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Detection of Mycobacterium avium subsp. paratuberculosis in goat and sheep flocks in Mexico
Small Ruminant Research 72 (2007) 209–213
Short communication
Detection of Mycobacterium avium subsp. paratuberculosis in goat and sheep flocks in Mexico I. Est´evez-Denaives a,b , R. Hern´andez-Castro a , A.M. Trujillo-Garc´ıa c , G. Ch´avez-Gris b,∗ a
c
Departamento de Microbiolog´ıa e Inmunolog´ıa, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, Mexico b Departamento de Patolog´ıa, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´ onoma de M´exico, Ciudad Universitaria, Coyoac´an, 04510 M´exico, DF, Mexico Departamento de Ruminantes, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, Mexico Received 29 November 2005; received in revised form 24 August 2006; accepted 4 October 2006 Available online 15 November 2006
Abstract The slow growth of Mycobacterium avium subsp. paratuberculosis (Map) has hindered its investigation. No data concerning the presence of paratuberculosis in Mexico are currently available. The aim of this study was to identify M. avium subsp. paratuberculosis infection in sheep and goat flocks from several states in Mexico. Twenty-two animals, all manifesting clinical signs of paratuberculosis were obtained from six different Mexican states. Of these, 14 sheep and 8 goats were serologically diagnosed and multibacillary type lesions were identified. On the basis of mycobacteria concentration technique used for ileal mucosa, Map identification was confirmed by means of PCR and bacterial culture. In addition, samples were classified according to the abundance of acid-fast bacteria (AFB) identified in the intestinal mucosa and by the final concentrate of mycobacteria and the amount of DNA obtained. All correlations concerning the abundance of AFB in tissue, AFB recovered in the final concentrate and the amount of DNA obtained were recorded. These findings will allow to predict the amount of DNA which can be obtained by referring to the abundance of acid-fast organisms found in the tissue and, therefore, define the potential of using this method for the purpose of Map characterization. The results of this study indicate that paratuberculosis is widespread among goats and sheep in Mexico. © 2006 Published by Elsevier B.V. Keywords: Paratuberculosis; Johne’s disease; Goat; Sheep; Mexico; PCR
1. Introduction Paratuberculosis (PTB), or Johne’s disease (JD), is a chronic enteropathy which occurs in ruminants and is caused by infection with Mycobacterium avium
∗ Corresponding author. Tel.: +52 55 56 22 58 88; fax: +52 55 56 16 67 95. E-mail address: [email protected] (G. Ch´avez-Gris).
0921-4488/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2006.10.017
subsp. paratuberculosis (Map). Characteristic symptoms include; diarrhea, progressive weight loss and death among adult animals and the disease has been reported in many countries. Besides this, Map has been reported as able to survive milk pasteurization (Grant et al., 2002) and has been implicated as the etiologic agent of Crohn’s disease in humans (Sechi et al., 2001). Diagnosis of Map infections by means of serology requires further confirmation, in order to avoid the possibility of false-negative results, which are often
210
I. Est´evez-Denaives et al. / Small Ruminant Research 72 (2007) 209–213
recorded at the early stages of infection and crossreactions with genetically related mycobacterial species (Clarke et al., 1996; P´erez et al., 1997; Burrells et al., 1998). Histopathological findings have been proposed as good parameters of paratuberculosis infection in small ruminants. Among sheep and goats which are clinically affected with paratuberculosis, two distinct forms of microscopical pathology were identified (P´erez et al., 1996; Clarke and Little, 1996; Corpa et al., 2000), related to either a high or a low degree of mycobacterial colonization in clinical cases (“multibacillary” or “paucibacillary” presence). Culture is the most reliable method for identifying the disease, but the extended time lapse (of at least 6 weeks) required to culture the organism and its low sensitivity may be considered disadvantages. Moreover, the isolation of Map from infected sheep is difficult (Collins et al., 1990; P´erez et al., 1996; Burrells et al., 1998; Corpa et al., 2000). Current molecular identification protocols for Map rely on the presence of the IS900 insertion element (Clarke and Little, 1996; Burrells et al., 1998; Grant et al., 2002; Ikonomopoulos et al., 2004). No data are available referring to the prevalence of paratuberculosis in cattle, sheep or goats in Mexico. The aim of this study was to diagnose M. avium subsp. paratuberculosis infection in sheep and goat flocks from several Mexican states, using serology and histopathology. Based on a mycobacteria concentration technique used for ileal mucosa, this identification was confirmed by PCR and bacterial culture.
into three groups according to the abundance and distribution of acid-fast organisms: Group 1: AFB in the lamina propia in diffuse form; Group 2: AFB in the medial area and at the top of villosity, in focal form; Group 3: AFB at the top of villosity in a dispersed form.
