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Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds
Available online at www.sciencedirect.com
Preventive Veterinary Medicine 86 (2008) 8–13 www.elsevier.com/locate/prevetmed
Short communication
Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds Masoud Haghkhah a, Maryam Ansari-Lari b,*, Amir Mansour Novin-Baheran a, Ayatollah Bahramy a a b
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345, Iran Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Besat Street, Shiraz 71345, Iran Received 13 August 2007; received in revised form 21 March 2008; accepted 27 March 2008
Abstract A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection. Bulk-tank milk samples were collected from 110 dairy herds in the 3 districts (Shiraz, Marvdasht and Sepidan) of the province. Among study populations, 12 herds (11%, 95%CI: 5–17%) were positive for MAP infection based on IS900 nested PCR. The prevalence of positive milk samples in the three districts of Fars province was different ranging from 8.6% to 23% which was not statistically significant (P = 0.19). It is recommended to conduct further epidemiologic studies to determine cow-level prevalence and risk factors for infection, and to evaluate the economic consequences of the MAP infection in the region. # 2008 Elsevier B.V. All rights reserved. Keywords: Iran; Johne’s disease; Mycobacterium avium subspecies paratuberculosis; Prevalence
1. Introduction Johne’s disease is a chronic disease of ruminants worldwide caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). The Johne’s disease in the dairy community has long been considered as an economically important disease by reports of the negative impact on milk production and the overall economic health of dairies (Benedictus et al.,
* Corresponding author. Tel.: +98 711 2286950; fax: +98 711 2286940. E-mail addresses: [email protected], [email protected] (M. Ansari-Lari). 0167-5877/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2008.03.010
M. Haghkhah et al. / Preventive Veterinary Medicine 86 (2008) 8–13
9
1987; Johnson et al., 2001; Beaudeau et al., 2007; Gonda et al., 2007). This disease has gained notoriety in recent years because of concern about a possible relationship to Crohn’s disease, an intestinal disorder in humans (Greenstein and Collins, 2004; Gaya et al., 2004; Romero et al., 2005). These suggestions call for further knowledge on the prevalence of the infection in the cattle population around the world. Surveys for MAP are difficult to accomplish because of the chronic nature of the infection and the lack of good testing methods for animals in the pre-clinical stages. Diagnostic tests such as ELISA, agar gel immunodiffusion (AGID) test and fecal culture are being used commonly. Also PCR methods, targeting MAP-specific insertion sequence (IS900) or other species-specific genes have been developed to increase sensitivity and specificity of diagnosis (Millar et al., 1995; Englund et al., 1999; Djonne et al., 2003). Presence of the pathogen in milk has been confirmed by several researchers (Sweeney et al., 1992; Grant, 2003). It was shown that M. paratuberculosis could be detected directly from quarter milk and bulk-tank milk by IS900 PCR (Pillai and Jayarao, 2002; Jayarao et al., 2004; Buergelt and Williams, 2004). Therefore, application of PCR for milk samples from individual cows or at herd level as bulk-tank milk have proven useful for prevalence estimation of infection with M. paratuberculosis by some studies (Corti and Stephan, 2002; Grant et al., 2002; Stabel et al., 2002). Prevalence of infection in many countries in the world is unknown, but herd prevalence of Johne’s disease in Europe is reported to be between 7% and 55%, in the United States nearly 40% and in Australia in ranges between 9% and 22% (Manning and Collins, 2001). In Iran, the first report of paratuberculosis comes back to about 40 years ago by Talatchian (1965) which showed that the origin of infection was imported animals. There are several other reports from various parts of the country about the occurrence of disease and its undesirable consequences in cattle, sheep, and goats (Anzaby et al., 2006; Pourjafar and Badiei, 2005; Kasravi and Nowrouzian, 2004; Khodakaram Tafti and Rashidi, 2000). A test-and-slaughter policy is applied at the request of farmers, and dairy farms and registered flocks are covered by insurance to provide compensation. It is suggested that the incidence of M. paratuberculosis in Iran is low. However, M. paratuberculosis prevalence studies to estimate the herd-level prevalence in dairy cattle are limited. The aim of the present study was to estimate the current herd-level prevalence of M. paratuberculosis infection in dairy herds located in Fars province, southern Iran using IS900 nested PCR on bulk-tank milk samples. 