in Norddeutschland sowie in Tankmilchproben aus Herden, in denen sich diese chronisch kranken Tiere befinden, zu beurteilen.
Das VetMAX™-MAP-Real-Time-PCR-Kit arbeitet mit einer individuellen Transposonsequenz (ISMAP02) als Target für sensitiv spezifische Nachweise. Die analytische Sensitivität des Testsystems wurde anhand des MAP-Typstammes ATCC 19698T (DSM 44133)T bestimmt, die Spezifität mit 15 weiteren MAP-Isolaten, 13 Isolaten von Nicht-MAP-Mykobakterien und 8 weiteren Isolaten verwandter Nicht-Mykobakterien. Dabei wurden alle Stämme sicher als MAP bzw. als Nicht-MAP erkannt. Sechs Experimente mit künstlicher Kontamination wurden mit 50 ml-Proben von Rohmilch und rekonstituierter Säuglingsnahrung durchgeführt. Jede Probe wurde mit einer Verdünnung in Zehnerschritten mit 100 bis 105 MAP-Zellen/ml künstlich kontaminiert. Rohmilch und Säuglingsmilch wurden jeweils an unterschiedlichen Tagen untersucht. Die Nachweiswahrscheinlichkeit in Rohmilch
72
mit 1,4 × 101 MAP-Zellen/50 ml betrug dabei 16,6 %, die von 1,7 × 101 MAP-Zellen/50 ml in der Säuglingsnahrung 91,6 %.
Darüber hinaus wurde das Testsystem an Feldproben zur Diagnostik von MAP verwendet.
Insgesamt wurden 928 Kot-, 922 Gesamtviertelgemelks- und 92 Tankmilchproben aus 58 Fall- und 35 Kontrollherden (die eine Anzahl von Kriterien bezüglich chronischer Erkrankungen erfüllen mussten bzw. nicht aufweisen durften) untersucht. Mittels Real-Time-PCR wurden 11 (18,96) der Fall- und 6 (17,14 %) der Kontrollherden positiv auf MAP getestet. Die Odds Ratio als Maß des Zusammenhanges zwischen MAP-positiven Ergebnissen aus Milch und aus Kotproben sowie dem Krankheitsstatus der Herde belief sich auf OR = 1,9 (95% CI = 0,75,4) bzw. OR = 5,7 (95% CI = 2,0 16,2). Alle Tankmilchproben waren MAP-negativ. Die Ergebnisse für Kot- und Milchproben stimmten weitestgehend überein (kappa = 0,27).
Zusammenfassend ist festzuhalten, dass die Validität des kommerziellen Real-Time-PCR-Kits zum Nachweis von MAP in Milch durch die Ergebnisse dieser Studie bestätigt werden konnte. Diese belegen, dass Real-Time-PCR-Ergebnisse MAP-positive Tiere detektieren können, wobei sich Kotproben als aussagekräftiger als Gesamtviertelgemelke erwiesen, wenn der Test an erkrankten Kühen durchgeführt wird.
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