The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n = 215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).     

Mycobacterium bovis infection in the brush-tailed possum (Trichosurus vulpecula): II. Comparison of experimental infections with an Australian cattle strain and a New Zealand possum strain

Experimentally induced M. bovis infection in brush-tailed possums (Trichosurus vulpecula) with a possum-adapted strain from New Zealand, produced a more rapidly progressive, severe disease than did that induced by an Australian cattle strain. Possums infected with either strain excreted large numbers of mycobacteria in urine, faeces and discharges from abscesses. Aerosol transmission of infection occurred only with the New Zealand possum strain. The infectious aerosol most probably was of respiratory origin but urine may have been the source.All possums had focal interstitial nephritis consistent with Leptospira infection. Histopathological evidence of severe stress was observed in only one possum; the remainder had either mild or no stress-associated lesions.     

The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

Aim. To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease.

Methods. Thirty-three adult possums were inoculated via the endo-bronchial route with 20–100 colony forming units of Mycobacterium bovis. The possums were killed at 1, 2, 3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopatbologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally.

Results. Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised.

Conclusions. Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.     

Tuberculosis in a free-living brown hare (Lepus europaeus occidentalis)

A free-living brown hare (Lepus europaeus occidentalis) trapped during a survey of tuberculous possums in the Ahaura Valley, Westland, was found to have both microbiological and histopathological evidence of infection with Mycobacterium bovis. The mesenteric lymph nodes were enlarged and showed extensive caseation, tuberculous nodules were present in the liver and kidney, and there was a proliferative pleurisy. Histologically, characteristic tuberculous granulomatous foci were seen in the mesenteric lymph nodes, liver, kidney, pleura and lungs, often in association with small numbers of acid-fast organisms. This is the first report of naturally occurring tuberculosis due to M. bovis in the brown hare.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

Experimental infection with BCG as a model of tuberculosis in the brushtail possum (Trichosurus vulpecula)

AIMS: To study the development and progression of lesions produced following experimental inoculations of possums with Bacille Calmette-Guérin (BCG) Pasteur Strain 1173P2 and to compare these with lesions that occurred following natural Mycobacterium bovis infection.

METHODS: Possums were inoculated with 5 x 10⁶ colony forming units (cfu) of BCG via the intra-dermal (I/D) route into the dorsum of the neck (n=38) or the left brachium (n=7), orally (n=10), via the endo-bronchial (E/B) route (n=12), or intravenously (I/V) (n=10, half of which received 5 x 10⁶ cfu and half of which received 5 x 10⁷ cfu of BCG). The possums were humanely killed between 1–4 weeks post inoculation (p.i.), and the nature and distribution of lesions examined grossly and histopathologically.

RESULTS: The distribution of lesions following I/D inoculation via either route was similar to that of the natural disease, but there were few lesions in the lung. Endo-bronchial inoculation resulted in pulmonary disease but produced few lesions outside the respiratory tract. Lesions produced by I/V inoculation were similar in distribution to those seen in terminally ill tuberculous possums. No lesions were produced following oral inoculation. Regression of lesions commenced after 3 weeks p.i.

CONCLUSIONS: Although the phenomenon of lesion resolution restricts the use of BCG to the study of early lesion development, it avoids the overwhelming disease induced using M. bovis and thus allows the early phases of the development and progression of tuberculosis in this species to be observed. Intra-dermal inoculation produced evidence that infection through the skin is associated with lesions in superficial lymph nodes, whereas pulmonary disease was associated with E/B inoculation. The results are consistent with the hypothesis that both percutaneous and respiratory routes are important in natural infection of possums with M. bovis.     

A study of cattle-to-cattle transmission of Mycobacterium bovis infection

Twenty steers, positive to the single intradermal comparative tuberculin test (SICTT), were selected fromherds with a recent history of Mycobacterium bovis infection. Ten steers, negative to SICTT, were selected from herds with no history of M. bovis infection and served as in-contact animals. The animals were divided into 10 groups, each consisting of two SICTT-positive (reactor) animals and one in-contact animal. Each group was housed in an individual loose-box for a period of 1 year. Five of the groups were fed a restricted diet for part of the experiment. All cattle were slaughtered at the end of the study period and examined at post mortem. Transmission of infection to an in-contact animal occurred in four of the 10 groups. One of the four in-contact animals, which became infected, had a retropharyngeal lymph node tubercle and M. bovis was isolated from lymph nodes without visible lesions from the other three. Two of the infected in-contact animals without visible lesions did not show any detectable cell-mediated immune response. There was no evidence that dietary, restriction had any effect on transmission of disease.     