2. Materials and methods 2.1. Study population This is a cross-sectional study which was conducted in the Fars province, southern Iran to determine the prevalence of M. paratuberculosis-infected herd in the region. Fars province is one of the most important parts of the country contributing to dairy industry. Industrial dairy herds does not distributed evenly in the province. There are three main districts (Shiraz, Marvdasht, and Sepidan) in the province which comprised more than 80% of all dairy herds in the region. Therefore these districts were considered to be nearly representative of the province and study population selected from there. There are nearly 120 industrial dairy herds in these districts. Herd size ranging between 10 and 1200 cows, average parity is 2.7, 305-day milk production is 7193 kg (Ministry of Agriculture, Animal breeding Center website) and trading of live animals between the herds is not a common practice. Because a few herds did not want to participate, overall, 110 herds were included in the study. Selection of herds for study was accomplished with
10
M. Haghkhah et al. / Preventive Veterinary Medicine 86 (2008) 8–13
Fars province Agricultural Jihad Organization which has the name of owner and location for each herd in the province. 2.2. Extraction of DNA and PCR process During the visit to the farms (March–August 2006), milk samples were collected from wellmixed bulk-tank of study herds and brought cooled to the laboratory. For detection of MAP, 50 ml was stored at 20 8C up to time of investigation. Extraction of DNA was undertaken as described by Stabel et al. (2002) with minor modifications. After defreezing the milk samples with water bath, samples were vortexed. Then 500 ml of each sample was transferred to 1.5 ml tube, centrifuged with 1500 rpm for 6 min. The supernatant was discarded and 50 ml proteinase K (1 mg/ml) (Fermentes Co.) was added to each tube and again vortex was done. Samples were shaked overnight in 50 8C water bath. To each sample, 22 ml of mycobacterial lysis buffer (0.1 M NaCl; 10 mM Tris–HCl; 1 mM EDTA) and 10 ml NaOH 0.4 M was added, vortexed and placed in boiling water bath for 30 min. Equal volume of phenol, chloroform and isoamylalcohol (25:24:1) was added, vortexed and immediately centrifuged at 10,000 rpm for 5 min. Supernatant aqueous layer was transferred to new tubes. The DNA was precipitated by adding 2.2 v of cold 100% ethanol and 0.1 v of 3 M sodium acetate. Then samples were mixed gently and put in 20 8C for at least 1 h. The tubes then were centrifuged at 14,000 rpm for 16 min, the supernatants were discarded and pellet was dried at room temperature and finally resuspended in sterile distilled water. The DNA was frozen at 20 8C for later analysis. IS900 nested PCR was conducted as described by Corti and Stephan (2002) with minor modification. Resuspended DNA (3 ml) was used as the template for IS900 nested PCR. The primers P90, 50 -GAA GGG TGT TCG GGG CCG TCG CTT AGG-30 and P91, 50 -GGC GTT GAG GTC GAT CGC CCA CGT GAC-30 were used for initial amplification cycles. The PCR mix consisted of 10 ml reaction volume containing 20 pmol of each primer, 5 mM MgCl2, 40 mM dNTP, 0.5 U Taq polymerase in 1 reaction buffer (Cinagen, Tehran, Iran). Cycling was as follows: 94 8C for 5 min (1 cycle); 94 8C for 1 min, 59 8C for 1 min, 72 8C for 3 min (30 cycles); 72 8C for 7 min (1 cycle). For samples with 413 bp band and suspected samples, 3 ml from the primary amplification were used for nested PCR. The primers were AV1, 50 -ATG TGG TTG CTG TGT TGG ATG G-30 and AV2, 50 -CCG CCG CAA TCA ACT CCA G-30 . The reaction mixture was the same as single PCR. Cycling was as follows: 94 8C for 5 min (1 cycle); 94 8C for 1 min, 58 8C for 1 min, 72 8C for 3 min (35 cycles); 72 8C for 7 min (1 cycle). PCR products were visualized by gel electrophoresis on 1.5% agarose gel. Negative and positive bacterial controls were included for each PCR process. 2.3. Statistical analysis Confidence limits (95%) for prevalence were calculated using approximation to standard normal distribution. The unit of statistical analysis was herd. Comparison of prevalence between three districts was done using chi-square test and P-value less than 0.05 considered statistically significant. 3. Results A single band of 298 bp for each of the positive milk sample was detected by PCR amplification of the MAP-specific insertion sequence IS900 and subsequent agarose gel analysis
