Corresponding author: Machackova M. Phone Fax SUMMARY

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1 Infection of ruminants by uncultivable strains of Mycobacterium avium subsp. paratuberculosis in the Czech Republic Machackova M 1, Lamka J 1,2, Yayo Ayele W 1, Parmova I 3, Svastova P 1, Amemori T 1 and Pavlik I 1 1 Veterinary Research Institute, Hudcova 70, Brno, Czech Republic. 2 Charles University, Faculty of Pharmacy, Hradec Kralove, Czech Republic. 3 State Veterinary Diagnostic Institute, Prague, Czech Republic. Corresponding author: Machackova M. Phone Fax machackova@vri.cz Keywords: uncultivable strains, Johne s diseases, incubation period, cattle, sheep, goats, cervids, mouflons. SUMMARY During the survey of paratuberculosis ( ) in cattle, sheep, goats and wild ruminants: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), and mouflon (Ovis musimon), tissue samples of the small intestine and corresponding lymph nodes of 4212 animals were examined. From total number of examined animals, Ziehl-Neelsen microscopy of organs was positive in 308 ruminants. Subsequently culture results of these animals were positive in 271 cases (88.0 %). In 249 heads of cattle (Bos taurus), microscopy positive, Mycobacterium avium subsp. paratuberculosis (Map) was isolated from 226 (90.4 %) animals within 1.5 to 3 months of incubation: relatively fast-growing strains. On the contrary, from the 59 other ruminant species 25 (42.3 %) were found positive for Map within 2 and 3 months of incubation and 20 (33.8 %) within 4 and 8 months of incubation: relatively slow-growing strains. Tissue culture of Ziehl-Neelsen positive tissue samples from gastrointestinal tract from 14 (23.7 %) animals was negative in seven (50.0 %) mouflons, three (21.0 %) fallow deer, one (7.3 %) red deer and three (21.7 %) sheep (Ovis aries). Any relationship between occurrence of uncultivable Map strains and age and sex of ruminants was not revealed. From a total of 30 relatively slow-growing strains isolated from sheep, goats and mouflons, 13 were examined by standardised Restriction Fragment Length Polymorphism (RFLP) analysis. Four RFLP types were identified as cattle strains (n = 12): A- C10, B-C1, B-C2, and E-C1 and one RFLP type was found in sheep strain (n = 1): C-S1. No relationship was observed among RFLP types of above mentioned 13 relatively slow-growing strains and 604 relatively fast-growing strains in our database. In our study of incubation period, also another 382 ruminants were involved. These ruminants were positive in culture of gastrointestinal tract but Ziehl- Neelsen microscopy was negative in all samples. INTRODUCTION The main problem encountered in the diagnosis of paratuberculosis is the slow growth character and therefore difficult isolation of Mycobacterium avium subsp. paratuberculosis (Map) mainly in isolates from biological materials originated from sheep (Ovis aries), goats (Capra hircus), and some species of wild ruminants (3,12). Taylor (28,29) demonstrated the differences observed in growth characteristics of Map isolated from cattle (Bos taurus) and sheep in primary cultures. The out come of studies conducted to demonstrate adaptation of Map strains to defined host species were conflicting. Strains isolated from sheep caused clinical paratuberculosis in experimentally infected cattle (30) and vice versa (13). Strains isolated from goats elicited subclinical infection in experimentally infected calves, however in kids and rabbits (Oryctolagus cuniculus) clinical manifestation of the disease was observed (5,25,16). De Lisle et al. (8) demonstrated that sheep and cattle strains had the same virulence to red deer (Cervus elaphus), causing clinical paratuberculosis. Since the 1980s, different molecular biology methods were applied for the identification of Map isolates (2,4,8,9,10,17,18,19,22,24,31,32). Collins et al. (6) have differentiated isolates of Map from cattle, sheep and goats in to three groups by appliance of Restriction Fragment Length Polymorphism method (RFLP): RFLP types C (cattle), RFLP types S (sheep) and RFLP types I (intermediate). They identified isolates by RFLP using the restriction endonuclease BstEII, according to different localisation and number of 191