2. Materials and methods
Cell lysis and DNA extraction from mycobacterial concentrate was carried out as described by van Soolingen et al. (1994) for mycobacterial culture. The quality and quantity of DNA were evaluated in 1% agarose gel, stained with ethidium bromide and the 22 samples were classified into three groups according to their DNA band intensity.
2.1. Paratuberculosis diagnosis Twenty-two animals were selected from six different Mexican states: San Luis Potos´ı, Guanajuato, Quer´etaro, Distrito Federal, Estado de M´exico and Veracruz. All manifesting typical signs of paratuberculosis. These animals were diagnosed by a process of serology and multibacillary type lesions were identified. Serum samples were tested for antibodies against paratuberculosis, using either AGID or ELISA tests; employing a protoplasmic extract (PPA-3, Allied, USA) as antigen. For the ELISA test, serum samples were absorbed with a Mycobacterium phlei suspension (Allied, USA), and a 1:4500 dilution of a rabbit anti-goat IgG horseradish peroxidase conjugate (Sigma, USA) was employed. During the necropsy, specimens were collected for the purpose of histopathological examination. Tissues were fixed and stained with haematoxylin and eosin (HE) and Ziehl–Neelsen (ZN). Samples of small intestine with apparent gross thickening of the intestinal wall were also collected. Samples from the 22 animals having multibacillary lesions were classified
2.2. Mycobacterial concentration Mycobacterial concentration from the intestinal mucosa of infected animals was collected using a modified version of that described by Ratnamohan and Spencer (1986). The mucosal surface was rinsed with 200 g/ml ampicillin and removed from the submucosa by scraping. The mucosa was then homogenized, using a glass/glass tissue grinder (Ten Broeck). Eight grams of mucosal suspension were digested in 150 ml of 0.5% trypsin (Gibco, USA) in PBS. After centrifugation of the suspension at 800 × g for 20 min, the sediment was digested in 20 mg of lysozyme. The suspension was centrifuged as above, then the sediment was re-suspended in 10 ml of distilled water, before being centrifuged again. The presence of mycobacteria in the final pellet was confirmed by examining smears which were stained using the ZN technique. Due to the characteristic clump formation of mycobacteria, it was not possible to quantify the AFB. However, the AFB recovered in the final concentrate was estimated, as well as the presence of cellular debris and samples, which were classified into three categories: Group 1: abundant AFB distributed diffusely throughout the field; Group 2: AFB present in clumps, with cellular debris also present; Group 3: much cellular debris, and no clearly observed AFB. 2.3. Cell lysis and DNA extraction
2.4. PCR PCR assay was performed using specific primers for the insertion sequence IS900 (Pavlik et al., 1999): 5 CGCTGCTGGAGTTGATTG-3 and 5 -TTTCCTTCGGTGCGTTTTC-3 . The reaction was carried out on a final volume of 50 l and consisted of 10 l of 10× PCR× enhancer solution (Invitrogen, USA), 5 l of 10× reaction buffer, 1 l dNTP, 10 mM for each one, 2 l of 50 mM MgCl2 , 2 l from each primer (50 ng/l), 1 l of polymerase 5 U/l (Altaenzymes, Canada) and 4 l DNA. DNA was first denatured at 94 ◦ C for 4 min, then amplified with 30 cycles of (i) denaturation at 94 ◦ C for 55 s, (ii) primer annealing at 45 ◦ C for 51 s, and (iii) elongation at 72 ◦ C for 3 min, using a thermocycler (PCR Express, Hybaid, UK). After the last amplification cycle, the samples were incubated at 72 ◦ C for 8 min. The expected 972 bp PCR
I. Est´evez-Denaives et al. / Small Ruminant Research 72 (2007) 209–213
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Fig. 1. Photographs of: (a) ileum section showing abundance and distribution of acid-fast bacteria (AFB), Ziehl–Neelsen’s stain (ZN): 10×, 10×, 40×. (b) A smear showing AFB abundance obtained in the final mycobacterial concentrate, ZN: 100×. (c) Mycobacterial DNA. 0.8% agarose gel in TAE containing ethidium bromide.