Longevity of Mycobacterium bovis in brushtail possum (Trichosurus vulpecula) carcasses,and contact rates between possums and carcasses

Abstract

AIM: To determine, for a variety of environmental conditions, how long Mycobacterium bovis might remain viable inside the carcass of a brushtail possum (Trichosurus vulpecula) that died of bovine tuberculosis (Tb), and to measure the rate of contact between free-ranging possums and possum carcasses.

METHODS: Lesions of M. bovis were simulated by inoculating excised spleens weighing 0.5–1 g with 0.2 mL liquid culture containing approximately 5 x 10⁷ cfu M. bovis/mL. Simulated lesions were inserted into possum carcasses (n=48) at the peripheral lymph nodes. Carcasses were placed in the field at two sites (a tussock grassland and a podocarp-broadleaved forest site) and in two seasons (summer and winter) for up to 62 days. Survival rates of M. bovis were estimated by sampling the simulated lesions over time, and culturing the recovered lesion to determine if any viable M. bovis bacteria were present.

The time taken for a free-ranging possum to first encounter a dead possum in its home range was estimated by live-trapping possums and fitting them with proximity loggers (n=13). A ‘contact’ was recorded if these possums came within 40–50 cm of proximity loggers fitted to possum carcasses.

RESULTS: There were strong seasonal and site effects in the survival rate of M. bovis in possum carcasses. In the grassland habitat, no viable bacilli were cultured from any carcass after 3 days in summer, whereas in winter all samples were culture-positive for the first 20 days, and some were still positive after 27 days. The survival rates for forest habitat were intermediate between the results for grassland, and there were no culture-positive carcasses after 9 days in summer or 27 days in winter.

In summer, infected carcasses (n=6) were first encountered by possums a mean 1.9 (range 0.4–6.7) days after placement.

CONCLUSIONS: Possum carcasses were contacted by free-ranging possums within the period that viable M. bovis were shown to survive in a carcass. The risk of such infection is likely to be most significant in winter or in areas with microhabitats where the survival of M. bovis is high. However, the generally low survival rate of M. bovis in possum carcasses and the low frequency of possum-to-carcass contacts indicate this route of transmission alone could not maintain Tb in a possum population.     

Induction of immunity in calves to mycoplasma bovis infection of the respiratory tract

Immunity to colonization of the respiratory tract by Mycoplasma bovis (formerly Mycoplasma agalactiae subsp. bovis was induced in calves by inoculation of formalin inactivated organisms. Animals inoculated intramuscularly and then intratracheally with inactivated mycoplasmas had significantly fewer M. bovis in their lungs, compared with non-vaccinated animals, 3 weeks after intratracheal challenge with viable organisms. In contrast, there was no significant difference in the numbers of M. bovis isolated from the lungs of control animals and of calves given two intramuscular inoculations of inactivated organisms. These results indicate that stimulation of the local immune system is important in the development of resistance to M. bovis respiratory infection following vaccination with inactivated organisms.     

Experimental Mycobacterium bovis infection of cattle: Effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions

Groups of l8-month-old cattle were inoculated intratracheally with 5 X 10⁵ colony forming units (high dose) or 500 colony forming units (low dose) of Mycobacterium bovis to determine an appropriate dose to induce lesions similar to those seen in the natural disease. An additional group of 21–28 weeks pregnant cattle were inoculated with the high dose of M. bovis to determine if pregnancy increased the susceptibility of cattle to M. bovis infection. By 23–24 weeks after challenge, the high dose of M. bovis had induced extensive lung lesions, and tuberculous lesions were observed in the lymph nodes of the head, neck, and thoracic and abdominal cavities. In contrast, the low dose of M. bovis induced predominantly small lesions (< 1 cm diameter) which were localised to the lungs and pulmonary lymph nodes. The lesions induced by the low dose were similar to those seen in the natural disease in cattle. The majority of the high dose group cattle produced strong antibody responses to M. bovis culture filtrate, while only one low dose animal produced a detectable response. All of the M. bovis-inoculated cattle produced strong cellular immune responses to bovine PPD (skin test and interferon-γ responses). Pregnancy did not appear to affect the susceptibility to M. bovis infection, and immune responses of the cattle in this group at the end of the study were similar to those in the high dose non-pregnant group. However, from the first test after calving, the interferon-y responses of peripheral blood cultures to bovine PPD were low compared with the responses prior to calving.     

Preferential infection sites of Cysticercus bovis in cattle experimentally infected with Taenia saginata eggs

The preferential sites of infection of Cysticercus bovis were evaluated in the skeletal muscle and entrails of 25 cattle that were experimentally infected with Taenia saginata (2 × 10⁴ eggs). Two other animals were not inoculated (control). Ninety days after inoculation, all the cattle were euthanized. The carcasses were deboned and dissected into 26 anatomical sections (masseter muscles, brain, tongue, esophagus, heart, diaphragm, lungs, liver, kidneys, spleen, top sirloin butt, bottom sirloin butt, outside round, top (inside) round, transversus abdominus, top sirloin cap, strip loin, full tenderloin, eye of round, knuckle, shoulder clod, foreshank, shank, chuck, back ribs, and tail muscles). The dissected tissues were sliced into 5 mm sections. From the 25 cattle, 9258 C. bovis (cysticerci) were recovered; 75.02% (6946) of these were recovered from skeletal muscles and 24.98% (2312) from the entrails. A high parasitism level was found in the shoulder clod (12.55%), heart (11.02%), liver (9.48%), masseter muscles (8.51%), chuck (8.25%), strip loin and full tenderloin (7.26%), knuckle (6.63%), and back ribs (5.53%), totaling 69.23% (5738) of all of the detected cysticerci. On the other hand, there was a low C. bovis parasitism level in the brain, spleen, tail muscles, kidneys, esophagus, and diaphragm, representing just 3.9% of the total number of cysticerci. Given these results, we conclude that specific skeletal musculature regions, such as the shoulder blade, chuck, strip loin and full tenderloin, knuckle, back ribs and top round, which are not officially examined in many countries, are effective sites to efficiently screen C. bovis infection. To date, these regions have not been considered as preferential sites of C. bovis infection. Based on our work, however, these regions deserve greater attention from health inspectors because they contained a greater number of Cysticercus than the other regions of carcasses that are parasitized by T. saginata larvae.     

The pathogenesis of experimental endo-bronchial Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula)

AIM: To study the nature and development of experimentally induced respiratory tuberculosis in possums and compare the lesions observed with those seen in the natural disease. METHODS: Thirty-three adult possums were inoculated via the endo-bronchial route with 20-100 colony forming units of Mycobacterium bovis. The possums were killed at 1,2,3 and 4 weeks after inoculation and the nature and distribution of the lesions studied in detail histopathologically. Alveolar macrophages recovered from the infected possums were also studied ultrastructurally. RESULTS: Macroscopic lesions were largely confined to the respiratory tract, increasing in size and number with time. Histology greatly increased the detection of the total number of lesions. The most common sites affected outside the respiratory tract were the liver and hepatic lymph nodes, but lesions were less common in peripheral lymph nodes than is observed in the natural disease. Intra-pulmonary lesions were centred on blood vessels and their associated lymphatics. Peripheral blood lymphocyte blastogenic responses to M. bovis antigens were first detected at 3 weeks after inoculation, which was 1 week after lymphocyte infiltrations were detected in the lungs, but 1 week before the majority of infections became generalised. CONCLUSIONS: Differences in the nature of pulmonary lesions and the distribution of lesions were observed between experimentally induced and the natural disease. Rapid haematogenous and lymphatic spread occurs early in the experimentally induced disease.     