2 specific sequence IS900 in genome of Map. In 1999 twenty RFLP subtypes of C (C1-20), three RFLP subtypes of S (S1-3) and two RFLP subtypes of I (I1-2) types were known. Map of RFLP type C was isolated not only from cattle, also from sheep, goats, wild ruminants and rabbits. The RFLP type S was isolated from sheep, goats, cattle and deer (6,9,10,19,20). RFLP type I was isolated from sheep (6). Stevenson et al. (27) have demonstrated the close genotypic relationship of pigmented isolates of Map with the sheep isolate of RFLP type S using the methods RFLP and pulsed-field gel electrophoresis (PFGE). From a total of 1002 isolates examined by RFLP, 992 isolates of 28 RFLP types showed distinct colonies after three months of incubation identified as relatively fast-growing isolates. Ten isolates of five RFLP types with an incubation period of four up to eight months before the observation of visible colonies were designated as relatively slow-growing isolates. It was not found any relation between the RFLP type and the growth rate (19). The objective of this study was to find the relationship between uncultivable Map isolates, the host animals, duration of growth of the organism and RFLP type of selected Map isolates. MATERIALS AND METHODS Origin of the gastrointestinal specimens. Domestic and wild ruminants were originated from 67 foci of paratuberculosis: cattle (n = 41), sheep (n = 2), goats (n = 1), cervids (n =15) and mouflons (n = 8) during the period (Table 1). Gastrointestinal tissue of 4212 ruminants was obtained from 2010 heads of cattle, 348 sheep and goats, 368 red deer, 905 roe deer (Capreolus capreolus), 338 fallow deer (Dama dama) and 243 mouflons (Ovis musimon). Twenty six of sampled mouflons and 19 of sampled fallow deer were originated from the same game park. Pathological-anatomical, microscopic (Ziehl- Neelsen staining) and culture examination of the specimens were performed in all samples. Table 1. Outbreaks of paratuberculosis in ruminants. Animal species Farms Game Wild Total parks nature Cattle Sheep and goats Cervids Mouflons Total Laboratory examinations. Specimens were collected as a part of the intestine (terminal jejunum or ileocaecal valve) and corresponding lymph nodes. After pathological examination of organs specimens were stored at 20 C. A minimum number of four specimens derived from two mucosal samples and two corresponding lymph nodes were examined microscopically stained according to Ziehl-Neelsen. Subsequently, 1 g of tissue from domestic and wild ruminants were homogenised by a stomacher (Lab Blender, Germany) and the resulting tissue suspension was decontaminated in 0.75 % HPC for 24 h (domestic ruminants samples) and 72 h (wild ruminants samples), respectively (21). These samples obtained from cattle were inoculated on to three tubes with Herrold egg yolk medium (HEYM) with Mycobactin J. Because sodium pyruvate has toxic effect on strains of Map isolated from wild ruminants and sheep (12), specimens derived form these animals were cultured on three vials of HEYM with Mycobactin J but one vial of HEYM with Mycobactin J and without sodium pyruvate. Evaluation of length of time for growth. Inoculates were incubated at 37 ºC for 12 months. Cultures were read every third week for four months and after that every month for the following 12 months of incubation. Map growing within 3 months were indicated as relatively fast-growing isolates and Map growing later on as relatively slow-growing isolates (19). Identification of Map isolates. All 653 isolates were identified by test of dependence on Mycobactin J (subculture on 3 HEYM with Mycobactin J and one HEYM without Mycobactin J). Some of the strains with worse growth character in primoculture were identified by IS900 PCR method (14). Differentiation of Map isolates. Selected 617 isolates from tissue of ruminants were differentiated by standardised RFLP method by using the restriction endonucleases BstEII and PstI (19). RESULTS Map was detected in the gastrointestinal tract of 653 ruminants (Table 2). A total of 623 isolates were categorised to the group relatively fast-growing and 30 isolates were categorised to the group relatively slow-growing isolates. Isolation of Map from ruminants with positive microscopic smear findings of acid fast rods (AFR) in the gastrointestinal tract (Figure 1, Table 2). All Map isolates from cattle (n = 226), goats (n = 2), and cervids (n = 13) belonged to the group of relatively fast-growing isolates. At the second month of incubation, Map was isolated from 140 of cattle (62 %) and both goats. In six cervids (85.0 %) Map was isolated as early as at the first month of incubation. Ovine isolates (n = 23) were found to occur in both groups relatively slow-growing and relatively fast-growing isolates. In only one sheep, Map was isolated at the first month of the incubation period, however 192

3 Table 2. Laboratory examination of ruminant s gastrointestinal tract. Z-N: Ziehl-Neelsen staining. C.: culture. Animal species Nº of animals Total Nº of C+ve (%) Examined Direct Z-N positive Direct Z-N negative Total Nº. C.+ve (%) Nº C.+ve (%) Cattle (90.4) (18.4) 551 (27.4) Sheep and goats (89.3) (2.5) 33 (9.5) Red deer (90.9) (6.1) 32 (8.7) Fallow deer (50.0) (2.1) 10 (3.0) Mouflon (50.0) (4.3) 17 (7.0) Roe deer (1.1) 10 (1.1) Total (88.0) (9.7) 653 (15.5) the majority of isolates (n = 15) were visible between the 4 th to 5 th months of incubation. In four infected sheep, rough and small colonies were predominant between the 6 th to 8 th months of the incubation period. In seven mouflons, relatively fast-growing isolates were detected in six animals, as well in one mouflon Map was isolated at the fifth months of incubation. % cattle 226 sheep+goats 25 cervids13 mouflons 7 detected in 23 (88.5 %) animals. From 17 cervids Map was isolated in 13 (76.5 %) animals. Culture examination of three fallow deer and one red deer with clinical disease, originated from abroad from region of unknown epidemiological situation, was negative. From a total of 14 mouflons, Map was detected only in seven animals. Massive infection of the gastrointestinal tract was observed in 9 (64.2 %) mouflons and 5 (83.3 %) fallow deer. Nevertheless, an attempt made to isolate Map after 12 months of incubation was not successful in such infected mouflons (n = 5) and fallow deer (n = 2). All seven animals infected with uncultivable Map originated from the same game park months Figure 1. Incubation period of Map isolated from the GIT. Z-N positive ruminants (n = 271) % 40 cattle 325 sheep+goats 8 cervids 39 mouflon 10 Map isolates from ruminants with negative microscopic smear findings (AFR) in the gastrointestinal tract (Figure 2, Table 2). Isolates originated from cattle (n = 325) and cervids (n = 39) belonged to the group relatively fast-growing isolates. In 217 individuals of cattle the first visible colonies were observed at the second month of incubation and in 20 cervids at the first month of incubation. On the contrary, sheep (n = 8) and mouflon (n = 10) isolates belonged to both groups of relatively fast-growing and relatively slow-growing isolates. In sheep 50 % of isolates belonged to the group of relatively fast-growing strains and the other 50 % belonged to the group of relatively slowgrowing isolates. In mouflons, however, four isolates belonged to the relatively fastgrowing and the remaining six isolates to the relatively slow-growing isolates. Performance of detection of Map isolates from the gastrointestinal tract of animals with positive AFR findings (Table 2). From a total of 249 heads of cattle, Map was isolated in 225 (90.4 %) animals. In both goats isolates, were obtained from the mucosa of the small intestine as well as the mesenteric lymph nodes. In 26 sheep Map was months Figure 2. Incubation period of Map isolated from the GIT. Z-N negative ruminants (n = 382). Relationship between RFLP types and the growth character (Figure 3). Using the RFLP method, 604 relatively fast-growing and 13 relatively slow-growing Map isolates were differentiated. In cattle (n = 519) five RFLP types (A-C10, B-C1, B-C2, B-C9 and E-C1) of only relatively fast-growing Map isolates were observed. In 29 isolates from sheep and goats, five RFLP types (A-C10, B-C1, B-C2, E-C1 and C-S1) were identified from which four RFLP types (A-C10, B-C2, E-C1 and C-S1) were observed in relatively slow-growing (n = 12) isolates. In 68 wild ruminants relatively fastgrowing isolates of RFLP types B-C1, B-C9, D- C12 and M-C16 were identified. In mouflons, however, one relatively slow-growing isolate of RFLP type B-C1 was detected. 193