amplified products were analyzed in 2% agarose gels containing ethidium bromide. 2.5. Mycobacterial culture Each mycobacterial concentrate was treated with 0.76% hexadecyl pyridinium chloride (Sigma, USA) and cultured in screw capped bottles containing L¨owenstein–Jensen medium (Difco, USA) with and without mycobactin J (Allied, USA), as described by Juste et al. (1991). The 22 samples were incubated at 37 ◦ C for 34 weeks.
3. Results Fourteen sheep and eight goats suspected to paratuberculosis were obtained from six different Mexican states in Mexico. Diagnosis was established by means of serology and multibacillary type lesions were iden-
tified. The 22 multibacillary samples showed varying abundance and distribution of AFB in intestinal sections (Fig. 1(a)). There was also variability concerning the results of mycobacterial concentration from the intestinal mucosa of infected animals. Twelve samples were included in Group 1 (54.54%), four samples were included in Group 2 (18.18%) and six samples corresponded to Group 3, that represent 27.27%. Differences in AFB abundance and the presence of cellular debris are shown in Fig. 1(b) and (c) depicts the average amount of DNA obtained from each group. This classification revealed a relationship between the abundance of AFB in intestinal mucosa, the amount of AFB recovered in the final concentrate and the amount of DNA obtained. Diagnosis was further confirmed by means of PCR (Fig. 2). All the 22 DNA samples obtained from mycobacterial concentrate were PCR-positive.
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Fig. 2. Agarose gel electrophoresis of IS900 PCR products. (a) Lane 1: molecular size marker 1 kb DNA ladder; lane 2: positive control; lanes 3–13: DNA products of PCR performed on mycobacterial concentrate samples. (b) Lane 1: molecular size marker 1 kb DNA ladder; lane 2: positive control; lanes 3–13: DNA products of PCR performed on mycobacterial concentrate samples; lane 14: negative control.
Finally, Map was successfully isolated from medium, containing mycobactin J taken from all the animals with multibacillary lesions. The absence of growth in media without added mycobactin indicated that other species of Mycobacterium were either absent or not viable. 4. Discussion The prevalence of paratuberculosis in Mexico is currently unknown. Moreover, large numbers of head of cattle, sheep and goats have recently been introduced from Europe, United States of America, Canada, New Zealand and Australia. As paratuberculosis diagnosis is not an important prerequisite and some of these animals may not be obvious carriers of this microorganism, they can subsequently transmit the infection to the ruminant population in Mexico. The serological results and histological findings obtained in this study confirm the presence of paratuberculosis in goat or sheep flocks from six different Mexican states. The multibacillary form of this pathology was recognized, which confirms observations made in previous studies (P´erez et al., 1996; Clarke and Little, 1996; Corpa et al., 2000). Different methods have been described for the recovery of M. avium subsp. paratuberculosis both from the tissue and faeces of infected sheep for the purpose of obtaining species-specific antigens (Ratnamohan and
Spencer, 1986) to be used in specific DNA detection by PCR (Challans et al., 1994; Garrido et al., 2000) or for Map characterization (Collins et al., 1990; Choy et al., 1998). These techniques allow large quantities of bacteria to be obtained without depending on the long incubation periods required to propagate a successful culture. However, these authors did not quantify the amount of AFB in the samples or in the final pellet. The trend for this organism to occur in clumps makes it difficult to estimate the numbers of cells in the samples. In this study an analysis of the concentration of mycobacteria from frozen tissue samples was performed and the Ziehl–Neelsen stain confirmed the presence of AFB in the mycobacterial pellet. DNA was successfully extracted from in vivo-derived Map, and although the yield of bacterial DNA from these samples varied for each individual host, when these were classified, it became evident that a relationship exists between the abundance of acid-fast organisms in the tissue, the AFB recovered in the final concentrate and the amount of DNA obtained. These findings suggest that the DNA recovered from bacteria consisted predominantly of AFB. Furthermore, this classification will allow to predict the amount of DNA that can be obtained, by measuring the abundance of acid-fast organisms in the tissue and, thus, to define the extent to which this method can be used for Map characterization. Following this procedure for the concentration of mycobacteria, tissue samples collected
