Proceedings of the 10th International Colloquium on Paratuberculosis

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#231 Application <strong>of</strong> a rapid and sensitive combined phage-PCR method for <strong>the</strong> detection <strong>of</strong> Mycobacterium avium subspecies paratuberculosis in raw milk George Botsaris a , Maria Liapi b , Charalambos Kakogiannis b , Christine Dodd a and Ca<strong>the</strong>rine Rees a a Food Sciences Division, Univ. <strong>of</strong> Nottingham, Sutton Bonington Campus, Leics, LE12 5RD, UK b Cyprus Veterinary Services, 1417 Athalassas Av, Nicosia, Cyprus ABSTRACT The objective <strong>of</strong> this study was to evaluate a new combined phage-PCR method by investigating levels <strong>of</strong> MAP in milking herds in <strong>the</strong> UK and throughout <strong>the</strong> Republic <strong>of</strong> Cyprus. Bulk milk samples were collected from 54 UK farms and 225 dairy farms in Cyprus and were tested using both <strong>the</strong> phage assay and a conventional culture method for <strong>the</strong> presence <strong>of</strong> MAP. The identity <strong>of</strong> MAP cells detected was confirmed in both cases by IS900 PCR. None <strong>of</strong> <strong>the</strong> UK samples were culture positive whereas 1 was phage-PCR positive. In Cyprus MAP 50 <strong>of</strong> <strong>the</strong> 225 samples were MAP positive using <strong>the</strong> combined phage-PCR and MAP was cultured from 2 samples, despite <strong>the</strong> fact that <strong>the</strong> animals tested were not displaying clinical symptoms <strong>of</strong> Johne’s disease. Total viable count was also determined as an indicator <strong>of</strong> <strong>the</strong> general hygiene status <strong>of</strong> <strong>the</strong> samples. Comparison <strong>of</strong> MAP status with TVC suggests that <strong>the</strong>se were not introduced by faecal contamination. Typing <strong>of</strong> <strong>the</strong> two MAP strains isolated from cow’s milk in Cyprus by culture was performed using REA IS1311 PCR and identified one <strong>of</strong> <strong>the</strong>se to be an S strain and <strong>the</strong> o<strong>the</strong>r C strain. The results confirm that <strong>the</strong> phage-based detection test is more sensitive than conventional culture and demonstrate <strong>the</strong> efficacy and practicality <strong>of</strong> <strong>the</strong> phage-based test as a routine rapid method for <strong>the</strong> detection <strong>of</strong> viable MAP in milk. INTRODUCTION Detection <strong>of</strong> Mycobacterium avium subsp. paratuberculosis (MAP) in milk currently relies on culture, immunoassays and molecular techniques. The culture based techniques require a very long incubation period <strong>of</strong> about 3 months and a chemical decontamination step which reduces <strong>the</strong> number <strong>of</strong> competitive microbes, but also reduces <strong>the</strong> number <strong>of</strong> viable Mycobacteria present in a sample, limiting <strong>the</strong> sensitivity <strong>of</strong> growth-based assays. Immunoassays have a very low sensitivity in milk and <strong>the</strong> molecular techniques have <strong>the</strong> disadvantage <strong>of</strong> not being able to differentiate live from dead cells. A new combined phage-PCR assay for <strong>the</strong> detection <strong>of</strong> MAP in milk was described by Stanley et al. (2007) based on <strong>the</strong> commercially available FASTplaqueTB TM assay for detecting tuberculosis in human sputum samples. This test detects <strong>the</strong> ability <strong>of</strong> a bacteriophage (D29) to infect MAP and provided successful identification <strong>of</strong> viable cells in milk in less than 24 hours. The advantage here is that only viable cells will support phage replication, providing live-dead differentiation, no chemical decontamination <strong>of</strong> samples is required and large volumes (up to 50 ml) can be sampled. Genotyping is <strong>the</strong>n performed by amplification <strong>of</strong> IS900 sequences from in <strong>the</strong> cells detected directly from <strong>the</strong> bacteriophage plaques (see Rees and Dodd, 2007). The aim <strong>of</strong> <strong>the</strong> work presented here was to apply <strong>the</strong> new method on real samples to assess <strong>the</strong> potential application <strong>of</strong> <strong>the</strong> test for <strong>the</strong> detection <strong>of</strong> MAP in milk and also compare <strong>the</strong> results with conventional culture for MAP. A viable count was also performed on <strong>the</strong> samples to investigate any potential relationship between <strong>the</strong> presence <strong>of</strong> MAP in milk with TVC which was used as a general indicator <strong>of</strong> <strong>the</strong> overall hygienic status <strong>of</strong> <strong>the</strong> samples. 25
