Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.

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During a survey of carnivores and omnivores for bovine tuberculosis conducted in Michigan (USA) since 1996, Mycobacterium bovis was cultured from lymph nodes pooled from six coyotes (Canis latrans) (four adult female, two adult male), two adult male raccoons (Procyon lotor), one adult male red fox (Vulpes vulpes), and one 1.5-yr-old male black bear (Ursus americanus). One adult, male bobcat (Felis rufus) with histologic lesions suggestive of tuberculosis was negative on culture but positive for organisms belonging to the Mycobacterium tuberculosis complex when tested by polymerase chain reaction. All the tuberculous animals were taken from three adjoining counties where M. bovis is known to be endemic in the free-ranging white-tailed deer (Odocoileus virginianus) population. There were two coyotes, one raccoon, one red fox, and one bobcat infected in Alpena county. Montmorency County had two coyotes and one raccoon with M. bovis. Two coyotes and a bear were infected from Alcona County. These free-ranging carnivores/omnivores probably became infected with M. bovis through consumption of tuberculous deer. Other species included in the survey were opossum (Didelphis virginiana), gray fox (Urocyon cinereoargenteus), and badger (Taxidea taxus); these were negative for M. bovis.

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The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3) (low dose) or 2 x 10(5) (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.

Mycobacterium awium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. awium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. awium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. awium complex and M. paratubemulosis clinical isolates with T W e r i v e d luciferase reporter phages demonstrated that the majority of these isolates were susceptible t o TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied t o isolate kanamycin-resistant transformants of M. a wium complex and M. paratubemulosir with ischerichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformanb without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species. were grown standing at 37 OC in Middlebrook 7H9 broth, adjusted to pH 5.9, and supplemented with oleic acid/albumin/dextrose complex and 005 % Tween 80. Ferric mycobactin J (Allied Monitor) at 1.0 pg ml-' was added for M. paratuberculosis cultures. For solid media, Tween was omitted and Bacto Agar was added to 7H9 Middlebrook medium at 15 g 1-l. High-titre lysates were prepared and purified by caesium chloride equilibrium density centrifugation, as previously described (Jacobs et al., 1991). Mycobacteriophage infection assays.Phage lysates (0.1 ml of duplicate 10-fold serial dilutions) were incubated with 0.2 ml of fresh mycobacterial cultures, corresponding to approximately 3.0 x lo7 c.f.u., for 30 min at room temperature. Middlebrook 7H9 soft agar (0.7 %) was added, and the cells were plated on 7H9 Middlebrook medium by the soft agar layer method as described by Adams (1959) and incubated at 37 OC until either plaques or confluent lawns developed (1 to 3 d for M. smegmatis, 2 to 4 weeks for M. avium complex strains and M. paratuberculosis). Phage titres were determined at dilutions that gave single isolated plaques to exclude the possibility of lysis from without.Infection with luciferase reporter phasmids and luciferase assays. Infection with luciferase reporter phages and luciferase assays were performed as described by Jacobs et al. (1993). Mycobacterial cultures were grown to exponential phase (OD,,, 0-2; approximately 6.0 x lo7 c.f.u. ml-l) in Middlebrook 7H9 medium, washed three times in growth medium without Tween, 1984), was likely ...

Objective—To investigate the infection of calves with Mycobacterium bovis through oral exposure and transmission of M bovis from experimentally infected white-tailed deer to uninfected cattle through indirect contact. Animals—24 11-month-old, white-tailed deer and 28 6-month-old, crossbred calves. Procedure—In the oral exposure experiment, doses of 4.3 × 106 CFUs (high dose) or 5 × 103 CFUs (low dose) of M bovis were each administered orally to 4 calves; as positive controls, 2 calves received M bovis (1.7 × 105 CFUs) via tonsillar instillation. Calves were euthanatized and examined 133 days after exposure. Deer-to-cattle transmission was assessed in 2 phases (involving 9 uninfected calves and 12 deer each); deer were inoculated with 4 × 105 CFUs (phase I) or 7 × 105 CFUs (phase II) of M Bovis. Calves and deer exchanged pens (phase I; 90 days' duration) or calves received uneaten feed from deer pens (phase II; 140 days' duration) daily. At completion, animals were euthanatized and tissues were collected for bacteriologic culture and histologic examination. Results—In the low- and high-dose groups, 3 of 4 calves and 1 of 4 calves developed tuberculosis, respectively. In phases I and II, 9 of 9 calves and 4 of 9 calves developed tuberculosis, respectively. Conclusions and Clinical Relevance—Results indicated that experimentally infected deer can transmit M bovis to cattle through sharing of feed. In areas where tuberculosis is endemic in free-ranging white-tailed deer, management practices to prevent access of wildlife to feed intended for livestock should be implemented. (Am J Vet Res 2004;65:1483–1489)