I. Est´evez-Denaives et al. / Small Ruminant Research 72 (2007) 209–213
from Groups 1 and 2 are likely to be candidates for Map characterization. The PCR assay produced the expected result for 100% of samples, indicating that M. avium subsp. paratuberculosis was present among the population of AFB, which had been recovered from the intestinal mucosa. Several authors have reported false negative results when this technique was applied to clinical samples, and this has been attributed to the presence of inhibitors in the PCR as well as to inefficient extraction of mycobacteria from the samples, particularly if only a small quantity of organisms is involved (Challans et al., 1994; Clarke and Little, 1996; Burrells et al., 1998; Garrido et al., 2000). L¨owenstein–Jensen medium with mycobactin J was found to be effective for the purpose of isolating M. avium subsp. paratuberculosis from all animals manifesting multibacillary lesions, as reported by Juste et al. (1991), P´erez et al. (1996) and Corpa et al. (2000). Although culture propagation is a slow process, the technique proved to be as sensitive as the detection of specific DNA, recovered from the intestinal mucosa and thus confirmed the diagnosis of the disease. The results of this study indicate that paratuberculosis is widespread in Mexico among goat and sheep flocks. Acknowledgements The study was supported by CONACYT (grant 25395-B) and PAPIIT (grant IN22 1999). I. Est´evez Denaives received financial support from CONACYT (162910). Marycruz Dom´ınguez Punaro, Antonio G´omez Gonz´alez, Miguel Batres Govea and Javier Aranda are acknowledged for providing samples used in this study and Luis Antonio Morales Arreola and Jaime Eugenio C´ordova L´opez for sample preparation. References Burrells, C., Clarke, C.J., Colston, A., Kay, J.M., Porter, J., Little, D., Sharp, J.M., 1998. A study of immunological responses of sheep clinically-affected with paratuberculosis (Johne’s disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology. Vet. Immunol. Immunopathol. 66, 343–358. Challans, J.A., Stevenson, K., Reid, H.W., Sharp, J.M., 1994. A rapid method for the extraction and detection of Mycobacterium avium subspecies paratuberculosis from clinical specimens. Vet. Rec. 134, 95–96.
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Choy, E., Whittington, R.J., Marsh, I., Marshall, J., Campbell, M.T., 1998. A method for purification and characterisation of Mycobacterium avium subsp. paratuberculosis from the intestinal mucosa of sheep with Johne’s disease. Vet. Microbiol. 64, 51–60. Clarke, C., Patterson, I., Armstrong, K., Low, J., 1996. Comparison of the absorbed ELISA and agar gel immunodiffusion test with clinicopathological findings in ovine clinical paratuberculosis. Vet. Rec. 139, 618–621. Clarke, C.J., Little, D., 1996. The pathology of ovine paratuberculosis: gross and histological changes in the intestine and other tissues. J. Comp. Pathol. 114, 419–437. Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591–1596. Corpa, J.M., Garrido, J., Garcia Marin, J.F., P´erez, V., 2000. Classification of lesions observed in natural cases of paratuberculosis in goats. J. Comp. Pathol. 122, 255–265. Garrido, J.M., Cortabarria, N., Oguiza, J.A., Aduriz, G., Juste, R.A., 2000. Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis. Vet. Microbiol. 77, 379–386. Grant, I.R., Ball, H.J., Rowe, M.T., 2002. Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurized cows’ milk from approved dairy processing establishments in the United Kingdom. Appl. Environ. Microbiol. 