4 Figure 3. Incubation period of different RFLP types of Map. DISCUSSION In the isolation of Map from tissue samples of different species of ruminants, samples derived from sheep were found difficult to culture (Table 2). Similar problems in isolating Map were observed in the U.S.A., Europe, Australia and New Zealand (3,6,8,12,15,19,27,28). We have observed high occurrence of uncultivable strains in specimens collected from mouflons, which are species related to sheep (Table 2). Therefore it can be inferred that these uncultivable strains are transmissible to other ruminant s species. Contrary to the work of Juste et al. (12) we did not confirm the highest detection rate of Map from tissues of wild ruminants and small ruminants on HEYM with Mycobactin J and without sodium pyruvate. Opinions on whether different types of strains of Map may be transmitted between ruminants kept on common pasture are different. Taylor (28), Armstrong (1) and Seaman and Thompson (26) do not assume sheep to be the source of infection of Map for cattle. Riemann et al. (23), Jessup et al. (11), De Lisle et al. (9), Pavlik et al. (18), Cook et al. (7), Greig et al. (10) and Pavlik et al. (17) describe inter-species transmission of paratuberculosis. In one of our studied farm, where mouflons and fallow deer were kept together, we detected uncultivable strains of Map in both species. From the available history it was not possible to find out which species of ruminants might have been a source of the infection. The observation that no relationship was observed between RFLP types and the growth characteristics of Map isolates in vitro study coincides with the work of Pavlik et al. (19). Only a RFLP type C-S1, sheep strain, with visible colonies after four months of incubation represented the relatively slow-growing isolates. Bauerfeind et al. (2) described relatively slow-growing isolate of RFLP type H-C1, which later on was detected in cattle as a relatively fast-growing isolate of the same RFLP type H-C1 (19). As the predominant isolates detected in sheep were bovine RFLP types we were expecting the growth of visible colonies within three months, nevertheless the incubation period of some isolates of some RFLP types (A-C10, B-C2 and E-C1) was longer than three months. The only Map which was representative of the relatively slow-growing isolate was the sheep isolate of RFLP type C- S1. CONCLUSIONS It has been ensued from our results that two types of isolates of Map occur in ruminants according to the incubation period: relatively fast-growing and relatively slow growing isolates, which are difficult to detect in vitro. Relatively fast-growing isolates were found mostly in cattle and cervids, however, relatively slow-growing isolates were detected in sheep and mouflons. The heavy infection of intestine detected in sheep and in mouflons was caused by Map isolates, which were impossible to detect in vitro even after 12 months of incubation. Occurrence of these uncultivable Map organisms was found out in one game park where mouflons and fallow deer were kept together. It is necessary for diagnostics of such cases of infection to apply methods which enable to detect DNA of 194