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Table 1 Distribution of Mycobacterium paratuberculosis infection in 110 dairy herds in the 3 districts of Fars province, southern Iran based on IS900-PCR in bulk-tank milk samples (March–August 2006) Area
Shiraz Marvdasht Sepidan
PCR positive
PCR negative
N
%
N
%
5 3 4
8.6 8.6 23.5
53 32 13
91.4 91.4 76.5
of the amplified products. Among 110 bulk-tank milk samples, 12 (11%, 95%CI: 5–17%) were positive in IS900 nested PCR. Distribution of herds in three districts according to PCR results is shown in Table 1. The prevalence of positive milk samples in the three district of Fars province was different ranging from 8.6% to 23% which was not significant (x2 = 3.3, d.f. = 2, P = 0.19). 4. Discussion We found a herd-level prevalence of MAP infection in the Fars province, southern Iran 11% based on IS900 nested PCR on bulk-tank milk samples. Some other studies used bulk-tank milk PCR method for diagnosis of MAP. Grant et al. (2002) performed an immunomagnetic PCR on 244 bulk-tank milk samples from the United Kingdom and detected a herd-level prevalence of 7.8%. In another study, Corti and Stephan (2002) examined bulk-tank milk samples from different regions in Switzerland and showed that 19.7% of the 1384 milk samples were IS900 PCR positive. Traditionally, fecal culture for MAP is considered as the gold standard for diagnosis. However, fecal culture is time-consuming and detects only 38–50% of cows infected (Stabel, 1998; Whitlock et al., 2000). Serologic tests such as ELISA are even less sensitive than fecal culture, particularly in apparently healthy or subclinically infected animals (Sweeney et al., 1995; Stabel, 1998; Buergelt and Williams, 2004). Use of PCR methods in contrast to culture and serological tests, allowed to detect nonviable as well as viable micro-organisms and would be a more sensitive detection method (Stabel et al., 2002; Millar et al., 1995; Englund et al., 1999; Djonne et al., 2003). Therefore, in comparison with serologic or culture methods, detection of MAP directly from bulk-tank milk by IS900 PCR could be considered a valuable test for the estimation of herd-level prevalence. Information about bulk-tank milk PCR sensitivity and specificity in the literature is not consistent (Pillai and Jayarao, 2002; Jayarao et al., 2004; Stable, 2000; Stabel et al., 2002). Therefore we could not estimate the true prevalence for MAP infection. However, the apparent herd prevalence reported here may be biased toward an underestimate of the true prevalence. It has been shown that excretion of MAP in the milk is low and intermittent (Sweeney et al., 1992; Corti and Stephan, 2002); it is probable that at the time of sample collection infected animals have not shed MAP in the milk. Therefore it is possible that the true prevalence of MAP-infected herds is higher than we have found. On the other hand, the possibility that a herd being classified as infected even though it is free of MAP could not be eliminated. Mycobacterium paratuberculosis has been detected in dairy herds throughout Iran (Talatchian, 1965; Anzaby et al., 2006; Pourjafar and Badiei, 2005; Kasravi and Nowrouzian, 2004; Khodakaram Tafti and Rashidi, 2000). Most of previous reports were based on
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slaughterhouse samples or related to the consequences of the disease. Actually, there are no other comparative data on the detection of MAP in milk samples for estimation of herd-level prevalence. The current strategy for control of paratuberculosis in Iran is voluntary, and relies on individual producers to decide if Johne’s disease is a significant problem in their own herds and need aggressive control efforts. Some efforts are recently under conduction to give a national strategy for the control of the disease. Nevertheless until in-depth studies of the epidemiology and economic’s of Johne’s disease including individual-level prevalence throughout the country are performed, it remains to be determined whether MAP has a large enough economic and public health impact to justify a mandatory control program. 