68, 2428–2435. Ikonomopoulos, J., Gazouli, M., Pavlik, I., Bartos, M., Zacharatos, P., Xylouri, E., Papalambros, E., Gorgoulis, V., 2004. Comparative evaluation of PCR assays for the robust molecular detection of Mycobacterium avium subsp. paratuberculosis. J. Microbiol. Meth. 56, 315–321. Juste, R.A., Marco, J.C., Saez de Ocariz, C., Aduriz, J.J., 1991. Comparison of different media for the isolation of small ruminant strains of Mycobacterium paratuberculosis. Vet. Microbiol. 28, 385–390. Pavlik, I., Horvathova, A., Dvorska, L., Bartl, J., Svastova, P., du Maine, R., Rychlik, I., 1999. Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. J. Microbiol. Meth. 38, 155–167. P´erez, V., Garcia Marin, J.F., Badiola, J.J., 1996. Description and classification of different types of lesion associated with natural paratuberculosis infection in sheep. J. Comp. Pathol. 114, 107–122. P´erez, V., Tellechea, J., Badiola, J.J., Gutierrez, M., Garcia Marin, J.F., 1997. Relation between serologic response and pathologic findings in sheep with naturally acquired paratuberculosis. Am. J. Vet. Res. 58, 799–803. Ratnamohan, T.N., Spencer, T.L., 1986. A technique for the purification of Mycobacterium paratuberculosis from the ileal mucosa of infected cattle. Aust. Vet. J. 63, 185–187. Sechi, L.A., Mura, M., Tanda, F., Lissia, A., Solinas, A., Fadda, G., Zanetti, S., Manuela, M., Francesco, T., Amelia, L., Antonello, S., Giovanni, F., Stefania, Z., 2001. Identification of Mycobacterium avium subsp. paratuberculosis in biopsy specimens from patients with Crohn’s disease identified by in situ hybridization. J. Clin. Microbiol. 39, 4514–4517. van Soolingen, D., de Haas, P.E.W., Hermans, P.W.M., van Embden, J.D.A., 1994. DNA fingerprinting of Mycobacterium tuberculosis. Meth. Enzymol. 235, 196–205.
Short communication
Detection of Mycobacterium avium subsp. paratuberculosis in goat and sheep flocks in Mexico I. Est´evez-Denaives a,b , R. Hern´andez-Castro a , A.M. Trujillo-Garc´ıa c , G. Ch´avez-Gris b,∗ a
c
Departamento de Microbiolog´ıa e Inmunolog´ıa, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, Mexico b Departamento de Patolog´ıa, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´ onoma de M´exico, Ciudad Universitaria, Coyoac´an, 04510 M´exico, DF, Mexico Departamento de Ruminantes, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut´onoma de M´exico, Mexico Received 29 November 2005; received in revised form 24 August 2006; accepted 4 October 2006 Available online 15 November 2006
Abstract The slow growth of Mycobacterium avium subsp. paratuberculosis (Map) has hindered its investigation. No data concerning the presence of paratuberculosis in Mexico are currently available. The aim of this study was to identify M. avium subsp. paratuberculosis infection in sheep and goat flocks from several states in Mexico. Twenty-two animals, all manifesting clinical signs of paratuberculosis were obtained from six different Mexican states. Of these, 14 sheep and 8 goats were serologically diagnosed and multibacillary type lesions were identified. On the basis of mycobacteria concentration technique used for ileal mucosa, Map identification was confirmed by means of PCR and bacterial culture. In addition, samples were classified according to the abundance of acid-fast bacteria (AFB) identified in the intestinal mucosa and by the final concentrate of mycobacteria and the amount of DNA obtained. All correlations concerning the abundance of AFB in tissue, AFB recovered in the final concentrate and the amount of DNA obtained were recorded. These findings will allow to predict the amount of DNA which can be obtained by referring to the abundance of acid-fast organisms found in the tissue and, therefore, define the potential of using this method for the purpose of Map characterization. The results of this study indicate that paratuberculosis is widespread among goats and sheep in Mexico. © 2006 Published by Elsevier B.V. Keywords: Paratuberculosis; Johne’s disease; Goat; Sheep; Mexico; PCR
1. Introduction Paratuberculosis (PTB), or Johne’s disease (JD), is a chronic enteropathy which occurs in ruminants and is caused by infection with Mycobacterium avium
∗ Corresponding author. Tel.: +52 55 56 22 58 88; fax: +52 55 56 16 67 95. E-mail address: [email protected] (G. Ch´avez-Gris).
