Proceedings of the 10th International Colloquium on Paratuberculosis

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A total <strong>of</strong> 206 quarter milk samples were aseptically collected, and bulk tank milk samples from each <strong>of</strong> <strong>the</strong> 16 dairy herds in <strong>the</strong> region <strong>of</strong> Viçosa, MG, totaling 16 bulk tank milk samples, were collected. All <strong>the</strong> samples were processed and PCR was performed according to Sivakumar et al. (2005). A wild certified MAP strain was used as positive control; milli-Q water was used as negative control and �X174/HaeIII as molecular marker. In order to confirm <strong>the</strong> identity <strong>of</strong> <strong>the</strong> amplified fragments (626bp), <strong>the</strong>y were cloned in <strong>the</strong> pGEM vector using <strong>the</strong> pGem T-Easy TM Vector System. Plasmid DNA was purified with Wizard® Plus SV Minipreps DNA Purification System. DNA sequencing, adapted by Sanger et al. (1977), was performed to confirm <strong>the</strong> sequences, by using <strong>the</strong> M13 forward and reverse primers. The obtained sequences were edited by <strong>the</strong> DNA MAN s<strong>of</strong>tware and compared to IS900 (Green et al., 1989) deposited in Genbank under <strong>the</strong> accession number X16293.1, using <strong>the</strong> Basic Local Alignment Search Tool (BLAST) s<strong>of</strong>tware, available at National Center for Biotechnology Information – NCBI website (http://www.ncbi.nlm.nih.gov). Multiple alignments were obtained using <strong>the</strong> s<strong>of</strong>tware ClustalW and from <strong>the</strong>se, genetic groups were performed using <strong>the</strong> s<strong>of</strong>tware MEGA version 4.0 Windows, by <strong>the</strong> UPGMA method. Statistical analysis was performed using 1000 replications <strong>of</strong> bootstrap. RESULTS AND DISCUSSION Eight quarter milk samples (3.6%) and none <strong>of</strong> <strong>the</strong> bulk tank milk samples amplified fragments <strong>of</strong> <strong>the</strong> expected size, 626bp (Figure 1). Out <strong>of</strong> <strong>the</strong> cloned samples, three (37.5%) and <strong>the</strong> positive control were successfully sequenced, but it was not possible to sequence <strong>the</strong> o<strong>the</strong>rs. The genetic analysis showed 99% <strong>of</strong> identity between <strong>the</strong> sequences <strong>of</strong> this study and <strong>the</strong> sequence X16293.1; 90% with M. avium (AF527957.1); 91-92% with Mycobacterium sp. (AF455252) not-yet-identified; and 74% with Streptomyces turgidiscabies (AY707080.1). Among <strong>the</strong> compared sequences, 11 were randomly selected to carry out a genetic sequence alignment among <strong>the</strong> 626 nucleotides, toge<strong>the</strong>r with <strong>the</strong> four sequences <strong>of</strong> this study (Figure 2). From this alignment, a gene cluster map among <strong>the</strong> 15 gene sequences was built, showing high levels <strong>of</strong> similarity, since <strong>the</strong> sequences were pooled toge<strong>the</strong>r within <strong>the</strong> same tree branch (Figure 3). Only one sequence showed lower similarity and is located in ano<strong>the</strong>r branch, probably for being a not-yet-identified Mycobacterium species. Figure 1: PCR products visualized in 1% agarose gel electrophoresis, using primer pairs BN1/BN2. 1, 2, 3, 4, 5, 6, 7 and 8) amplified samples; 9) positive control; 10) negative control: milli-Q water; M) molecular marker: �X174/HaeIII. Figure 2: Part <strong>of</strong> <strong>the</strong> genetic alignment between DNA fragments sequenced and sequences existing in GenBank. 275 2
Figure 3: Gene cluster map between genetic sequences <strong>of</strong> MAP described in this study (MAP-UFRGS; MAP-UFV/07; MAP-UFV/09 and MAP-UFV/J) and sequences available on GenBank (X16293, AF416985, AJ250015, AJ250022, AJ250023; AJ250020, AY660658, AJ251436, AF305073, AY660657 and AF455252). Method: UPGMA; Statistical Support: 1000 replications <strong>of</strong> bootstrap. CONCLUSION Results confirmed presence <strong>of</strong> MAP in <strong>the</strong> analyzed samples that showed identity with <strong>the</strong> insertion sequence IS900. This study confirmed MAP in <strong>the</strong> tested milk samples, providing key information about presence <strong>of</strong> paratuberculosis in dairy herds in <strong>the</strong> region <strong>of</strong> Viçosa, Minas Gerais State, Brazil. Since <strong>the</strong>re is a lack <strong>of</strong> <strong>of</strong>ficial paratuberculosis surveys and control programs in Brazil, fur<strong>the</strong>r studies will be needed to support <strong>the</strong> adoption <strong>of</strong> national paratuberculosis control measures. The dilution that occurs in bulk tank samples makes <strong>the</strong>m non-ideal for PCR analysis, despite being <strong>the</strong> type <strong>of</strong> sample chosen to perform a herd screening. This is <strong>the</strong> first report <strong>of</strong> MAP detected in raw milk samples from dairy cattle in Brazil and it is reasonable to consider its as a first survey on <strong>the</strong> disease in <strong>the</strong> state <strong>of</strong> Minas Gerais, Brazil. ACKNOWLEDGMENTS The authors would like to thank both Pr<strong>of</strong>. Marcos Jose Pereira Gomes, from Universidade Federal do Rio Grande do Sul, for providing a wild certified MAP strain, and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for financing this study as part <strong>of</strong> <strong>the</strong> project (EDT-2412/05); “Detection <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (MAP) by PCR in bovine raw milk samples”. REFERENCES Abubakar, I., D. J. Myhill, A. R. Hart, I. R. Lake, I. Harvey, J. M. Rhodes, R. Robinson, A. J. Lobo, C. S. J. Probert, and P. R. Hunter. 2007. A case-control study <strong>of</strong> drinking water and dairy products in Crohn's Disease--fur<strong>the</strong>r investigation <strong>of</strong> <strong>the</strong> possible role <strong>of</strong> Mycobacterium avium paratuberculosis. Am. J. Epidemiol. 165(7):776-783. Carvalho, I. A., Silva Jr., A., Campos, V. E. B., Moreira, M. A. S. 2009. Short Communication: Detection <strong>of</strong> Mycobacterium avium Subspecies paratuberculosis (MAP) by PCR in Bovine Milk in Brazil. J. Dairy Sci. 92:5408-5410. Collins, M. T. 2003. Update on paratuberculosis: I. Epidemiology <strong>of</strong> Johne's disease and <strong>the</strong> biology <strong>of</strong> Mycobacterium paratuberculosis. Ir. Vet. J. 56(11):565-574. 276 3
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
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Scott Wells - Planning Committee Ch
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Monday, August 10, 2009 - All sessi
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Kari Røste Lybeck, Anne Kristine S
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1120-1140 #88: Influence of
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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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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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Proceedings of the 10th International Colloquium on Paratuberculosis

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