Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS901 RFLP typing, and virulence for poultry.

Avian tuberculosis was detected in one flock of 38 water birds of the families Ardeideae (n = 20) and Threskiornithidae (n = 18). Mycobacterium avium subsp. avium (MAA, serotype 1, genotype IS901+ and IS1245+) was more often (p = 0.01) detected in tissue and/or faecal samples in 18 (90.0%) birds form the Ardeideae family: little egret (Egretta garzetta), buff-backed heron (Bubulcus ibis), great white egret (Egretta alba), and bittern (Botaurus stellaris) in comparison to two (11.1%) birds from the Threskiornithidae family: sacred ibis (Threskiornis aethiopicus). Avian tuberculosis was not diagnosed in spoonbills (Platalea leucorodia). Tuberculous lesions were found in nine birds. MAA isolates of IS901 RFLP type F-C3 were present in all of the 20 infected birds and in all environmental isolates. A mixed infection with the MAA isolates of three RFLP types F-C3 (tissue isolate), G-C3, and T-C3 (faecal isolates) was found in one sacred ibis. All 20 tissue isolates of IS901 RFLP type F-C3 from 20 birds and 8 environmental MAA isolates were fully virulent in pullets, whilst the isolates of RFLP types G-C3 and T-C3 were non-virulent in pullets. All of the tested MAA isolates had the same IS1245 RFLP "bird profile". In 12 of 20 infected birds with MAA M.a. hominissuis isolates of serotypes 4, 8, 9 and genotype IS901- and IS1245+ were detected and in 8 other birds mycobacteria not belonging to the M. avium complex were found. The presence of MAA in the environment may be a source for further spread of the causal agent of avian tuberculosis among other groups of animals in zoological gardens, farm animals, and also among their keepers.

Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds.

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.

Earthworms (Oligochaeta, Lumbricidae) and mycobacteria.

The objective of the study was to define the role of earthworms in the survival of mycobacteria in animal populations. In 13 sampling sites mycobacteria were detected in 53 (5.5%) samples of faeces and parenchymatous tissues from animals, in 25 (7.3%) environmental and in nine (8.2%) earthworm samples. In cattle and goat farms affected by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) of IS900 restriction fragment length polymorphism (RFLP) type B-C1 was isolated from 37 (4.6%) faecal samples, three (1.4%) environmental and one (3.1%) earthworm sample. Investigations of aviaries affected by avian tuberculosis detected M. avium of genotype IS901+ and IS1245+ in six (7.9%) bird's faecal and in four (4.4%) environmental samples. M. avium (genotype IS901- and IS1245+) was detected in four (4.4%) and M. abscessus in one (1.1%) environmental sample. M. avium of genotype IS901- and IS1245+ and M. gastri were isolated from three (6.4%) earthworm samples. In pig farm with mycobacteriosis M. avium of genotype IS901- and IS1245+ was detected in five (20.0%) faecal samples from pigs and in four (12.9%) environmental samples. M. scrofulaceum was isolated in one (4.6%) sample of Lumbricus rubellus. In laboratory experiments identical RFLP types of M. paratuberculosis were isolated from bodies and faeces of earthworms 1-2 days after the last contact with the faeces contaminated with the same RFLP type of M. paratuberculosis. The results suggest that earthworms may become vectors of mycobacteria.

Paratuberculosis in farmed and free-living wild ruminants in the Czech Republic (1999-2001).