0921-4488/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.smallrumres.2006.10.017
subsp. paratuberculosis (Map). Characteristic symptoms include; diarrhea, progressive weight loss and death among adult animals and the disease has been reported in many countries. Besides this, Map has been reported as able to survive milk pasteurization (Grant et al., 2002) and has been implicated as the etiologic agent of Crohn’s disease in humans (Sechi et al., 2001). Diagnosis of Map infections by means of serology requires further confirmation, in order to avoid the possibility of false-negative results, which are often
210
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recorded at the early stages of infection and crossreactions with genetically related mycobacterial species (Clarke et al., 1996; P´erez et al., 1997; Burrells et al., 1998). Histopathological findings have been proposed as good parameters of paratuberculosis infection in small ruminants. Among sheep and goats which are clinically affected with paratuberculosis, two distinct forms of microscopical pathology were identified (P´erez et al., 1996; Clarke and Little, 1996; Corpa et al., 2000), related to either a high or a low degree of mycobacterial colonization in clinical cases (“multibacillary” or “paucibacillary” presence). Culture is the most reliable method for identifying the disease, but the extended time lapse (of at least 6 weeks) required to culture the organism and its low sensitivity may be considered disadvantages. Moreover, the isolation of Map from infected sheep is difficult (Collins et al., 1990; P´erez et al., 1996; Burrells et al., 1998; Corpa et al., 2000). Current molecular identification protocols for Map rely on the presence of the IS900 insertion element (Clarke and Little, 1996; Burrells et al., 1998; Grant et al., 2002; Ikonomopoulos et al., 2004). No data are available referring to the prevalence of paratuberculosis in cattle, sheep or goats in Mexico. The aim of this study was to diagnose M. avium subsp. paratuberculosis infection in sheep and goat flocks from several Mexican states, using serology and histopathology. Based on a mycobacteria concentration technique used for ileal mucosa, this identification was confirmed by PCR and bacterial culture.
into three groups according to the abundance and distribution of acid-fast organisms: Group 1: AFB in the lamina propia in diffuse form; Group 2: AFB in the medial area and at the top of villosity, in focal form; Group 3: AFB at the top of villosity in a dispersed form.
2. Materials and methods
Cell lysis and DNA extraction from mycobacterial concentrate was carried out as described by van Soolingen et al. (1994) for mycobacterial culture. The quality and quantity of DNA were evaluated in 1% agarose gel, stained with ethidium bromide and the 22 samples were classified into three groups according to their DNA band intensity.
2.1. Paratuberculosis diagnosis Twenty-two animals were selected from six different Mexican states: San Luis Potos´ı, Guanajuato, Quer´etaro, Distrito Federal, Estado de M´exico and Veracruz. All manifesting typical signs of paratuberculosis. These animals were diagnosed by a process of serology and multibacillary type lesions were identified. Serum samples were tested for antibodies against paratuberculosis, using either AGID or ELISA tests; employing a protoplasmic extract (PPA-3, Allied, USA) as antigen. For the ELISA test, serum samples were absorbed with a Mycobacterium phlei suspension (Allied, USA), and a 1:4500 dilution of a rabbit anti-goat IgG horseradish peroxidase conjugate (Sigma, USA) was employed. During the necropsy, specimens were collected for the purpose of histopathological examination. Tissues were fixed and stained with haematoxylin and eosin (HE) and Ziehl–Neelsen (ZN). Samples of small intestine with apparent gross thickening of the intestinal wall were also collected. Samples from the 22 animals having multibacillary lesions were classified