MATERIALS AND METHODS Sampling For <strong>the</strong> UK samples 54 BTM samples were provided from <strong>the</strong> VLA in Sutton Bonington, UK. For <strong>the</strong> Cyprus survey samples were collected from from each <strong>of</strong> 225 registered dairy farms in Cyprus. Combined phage PCR assay BMT samples were processed by a modified version <strong>of</strong> <strong>the</strong> method described by Stanley et al. (2007). In <strong>the</strong> assay bacteria in <strong>the</strong> milk samples are collected by centrifugation and <strong>the</strong> cell pellets resuspended in 2 ml <strong>of</strong> Media Plus. Here a second wash step was included using <strong>the</strong> same centrifugation conditions and <strong>the</strong>n finally <strong>the</strong> pellet was resuspended in 1 ml Media Plus. Culture examination and identification <strong>of</strong> mycobacterial isolates Samples (30 ml) <strong>of</strong> BTM were decontaminated for 5 h with 0.75% hexadecyl-pyridinium chloride (HPC; Merck KGaA, Germany) and three samples were cultured on Herrold’s Egg Yolk Media (HEYM) containing 2 �g ml -1 <strong>of</strong> Mycobactin J; two slopes were purchased from Becton Dickinson (New Jersey, United States) and one slope was prepared as described previously (Ayele et al., 2005). Slopes were monitored after <strong>the</strong> first week <strong>of</strong> incubation to identify ei<strong>the</strong>r contaminated cultures or those with fast-growing mycobacteria and <strong>the</strong>n observed at two week intervals until <strong>the</strong>re was visible colony growth: incubation was carried out for not less than eight months at 37°C. From all primary cultures, presumptive colonies were stained using Ziehl-Neelsen (Z-N) for <strong>the</strong> presence <strong>of</strong> acid-fast bacilli (AFB). AFB isolates were identified as MAP by PCR as described previously (IS900, Whittington et al., 1998 and F57, Coetsier et al., 2000). IS1311 REA-PCR was used to distinguishing between cattle and sheep strains <strong>of</strong> MAP with PCR products being cleaved with HinfI (Marsh et al. 1999). Total viable count (TVC) TVC was performed on Milk Count Agar (MCA) using standard methods. Milk was diluted in MRD (Maximal Recovery Dilluent) and <strong>the</strong> dilutions were plated on MCA and incubated aerobically at 30 °C for 3 days. RESULTS AND DISCUSSION The results from <strong>the</strong> two surveys are presented in Table 1. From a total <strong>of</strong> 54 samples analyzed in <strong>the</strong> UK survey 19 (35.2%) were positive using <strong>the</strong> phage assay which indicates <strong>the</strong> presence <strong>of</strong> Mycobacteria spp, but one <strong>of</strong> <strong>the</strong>se samples (1.9%) was identified as MAP following PCR genotyping. A duplicate <strong>of</strong> <strong>the</strong> samples collected was cultured by <strong>the</strong> VLA and none <strong>of</strong> <strong>the</strong> samples were positive for MAP. A recent UK survey reported that 75% <strong>of</strong> dairy herds were seropositive but seroprevalance was only 0.12 and <strong>the</strong>refore levels <strong>of</strong> MAP expected in BTM would be low (Woodbine et al., 2009). From a total <strong>of</strong> 225 samples analysed for <strong>the</strong> Cyprus survey 218 (96.9%) were Mycobacteria-positive using <strong>the</strong> phage assay and 50 <strong>of</strong> <strong>the</strong>se (22.2%; 95% CI: 17.1% - 28.0%) were identified as MAP by IS900 PCR. In this case only two <strong>of</strong> <strong>the</strong> samples (0.9%) were culture positive. From <strong>the</strong> 225 cows’ BMT samples that were collected during this survey 50 (22.2%; 95% CI: 17.1% - 28.0%) were found to be MAP positive using <strong>the</strong> combined phage-PCR assay. The results <strong>of</strong> Slana et al. (2009) reported that 28.6% (95% CI: 95% CI: 22.5% – 34.3%) were MAP positive by IS900 qPCR, showing a good agreement with <strong>the</strong> level <strong>of</strong> prevalence recorded in <strong>the</strong> two different surveys undertaken. All BTM samples collected in <strong>the</strong> Cyprus were also cultured after decontamination and only two <strong>of</strong> <strong>the</strong>m (0.9%) produced colonies on HEYM slopes that were identified as MAP-positive by both IS900 and F57 qPCR. Both <strong>of</strong> <strong>the</strong> samples from which viable MAP were cultured were also phage-PCR positive. The two confirmed MAP isolates were analysed using IS1311 REA PCR and one <strong>of</strong> <strong>the</strong>se was identified as a MAP cattle strain and <strong>the</strong> o<strong>the</strong>r as a sheep strain. 26
Page 1 and 2: Book of Abstracts
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Host Response and Immunology Keynot
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#33 MERKAL AWARD LECTURE No interfe
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#75 Interleukin 17 and interleukin
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#173 Local and systemic roles for b
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#23 Role of Mycoba
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Host Response and Immunology Poster
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#28 Intestinal MAP Infection via Pe
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#73 Age susceptibility of</
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#95 Role of nitric
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#134 Analysis of <
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#157 Study of <str
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#163 Immunohistochemical expression
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#193 Development of</strong
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#209 Murine macrophages carrying an
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#224 Application of</strong
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Table 1 UK and Cyprus MAP survey Re