5. Conclusion The overall herd-level prevalence of Mycobacterium paratuberculosis in dairy herds investigated was approximately 11% based on IS900 nested PCR on bulk-tank milk samples. It is critical to conduct in-depth epidemiologic studies to identify cow-level prevalence and economic consequences of MAP infection throughout the country. Acknowledgments The authors would like to thank Prof. Leonardo Sechi, Department of Biomedical Sciences, University of Sassari, Italy, for providing the MAP for positive control in PCR, Miss. Masoudian for technical assistance and herd owners for their help and cooperation in collecting milk samples. This study was supported by Grant No. 84-VE-1780_C308 Shiraz University, Fars, Iran. References Anzaby, Y., Tabatabayi, A.H., Asgharzade, M., 2006. A survey of the infection status of Mycobacterium avium paratuberculosis in dairy cattle using PCR and culture of Tabriz region. Iran. J. Vet. Sci. 2, 297–310. Beaudeau, F., Belliard, M., Joly, A., Seegers, H., 2007. Reduction in milk yield associated with Mycobacterium avium subspecies paratuberculosis (Map) infection in dairy cows. Vet. Res. 38, 625–634. Benedictus, G., Dijkhhuizen, A.A., Stelwagen, J., 1987. Economic losses due to paratuberculosis in dairy cattle. Vet. Rec. 121, 142–146. Buergelt, C.D., Williams, J.E., 2004. Nested PCR on blood and milk for the detection of Mycobacterium avium subsp. paratuberculosis DNA in clinical and subclinical bovine paratuberculosis. Aust. Vet. J. 82, 497–503. Corti, S., Stephan, R., 2002. Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2, 15. Djonne, B., Jensen, M.R., Grant, I.R., Holstad, G., 2003. Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway. Vet. Microbiol. 20, 135–143. Englund, S., Ballagi-Pordany, A., Bolske, G., Johansson, K.E., 1999. Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagn. Microbiol. Infect. Dis. 33, 163–171. Gaya, D.R., Black, R.A., MacKenzie, J.F., 2004. Crohn’s disease and MAP. Lancet 364, 2179. Gonda, M.G., Chang, Y.M., Shook, G.E., Collins, M.T., Kirkpatrick, B.W., 2007. Effect of Mycobacterium paratuberculosis infection on production, reproduction and health traits in US Holsteins. Prev. Vet. Med. 80, 103–119. Grant, I.R., Ball, H.J., Rowe, M.T., 2002. Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cow’s milk from approved dairy processing establishments in the United Kingdom. Appl. Environ. Microbiol. 68, 2428–2435. Grant, I.R., 2003. Mycobacterium paratuberculosis and milk. Acta Vet. Scand. 44, 261–266. Greenstein, R.J., Collins, M.T., 2004. Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic? Lancet 31, 396–397.
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Jayarao, B.M., Pillai, S.R., Wolfgang, D.R., Griswold, D.R., Rossiter, C.A., Tewari, D., Burns, C.M., Hutchinson, L.J., 2004. Evaluation of IS900-PCR assay for detection of Mycobacterium avium subspecies paratuberculosis infection in cattle using quarter milk and bulk-tank milk samples. Foodborne Pathog. Dis. 1, 17–26. Johnson, Y.J., Kaneene, J.B., gardiner, J.C., Lloyd, J.W., Sprecher, D.J., Coe, P.H., 2001. The effect of subclinical Mycobacterium paratuberculosis infection on milk production in Michigan dairy cows. J. Dairy Sci. 84, 2188–2194. Kasravi, R., Nowrouzian, I., 2004. Clinical paratuberculosis as a cause for higher culling rate in cows with left displaced abomasum and diarrhoea in a Holstein dairy herd. J. Vet. Med. Ser. B 51, 302–303. Khodakaram Tafti, A., Rashidi, K., 2000. The Pathology of goat paratuberculosis: gross and histopathological lesions in the intestines and mesenteric lymph nodes. J. Vet. Med. B 47, 487–495. Manning, E.J.B., Collins, M.T., 2001. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev. Sci. Tech. Off. Int. Epiz. 20, 133–150. Millar, D.S., Withey, S.J., Tizard, M.L., Ford, J.G., Hermon-Taylor, J., 1995. Solid-phase hybridization capture of lowabundance target DNA sequences: application to the polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp. silvaticum. Anal. Biochem. 