2.2. Mycobacterial concentration Mycobacterial concentration from the intestinal mucosa of infected animals was collected using a modified version of that described by Ratnamohan and Spencer (1986). The mucosal surface was rinsed with 200 g/ml ampicillin and removed from the submucosa by scraping. The mucosa was then homogenized, using a glass/glass tissue grinder (Ten Broeck). Eight grams of mucosal suspension were digested in 150 ml of 0.5% trypsin (Gibco, USA) in PBS. After centrifugation of the suspension at 800 × g for 20 min, the sediment was digested in 20 mg of lysozyme. The suspension was centrifuged as above, then the sediment was re-suspended in 10 ml of distilled water, before being centrifuged again. The presence of mycobacteria in the final pellet was confirmed by examining smears which were stained using the ZN technique. Due to the characteristic clump formation of mycobacteria, it was not possible to quantify the AFB. However, the AFB recovered in the final concentrate was estimated, as well as the presence of cellular debris and samples, which were classified into three categories: Group 1: abundant AFB distributed diffusely throughout the field; Group 2: AFB present in clumps, with cellular debris also present; Group 3: much cellular debris, and no clearly observed AFB. 2.3. Cell lysis and DNA extraction
2.4. PCR PCR assay was performed using specific primers for the insertion sequence IS900 (Pavlik et al., 1999): 5 CGCTGCTGGAGTTGATTG-3 and 5 -TTTCCTTCGGTGCGTTTTC-3 . The reaction was carried out on a final volume of 50 l and consisted of 10 l of 10× PCR× enhancer solution (Invitrogen, USA), 5 l of 10× reaction buffer, 1 l dNTP, 10 mM for each one, 2 l of 50 mM MgCl2 , 2 l from each primer (50 ng/l), 1 l of polymerase 5 U/l (Altaenzymes, Canada) and 4 l DNA. DNA was first denatured at 94 ◦ C for 4 min, then amplified with 30 cycles of (i) denaturation at 94 ◦ C for 55 s, (ii) primer annealing at 45 ◦ C for 51 s, and (iii) elongation at 72 ◦ C for 3 min, using a thermocycler (PCR Express, Hybaid, UK). After the last amplification cycle, the samples were incubated at 72 ◦ C for 8 min. The expected 972 bp PCR
I. Est´evez-Denaives et al. / Small Ruminant Research 72 (2007) 209–213
211
Fig. 1. Photographs of: (a) ileum section showing abundance and distribution of acid-fast bacteria (AFB), Ziehl–Neelsen’s stain (ZN): 10×, 10×, 40×. (b) A smear showing AFB abundance obtained in the final mycobacterial concentrate, ZN: 100×. (c) Mycobacterial DNA. 0.8% agarose gel in TAE containing ethidium bromide.
amplified products were analyzed in 2% agarose gels containing ethidium bromide. 2.5. Mycobacterial culture Each mycobacterial concentrate was treated with 0.76% hexadecyl pyridinium chloride (Sigma, USA) and cultured in screw capped bottles containing L¨owenstein–Jensen medium (Difco, USA) with and without mycobactin J (Allied, USA), as described by Juste et al. (1991). The 22 samples were incubated at 37 ◦ C for 34 weeks.
3. Results Fourteen sheep and eight goats suspected to paratuberculosis were obtained from six different Mexican states in Mexico. Diagnosis was established by means of serology and multibacillary type lesions were iden-
tified. The 22 multibacillary samples showed varying abundance and distribution of AFB in intestinal sections (Fig. 1(a)). There was also variability concerning the results of mycobacterial concentration from the intestinal mucosa of infected animals. Twelve samples were included in Group 1 (54.54%), four samples were included in Group 2 (18.18%) and six samples corresponded to Group 3, that represent 27.27%. Differences in AFB abundance and the presence of cellular debris are shown in Fig. 1(b) and (c) depicts the average amount of DNA obtained from each group. This classification revealed a relationship between the abundance of AFB in intestinal mucosa, the amount of AFB recovered in the final concentrate and the amount of DNA obtained. Diagnosis was further confirmed by means of PCR (Fig. 2). All the 22 DNA samples obtained from mycobacterial concentrate were PCR-positive.
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Fig. 2. Agarose gel electrophoresis of IS900 PCR products. (a) Lane 1: molecular size marker 1 kb DNA ladder; lane 2: positive control; lanes 3–13: DNA products of PCR performed on mycobacterial concentrate samples. (b) Lane 1: molecular size marker 1 kb DNA ladder; lane 2: positive control; lanes 3–13: DNA products of PCR performed on mycobacterial concentrate samples; lane 14: negative control.