5 causal agent of paratuberculosis directly in tissue samples of intestine. ACKNOWLEDGMENTS We would like to thank M. Fisakova and Z. Rozsypalova for technical assistance. The research was supported partially by the grants No. QLRT Brussels, EC, further by grant QD1191 of Ministry of Agriculture, Czech Republic and No. J13/ of the Ministry of Education, Youth and Sports of the Czech Republic. REFERENCES 1. Armstrong MC Johne's disease of sheep in the South Island of New Zealand. N Z Vet J 4: Bauerfeind R, Benazzi S, Weiss R, Schliesser T, Willems H and Baljer G Molecular characterization of Mycobacterium paratuberculosis isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900. J Clin Microbiol 34: Carrigan MJ and Seaman JT The pathology of Johne's disease in sheep. Aust Vet J 67: Collins DM and De Lisle GW Restriction endonuclease analysis of varius strains of Mycobacterium paratuberculosis isolated from cattle. Am J vet Res 28: Collins P, Davies DC and Matthews PRJ Mycobacterial infection in goats: diagnosis and pathogenicity of the organism. Br Vet J 140: Collins DM, Gabric DM and De Lisle GW Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. Clin Microbiol 28: Cook WE, Cornish TE, Shielder S, Lasley B and Collins MT Radiometric culture of Mycobacterium avium paratuberculosis from the feces of tule elk. J Wild Dis 33: De Lisle GW, Collins DM and Huchzermeyer HAK Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization. Onderstepoort J Vet Res 59: De Lisle GW, Yates GF and Collins MD Paratuberculosis in farmed deer: case reports and DNA characterization of isolates of Mycobacterium paratuberculosis. J Vet Diagn Invest 5: Greig A, Stevenson K, Henderson D, Perez V, Hughes V, Pavlik I, Hines II ME, McKendrick I and Sharp JM Epidemiological study of paratuberculosis in wild rabbits in Scotland. J Clin Microbiol 37: Jessup DA, Abbas B and Behymer D Paratuberculosis in tule elk in California. JAVMA 179: Juste RA, Marco JC, De Ocariz CS and Aduriz JJ Comparison of different media for isolation of small ruminants strains of Mycobacterium paratuberculosis. Vet Microbiol 28: Kluge JP, Merkal RS, Monlux WS, Larsen AB, Kopecky KE, Ramsey FK and Lehmann RP Experimental paratuberculosis in sheep after oral, intratracheal, or intravenous inoculation: lesions and demonstration of the etiologic agent. Am J Vet Res 29: Kunze ZM, Portaels F and McFadden JJ Biologically distintict subtypes of Mycobacterium avium differ in possession of insertion sequence IS901. J Clin Microbiol 30: Merkal RS and McCullough WG A new Mycobactin, Mycobactin J, from Mycobacterium paratuberculosis. Current Microbiol 7: Mondal D and Sinha RP Pathogenicity of caprine strain of Mycobacterium paratuberculosis in rabbit. Ind J Anim Science 62: Pavlik I, Bartl J, Dvorska L, Svastova P, Du Maine R, Machackova M, Yayo Ayele W and Horvathova A. 2000a. Epidemilology of paratuberculosis in wild ruminants studied by restriction fragment lenghth polymorphism in the Czech Republic during the period Vet Microbiol 70: Pavlik I, Bejckova L, Pavlas M, Rozsypalova Z and Koskova S Characterisation by restriction endonuclease analysis and DNA hybridisation using IS900 of bovine, ovine, caprine and human mycobactin-dependent strains of Mycobacterium paratuberculosis isolated in various locations. Vet Microbiol 45: Pavlik I, Horvathova A, Dvorska L, Bartl J, Svastova P, Robin du Maine and Rychlik I. 1999a. Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis. J Microbiol Meth 38: Pavlik I, Matlova L, Bartl J, Svastova P, Dvorska L and Whitlock R. 2000b. Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle. Vet Microbiol 77: Pavlik I, Rozsypalova Z, Vesely T, Bartl J, Matlova L, Vrbas V, Valent L, Rajsky D, Mracko I, Hirko M and Miskovic P. 2000c. Control of paratuberculosis in five cattle farms by serological tests and faecal culture during the period Vet Med Czech 45: Pavlik I, Vesely T, Bartl J, Horvathova A, Matlova L, Vrbas V, Valent L, Miskovic P and Hirko M. 1999b. Reliability of diagnostic methods (clinical examination, faecal culture, skin and serologic tests) for paratuberculosis of cattle and sheep during the eradication and control programme. In: Proceedings of the Sixth International Colloquium on Paratuberculosis, th February, 1999, Melbourne, Victoria, Australia, ISBN : Riemann H, Zaman MR, Ruppanner R, Aalund O, Jorgensen JB, Worsaae H and 195

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