10, 325–330. Pillai, S.R., Jayarao, B.M., 2002. Application of IS900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk. J. Dairy Sci. 85, 1052–1057. Pourjafar, M., Badiei, K., 2005. Presence and trace element analysis in cattle affected by paratuberculosis in Yasouj, Iran. In: Proceedings of the Eight International Colloquium on Paratuberculosis, Spain. Romero, C., Hamdi, A., Valentine, J.F., Naser, S.A., 2005. Evaluation of surgical tissue from patients with Crohn’s disease for the presence of Mycobacterium avium subspecies paratuberculosis DNA by in situ hybridization and nested polymerase chain reaction. Inflamm. Bowel Dis. 11, 116–125. Stabel, J.R., 1998. Johne’s disease: a hidden threat. J. Dairy Sci. 81, 283–288. Stabel, J.R., Wells, S.J., Wagner, B.A., 2002. Relationships between fecal culture, ELISA, and bulk-tank milk test results for Johne’s disease in US dairy herds. J. Dairy Sci. 85, 525–533. Stable, J.R., 2000. Johne’s disease and milk: do consumers need to worry? J. Dairy Sci. 83, 1659–1663. Sweeney, R.W., Whitlock, R.H., Buckley, C.L., Spencer, P.A., 1995. Evaluation of a commercial enzyme-linked immunosorbent assay for the diagnosis of paratuberculosis in dairy cattle. J. Vet. Diagn. Invest. 7, 488–493. Sweeney, R.W., Whitlock, R.H., Rosenberger, A.E., 1992. Mycobacterium paratuberculosis cultured from milk and supramammary lymph nodes of infected asymptomatic cows. J. Clin. Microbiol. 30, 166–171. Talatchian, M., 1965. First report of Johne’s disease in Iran. Bull. Off. Int. Epizoot. 64, 779–782. Whitlock, R.H., Wells, S.J., Sweeney, R.W., Van Tiem, J., 2000. ELISA and fecal culture for paratuberculosis (Johne’s disease): sensitivity and specificity of each method. Vet. Microbiol. 77, 387–398.
Preventive Veterinary Medicine 86 (2008) 8–13 www.elsevier.com/locate/prevetmed
Short communication
Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds Masoud Haghkhah a, Maryam Ansari-Lari b,*, Amir Mansour Novin-Baheran a, Ayatollah Bahramy a a b
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345, Iran Department of Food Hygiene and Public Health, School of Veterinary Medicine, Shiraz University, Besat Street, Shiraz 71345, Iran Received 13 August 2007; received in revised form 21 March 2008; accepted 27 March 2008
Abstract A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection. Bulk-tank milk samples were collected from 110 dairy herds in the 3 districts (Shiraz, Marvdasht and Sepidan) of the province. Among study populations, 12 herds (11%, 95%CI: 5–17%) were positive for MAP infection based on IS900 nested PCR. The prevalence of positive milk samples in the three districts of Fars province was different ranging from 8.6% to 23% which was not statistically significant (P = 0.19). It is recommended to conduct further epidemiologic studies to determine cow-level prevalence and risk factors for infection, and to evaluate the economic consequences of the MAP infection in the region. # 2008 Elsevier B.V. All rights reserved. Keywords: Iran; Johne’s disease; Mycobacterium avium subspecies paratuberculosis; Prevalence
1. Introduction Johne’s disease is a chronic disease of ruminants worldwide caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). The Johne’s disease in the dairy community has long been considered as an economically important disease by reports of the negative impact on milk production and the overall economic health of dairies (Benedictus et al.,
* Corresponding author. Tel.: +98 711 2286950; fax: +98 711 2286940. E-mail addresses: [email protected], [email protected] (M. Ansari-Lari). 0167-5877/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.prevetmed.2008.03.010
M. Haghkhah et al. / Preventive Veterinary Medicine 86 (2008) 8–13
9
1987; Johnson et al., 2001; Beaudeau et al., 2007; Gonda et al., 2007). This disease has gained notoriety in recent years because of concern about a possible relationship to Crohn’s disease, an intestinal disorder in humans (Greenstein and Collins, 2004; Gaya et al., 2004; Romero et al., 2005). These suggestions call for further knowledge on the prevalence of the infection in the cattle population around the world. Surveys for MAP are difficult to accomplish because of the chronic nature of the infection and the lack of good testing methods for animals in the pre-clinical stages. Diagnostic tests such as ELISA, agar gel immunodiffusion (AGID) test and fecal culture are being used commonly. Also PCR methods, targeting MAP-specific insertion sequence (IS900) or other species-specific genes have been developed to increase sensitivity and specificity of diagnosis (Millar