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#228 Mycobacterium avium ssp. parat
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#145 MERKAL AWARD LECTURE Multinomi
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#50 Assessment of
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Table 1. Posterior median (CrIs) <s
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#55 Birth clusters of</stro
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from their dam, se
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#88 Influence of b
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#104 Relation between faecal shedde
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Epidemiology Poster Abstracts 109 1
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#22 The Prevalence of</stro
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CONCLUSION IS1311-based nested Ma/M
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#42 Seroprevalence survey o
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#77 Characterisation of</st
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Environmental samples were collecte
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REFERENCES 1) Chiodini RJ, Van Krui
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#113 Effect of soi
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#136 Error-adjusted, variance parti
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Latium Region Viterbo districts RES
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#144 The suitability of</st
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#178 Age structure of</stro
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A statistically significant lower f
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#183 Sero-prevalence of</st
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#218 Johne’s disease in t
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#244 A survey to estimate t
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Control Programs Keynote Lecture Re
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#202 Milk quality assurance for par
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#141 Control of pa
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#198 Use of small
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Control Programs Poster Abstracts 1
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RESULTS Loss of al
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#40 Managing Bovine Johnes Disease
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#74 Economic analysis of</s
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#87 Evaluation of
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separately for each existing herd <
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#98 An inter-laboratory ring-trial
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#120 Paratuberculosis vaccine inter
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The statement provides: • A stand
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COMPLEMENTARY ASPECTS On-Farm Disea
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#168 Paratuberculosis in Iran: past
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#180 Behavioural change in owners <
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the fact that <str
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#182 An integrated risk based appro
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etween 0 to 10, depending on <stron
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#203 Milk quality assurance for par
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#234 Johne’s disease vaccine: a c
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Pathogenomics and MAP Biology Conve
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#45 Identification of</stro
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#117 Adsorption of
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#222 Atypical structural features <
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Pathogenomics and MAP Biology Persp
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#52 Is ‘Indian Bison Type’ Myco
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#118 Cloning, expression and purifi
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#213 Construction the</stro
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Abstract not available at press tim
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#177 Associations between CARD15 po
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Perspectives Talk: Public Health My
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A total of 206 qua
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Feller, M., K. Huwiler, R. Stephan,
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In 2008, the Ameri
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#115 Crohn’s disease and ruminant
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International John
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#76 Towards improved reporting stan
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#97 The EU ParaTBTools Project - Th
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#241 US Vaccine Initiative through
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Paratuberculosis ELISA Reliable, si
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