Finally, Map was successfully isolated from medium, containing mycobactin J taken from all the animals with multibacillary lesions. The absence of growth in media without added mycobactin indicated that other species of Mycobacterium were either absent or not viable. 4. Discussion The prevalence of paratuberculosis in Mexico is currently unknown. Moreover, large numbers of head of cattle, sheep and goats have recently been introduced from Europe, United States of America, Canada, New Zealand and Australia. As paratuberculosis diagnosis is not an important prerequisite and some of these animals may not be obvious carriers of this microorganism, they can subsequently transmit the infection to the ruminant population in Mexico. The serological results and histological findings obtained in this study confirm the presence of paratuberculosis in goat or sheep flocks from six different Mexican states. The multibacillary form of this pathology was recognized, which confirms observations made in previous studies (P´erez et al., 1996; Clarke and Little, 1996; Corpa et al., 2000). Different methods have been described for the recovery of M. avium subsp. paratuberculosis both from the tissue and faeces of infected sheep for the purpose of obtaining species-specific antigens (Ratnamohan and
Spencer, 1986) to be used in specific DNA detection by PCR (Challans et al., 1994; Garrido et al., 2000) or for Map characterization (Collins et al., 1990; Choy et al., 1998). These techniques allow large quantities of bacteria to be obtained without depending on the long incubation periods required to propagate a successful culture. However, these authors did not quantify the amount of AFB in the samples or in the final pellet. The trend for this organism to occur in clumps makes it difficult to estimate the numbers of cells in the samples. In this study an analysis of the concentration of mycobacteria from frozen tissue samples was performed and the Ziehl–Neelsen stain confirmed the presence of AFB in the mycobacterial pellet. DNA was successfully extracted from in vivo-derived Map, and although the yield of bacterial DNA from these samples varied for each individual host, when these were classified, it became evident that a relationship exists between the abundance of acid-fast organisms in the tissue, the AFB recovered in the final concentrate and the amount of DNA obtained. These findings suggest that the DNA recovered from bacteria consisted predominantly of AFB. Furthermore, this classification will allow to predict the amount of DNA that can be obtained, by measuring the abundance of acid-fast organisms in the tissue and, thus, to define the extent to which this method can be used for Map characterization. Following this procedure for the concentration of mycobacteria, tissue samples collected
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from Groups 1 and 2 are likely to be candidates for Map characterization. The PCR assay produced the expected result for 100% of samples, indicating that M. avium subsp. paratuberculosis was present among the population of AFB, which had been recovered from the intestinal mucosa. Several authors have reported false negative results when this technique was applied to clinical samples, and this has been attributed to the presence of inhibitors in the PCR as well as to inefficient extraction of mycobacteria from the samples, particularly if only a small quantity of organisms is involved (Challans et al., 1994; Clarke and Little, 1996; Burrells et al., 1998; Garrido et al., 2000). L¨owenstein–Jensen medium with mycobactin J was found to be effective for the purpose of isolating M. avium subsp. paratuberculosis from all animals manifesting multibacillary lesions, as reported by Juste et al. (1991), P´erez et al. (1996) and Corpa et al. (2000). Although culture propagation is a slow process, the technique proved to be as sensitive as the detection of specific DNA, recovered from the intestinal mucosa and thus confirmed the diagnosis of the disease. The results of this study indicate that paratuberculosis is widespread in Mexico among goat and sheep flocks. Acknowledgements The study was supported by CONACYT (grant 25395-B) and PAPIIT (grant IN22 1999). I. Est´evez Denaives received financial support from CONACYT (162910). Marycruz Dom´ınguez Punaro, Antonio G´omez Gonz´alez, Miguel Batres Govea and Javier Aranda are acknowledged for providing samples used in this study and Luis Antonio Morales Arreola and Jaime Eugenio C´ordova L´opez for sample preparation. References Burrells, C., Clarke, C.J., Colston, A., Kay, J.M., Porter, J., Little, D., Sharp, J.M., 1998. A study of immunological responses of sheep clinically-affected with paratuberculosis (Johne’s disease). The relationship of blood, mesenteric lymph node and intestinal lymphocyte responses to gross and microscopic pathology. Vet. Immunol. Immunopathol. 66, 343–358. Challans, J.A., Stevenson, K., Reid, H.W., Sharp, J.M., 1994. A rapid method for the extraction and detection of Mycobacterium avium subspecies paratuberculosis from clinical specimens. Vet. Rec. 134, 95–96.
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