et al., 1995; Englund et al., 1999; Djonne et al., 2003). Presence of the pathogen in milk has been confirmed by several researchers (Sweeney et al., 1992; Grant, 2003). It was shown that M. paratuberculosis could be detected directly from quarter milk and bulk-tank milk by IS900 PCR (Pillai and Jayarao, 2002; Jayarao et al., 2004; Buergelt and Williams, 2004). Therefore, application of PCR for milk samples from individual cows or at herd level as bulk-tank milk have proven useful for prevalence estimation of infection with M. paratuberculosis by some studies (Corti and Stephan, 2002; Grant et al., 2002; Stabel et al., 2002). Prevalence of infection in many countries in the world is unknown, but herd prevalence of Johne’s disease in Europe is reported to be between 7% and 55%, in the United States nearly 40% and in Australia in ranges between 9% and 22% (Manning and Collins, 2001). In Iran, the first report of paratuberculosis comes back to about 40 years ago by Talatchian (1965) which showed that the origin of infection was imported animals. There are several other reports from various parts of the country about the occurrence of disease and its undesirable consequences in cattle, sheep, and goats (Anzaby et al., 2006; Pourjafar and Badiei, 2005; Kasravi and Nowrouzian, 2004; Khodakaram Tafti and Rashidi, 2000). A test-and-slaughter policy is applied at the request of farmers, and dairy farms and registered flocks are covered by insurance to provide compensation. It is suggested that the incidence of M. paratuberculosis in Iran is low. However, M. paratuberculosis prevalence studies to estimate the herd-level prevalence in dairy cattle are limited. The aim of the present study was to estimate the current herd-level prevalence of M. paratuberculosis infection in dairy herds located in Fars province, southern Iran using IS900 nested PCR on bulk-tank milk samples. 2. Materials and methods 2.1. Study population This is a cross-sectional study which was conducted in the Fars province, southern Iran to determine the prevalence of M. paratuberculosis-infected herd in the region. Fars province is one of the most important parts of the country contributing to dairy industry. Industrial dairy herds does not distributed evenly in the province. There are three main districts (Shiraz, Marvdasht, and Sepidan) in the province which comprised more than 80% of all dairy herds in the region. Therefore these districts were considered to be nearly representative of the province and study population selected from there. There are nearly 120 industrial dairy herds in these districts. Herd size ranging between 10 and 1200 cows, average parity is 2.7, 305-day milk production is 7193 kg (Ministry of Agriculture, Animal breeding Center website) and trading of live animals between the herds is not a common practice. Because a few herds did not want to participate, overall, 110 herds were included in the study. Selection of herds for study was accomplished with
10
M. Haghkhah et al. / Preventive Veterinary Medicine 86 (2008) 8–13
Fars province Agricultural Jihad Organization which has the name of owner and location for each herd in the province. 2.2. Extraction of DNA and PCR process During the visit to the farms (March–August 2006), milk samples were collected from wellmixed bulk-tank of study herds and brought cooled to the laboratory. For detection of MAP, 50 ml was stored at 20 8C up to time of investigation. Extraction of DNA was undertaken as described by Stabel et al. (2002) with minor modifications. After defreezing the milk samples with water bath, samples were vortexed. Then 500 ml of each sample was transferred to 1.5 ml tube, centrifuged with 1500 rpm for 6 min. The supernatant was discarded and 50 ml proteinase K (1 mg/ml) (Fermentes Co.) was added to each tube and again vortex was done. Samples were shaked overnight in 50 8C water bath. To each sample, 22 ml of mycobacterial lysis buffer (0.1 M NaCl; 10 mM Tris–HCl; 1 mM EDTA) and 10 ml NaOH 0.4 M was added, vortexed and placed in boiling water bath for 30 min. Equal volume of phenol, chloroform and isoamylalcohol (25:24:1) was added, vortexed and immediately centrifuged at 10,000 rpm for 5 min. Supernatant aqueous layer was transferred to new tubes. The DNA was precipitated by adding 2.2 v of cold 100% ethanol and 0.1 v of 3 M sodium acetate. Then samples were mixed gently and put in 20 8C for at least 1 