Due to the occurrence of the infection of Mycobacterium avium subspecies paratuberculosis among domestic ruminants and the rapid development of farmed deer industry and the market of cloven-hoofed game we have carried surveys of paratuberculosis, beginning in 1997, in the most common four species of wild ruminants in the Czech Republic [Pavlik et al., Vet. Microbiol. 77 (2000) 231-251]. From 1999 the prevalence of paratuberculosis has been slightly reduced in all three types of husbandry of wild ruminants. Nevertheless paratuberculosis has been diagnosed in wild ruminants in three districts, in four game parks and in five farms. M. a. paratuberculosis was isolated from 128 (5.3%) out of 2,403 wild ruminants of four animal species: 106 red deer, 2 roe deer, 4 fallow deer and 16 mouflons. In red deer farms, the highest number of clinical paratuberculosis cases was in yearling deer. RFLP type B-C1 of M. a. paratuberculosis predominated during the second period (1999-2001) in all types of husbandry with no relationship to wild ruminant species. New "cattle" RFLP types B-C5 and B-C16 of M. a. paratuberculosis were described in infected farmed red deer and one "intermediate" RFLP type R-I4 in fallow deer from one game park. The survival of M. a. paratuberculosis was found to be 4 months during winter in the pasture after destocking of all cattle infected with paratuberculosis. We found that non-vertebrates, wild ruminants or non-ruminant wildlife can be vectors and potentially become a risk factor in the spread of M. a. paratuberculosis infection.

Beetles as possible vectors of infections caused by Mycobacterium avium species.

Mycobacteria were not isolated from any of 229 beetle imagoes of 29 species originating from 14 distinct localities in the Czech and Slovak Republics: 186 imagoes (34 samples) and 43 imagoes (12 samples) from the wild and herds with paratuberculosis infected ruminants, respectively. From 75 environmental samples taken from barns with infected ruminants, Mycobacterium avium subsp. paratuberculosis was isolated from five scrapings of the floors in barns and a feed processing room. From bran and peat taken from pig farms, M. a. hominissuis was diagnosed in 13% of 72 samples and in 69% of 70 samples, respectively. M. a. avium was isolated from 2 (2.9%) and atypical mycobacteria from 12 (17.1%) peat samples. In the respective experiments, larvae of Tenebrio molitor Linnaeus and Zophobas atratus Fabricius were infected in vitro with isolates of M. a. paratuberculosis of IS900 RFLP type B-C1 and M. a. avium of IS901 RFLP type F-C3. T. molitor larvae were also infected with M. a. hominissuis by naturally contaminated bran and peat. M. a. paratuberculosis and M. a. avium were diagnosed in larvae of both species on days 1 to 3 post infection (p.i.). M. a. hominissuis was isolated from T. molitor larvae fed by bran on days 4 to 9 p.i. and from imagoes on day 35 p.i. and from larvae fed by peat on days 4 to 14 p.i. RFLP types of all the isolates identified before infection and after isolation from larvae were identical. Thus, beetles could mechanically transmit mycobacteria, this hazard should be considered for both the implementation of control measures and feeding captive animals with larvae.

Potential risk of Mycobacterium avium subspecies paratuberculosis spread by syrphid flies in infected cattle farms.

The syrphid Eristalis tenax Linnaeus (Diptera: Syrphidae) may be found in and around dung storage pits at cattle farms at various developmental stages of their life cycle. The purpose of this study was to investigate the occurrence of Mycobacterium avium ssp. paratuberculosis in 1044 E. tenax samples at various developmental stages, as well as fresh and stored dung originating from nine cattle farms. Mycobacterium fortuitum was isolated from one (1.5%) larva from the vicinity of three paratuberculosis-free herds of cattle. Mycobacterium a. paratuberculosis was isolated from 111 (21.4%) of E. tenax larvae collected from two of seven farms known to be infected with the causal agent of paratuberculosis. Mycobacteria were not isolated from any of the 340 pupae, 41 adults of 78 samples of exoskeletal exuviae. Mycobacterium a. paratuberculosis isolates from E. tenax larvae were of the IS900 restriction fragment length polymorphism (RFLP) type B-C1, identical to that detected in faecal samples from cattle herds infected with paratuberculosis. Larvae artificially infected with mycobacteria of IS900 RFLP type B-C9 did not contain statistically more CFU of identical IS900 RFLP type B-C9 in the intestinal tract and internal organs than on the body surface. These results show that M. a. paratuberculosis can survive in the intestinal tract and internal organs of E. tenax.

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis.

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis.

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.