h. The tubes then were centrifuged at 14,000 rpm for 16 min, the supernatants were discarded and pellet was dried at room temperature and finally resuspended in sterile distilled water. The DNA was frozen at 20 8C for later analysis. IS900 nested PCR was conducted as described by Corti and Stephan (2002) with minor modification. Resuspended DNA (3 ml) was used as the template for IS900 nested PCR. The primers P90, 50 -GAA GGG TGT TCG GGG CCG TCG CTT AGG-30 and P91, 50 -GGC GTT GAG GTC GAT CGC CCA CGT GAC-30 were used for initial amplification cycles. The PCR mix consisted of 10 ml reaction volume containing 20 pmol of each primer, 5 mM MgCl2, 40 mM dNTP, 0.5 U Taq polymerase in 1 reaction buffer (Cinagen, Tehran, Iran). Cycling was as follows: 94 8C for 5 min (1 cycle); 94 8C for 1 min, 59 8C for 1 min, 72 8C for 3 min (30 cycles); 72 8C for 7 min (1 cycle). For samples with 413 bp band and suspected samples, 3 ml from the primary amplification were used for nested PCR. The primers were AV1, 50 -ATG TGG TTG CTG TGT TGG ATG G-30 and AV2, 50 -CCG CCG CAA TCA ACT CCA G-30 . The reaction mixture was the same as single PCR. Cycling was as follows: 94 8C for 5 min (1 cycle); 94 8C for 1 min, 58 8C for 1 min, 72 8C for 3 min (35 cycles); 72 8C for 7 min (1 cycle). PCR products were visualized by gel electrophoresis on 1.5% agarose gel. Negative and positive bacterial controls were included for each PCR process. 2.3. Statistical analysis Confidence limits (95%) for prevalence were calculated using approximation to standard normal distribution. The unit of statistical analysis was herd. Comparison of prevalence between three districts was done using chi-square test and P-value less than 0.05 considered statistically significant. 3. Results A single band of 298 bp for each of the positive milk sample was detected by PCR amplification of the MAP-specific insertion sequence IS900 and subsequent agarose gel analysis
M. Haghkhah et al. / Preventive Veterinary Medicine 86 (2008) 8–13
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Table 1 Distribution of Mycobacterium paratuberculosis infection in 110 dairy herds in the 3 districts of Fars province, southern Iran based on IS900-PCR in bulk-tank milk samples (March–August 2006) Area
Shiraz Marvdasht Sepidan
PCR positive
PCR negative
N
%
N
%
5 3 4
8.6 8.6 23.5
53 32 13
91.4 91.4 76.5
of the amplified products. Among 110 bulk-tank milk samples, 12 (11%, 95%CI: 5–17%) were positive in IS900 nested PCR. Distribution of herds in three districts according to PCR results is shown in Table 1. The prevalence of positive milk samples in the three district of Fars province was different ranging from 8.6% to 23% which was not significant (x2 = 3.3, d.f. = 2, P = 0.19). 4. Discussion We found a herd-level prevalence of MAP infection in the Fars province, southern Iran 11% based on IS900 nested PCR on bulk-tank milk samples. Some other studies used bulk-tank milk PCR method for diagnosis of MAP. Grant et al. (2002) performed an immunomagnetic PCR on 244 bulk-tank milk samples from the United Kingdom and detected a herd-level prevalence of 7.8%. In another study, Corti and Stephan (2002) examined bulk-tank milk samples from different regions in Switzerland and showed that 19.7% of the 1384 milk samples were IS900 PCR positive. Traditionally, fecal culture for MAP is considered as the gold standard for diagnosis. However, fecal culture is time-consuming and detects only 38–50% of cows infected (Stabel, 1998; Whitlock et al., 2000). Serologic tests such as ELISA are even less sensitive than fecal culture, particularly in apparently healthy or subclinically infected animals (Sweeney et al., 1995; Stabel, 1998; Buergelt and Williams, 2004). Use of PCR methods in contrast to culture and serological tests, allowed to detect nonviable as well as viable micro-organisms and would be a more sensitive detection method (Stabel et al., 2002; Millar et al., 1995; Englund et al., 1999; Djonne et al., 2003). Therefore, in comparison with serologic or culture methods, detection of MAP directly from bulk-tank milk by IS900 PCR could be considered a valuable test for the estimation of herd-level prevalence. Information about bulk-tank milk PCR sensitivity and specificity in the literature is not consistent (Pillai and Jayarao, 2002; Jayarao et al., 2004; Stable, 2000; Stabel et al., 2002). Therefore we could not estimate the true prevalence for MAP infection. However, the apparent herd prevalence reported here may be biased toward an underestimate of the true prevalence. It has been shown that excretion of MAP in the milk is low and intermittent (Sweeney et al., 1992; Corti and Stephan, 2002); it is probable that at the time of sample collection infected animals have not shed MAP in the milk. Therefore it is possible that the true prevalence of MAP-infected herds is higher than we have found. On the other hand, the possibility that a herd being classified as infected even though it is free of MAP could not be eliminated. Mycobacterium paratuberculosis has been detected in dairy herds throughout Iran (Talatchian, 1965; Anzaby et al., 2006; Pourjafar and Badiei, 2005; Kasravi and Nowrouzian, 2004; Khodakaram Tafti and Rashidi, 2000). Most of previous reports were based on
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slaughterhouse samples or related to the consequences of the disease. Actually, there are no other comparative data on the detection of MAP in milk samples for estimation of herd-level prevalence. The current strategy for control of paratuberculosis in Iran is voluntary, and relies on individual producers to decide if Johne’s disease is a significant problem in their own herds and need aggressive control efforts. Some efforts are recently under conduction to give a national strategy for the control of the disease. Nevertheless until in-depth studies of the epidemiology and economic’s of Johne’s disease including individual-level prevalence throughout the country are performed, it remains to be determined whether MAP has a large enough economic and public health impact to justify a mandatory control program. 5. Conclusion The overall herd-level prevalence of Mycobacterium paratuberculosis in dairy herds investigated was approximately 11% based on IS900 nested PCR on bulk-tank milk samples. It is critical to conduct in-depth epidemiologic studies to identify cow-level prevalence and economic consequences of MAP infection throughout the country. Acknowledgments The authors would like to thank Prof. Leonardo Sechi, Department of Biomedical Sciences, University of Sassari, Italy, for providing the MAP for positive control in PCR, Miss. Masoudian for technical assistance and herd owners for their help and cooperation in collecting milk samples. This study was supported by Grant No. 84-VE-1780_C308 Shiraz University, Fars, Iran. References Anzaby, Y., Tabatabayi, A.H., Asgharzade, M., 2006. A survey of the infection status of Mycobacterium avium paratuberculosis in dairy cattle using PCR and culture of Tabriz region. Iran. J. Vet. Sci. 2, 297–310. Beaudeau, F., Belliard, M., Joly, A., Seegers, H., 2007. Reduction in milk yield associated with Mycobacterium avium subspecies paratuberculosis (Map) infection in dairy cows. Vet. Res. 38, 625–634. Benedictus, G., Dijkhhuizen, A.A., Stelwagen, J., 1987. Economic losses due to paratuberculosis in dairy cattle. Vet. Rec. 121, 142–146. Buergelt, C.D., Williams, J.E., 2004. Nested PCR on blood and milk for the detection of Mycobacterium avium subsp. paratuberculosis DNA in clinical and subclinical bovine paratuberculosis. Aust. Vet. J. 82, 497–503. Corti, S., Stephan, R., 2002. Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland. BMC Microbiol. 2, 15. Djonne, B., Jensen, M.R., Grant, I.R., Holstad, G., 2003. Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway. Vet. Microbiol. 20, 135–143. Englund, S., Ballagi-Pordany, A., Bolske, G., Johansson, K.E., 1999. Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagn. Microbiol. Infect. Dis. 33, 163–171. Gaya, D.R., Black, R.A., MacKenzie, J.F., 2004. Crohn’s disease and MAP. Lancet 364, 2179. Gonda, M.G., Chang, Y.M., Shook, G.E., Collins, M.T., Kirkpatrick, B.W., 2007. Effect of Mycobacterium paratuberculosis infection on production, reproduction and health traits in US Holsteins. Prev. Vet. Med. 80, 103–119. Grant, I.R., Ball, H.J., Rowe, M.T., 2002. Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cow’s milk from approved dairy processing establishments in the United Kingdom. Appl. Environ. Microbiol. 68, 2428–2435. Grant, I.R., 2003. Mycobacterium paratuberculosis and milk. Acta Vet. Scand. 44, 261–266. Greenstein, R.J., Collins, M.T., 2004. Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic? Lancet 31, 396–397.
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