Multiple sampling and discriminatory fingerprinting reveals clonally complex and compartmentalized infections by M. bovis in cattle

The combination of new genotyping tools and a more exhaustive sampling policy in the analysis of infection by Mycobacterium tuberculosis has shown that infection by this pathogen is more complex than initially expected. Mixed infections, coexistence of clonal variants from a parental strain, and compartmentalized infections are all different modalities of this clonal complexity. Until recently, genotyping of Mycobacterium bovis in animal populations was based on spoligotyping and analysis of a single isolate per infection; therefore, clonal complexity is probably underdetected. We used multiple sampling combined with highly discriminatory MIRU-VNTR to study compartmentalized infections by M. bovis in a low-tuberculosis prevalence setting. We spoligotyped the M. bovis isolates from two or more anatomic locations sampled from 55 animals on 39 independent farms. Compartmentalized infections, with two different strains infecting independent lymph nodes in the same animal, were found in six cases (10.9%). MIRU-VNTR analysis confirmed that the compartmentalization was strict and that only one strain was present in each infected node. MIRU-VNTR analysis of additional infected animals on one of the farms confirmed that the compartmentalized infection was a consequence of superinfection, since the two strains were independently infecting other animals. This same analysis revealed the emergence of a microevolved clonal variant in one of the lymph nodes of the compartmentalized animal. Clonal complexity must also be taken into consideration in M. bovis infection, even in low-prevalence settings, and analyses must be adapted to detect it and increase the accuracy of molecular epidemiology studies.     

Conjunctival vaccination of the brushtail possum (Trichosurus vulpecula) with bacille Calmette-Guérin

AIM: To determine the efficacy of conjunctival vaccination of captive brushtail possums (Trichosurus vulpecula) with bacille Calmette-Guérin (BCG), as measured by immunological responses to vaccination and response to intratracheal challenge with Mycobacterium bovis.

METHODS: Nine adult male brushtail possums were vaccinated by the instillation of a suspension of BCG strain Pasteur 1173P2 into the conjunctival sac of each eye. Each drop contained approximately 2.5 × 10⁵ colony forming units (cfu). At 8 weeks post-vaccination (pv) the vaccinated possums and 10 unvaccinated possums were challenged by intratracheal instillation of approximately 100 cfu of M. bovis. Cellular immune responses to bovine purified protein derivative (PPD) antigen were measured using the lymphocyte proliferation assay (LPA). Possums surviving to 50–51 days after challenge were euth anised and subjected to detailed post-mortem examination, including histopathology, to assess protection against tuberculosis. Sections of lung and spleen were cultured for M. bovis.

RESULTS: No conjunctival inflammation or other adverse reactions to the administration of the vaccine were evident macroscopically. The vaccinated group showed a systemic cellular immune response to bovine PPD antigen at 4 and 8 weeks pv, and the response at 8 weeks was significantly greater than at 4 weeks (p<0.05). Conjunctival vaccination induced significant levels of protective immunity, measured as less mass of tuberculous lesions in lung (p<0.05) and less dissemination of disease in vaccinated compared with unvaccinated possums (p<0.05).

CONCLUSIONS: Conjunctival vaccination with BCG induced a significant level of protective immunity against M. bovis infection in possums. This route of vaccination, together with intranasal aerosol vaccination, could be utilised in the delivery of an aerosolised vaccine using a device that sprays the vaccine suspension into the eyes and nose of possums.     

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation with Mycobacterium bovis: II. Progression of infection

The aim of the study was to describe, over a period of 24 weeks, the pathological and bacteriological changes in badgers experimentally infected with Mycobacterium bovis. The badgers were infected by endobronchial instillation of 2.5 × 10⁴ colony forming units (cfu) M. bovis. After infection, the badgers were examined at 3 weekly intervals when blood and tracheal aspirates were collected. At 6, 12, 18 and 24 weeks post-infection (pi) three animals were euthanized and a detailed pathological and bacteriological examination was performed to assess the nature of the experimental disease. During the course of the study only one badger developed clinical signs of disease: a subcutaneous swelling on its head, first observed at 18 weeks pi. At post-mortem examination gross and histological lesions of tuberculosis were observed and M. bovis was recovered from all, except one badger. In the majority of badgers the endobronchial route of inoculation resulted in the establishment of infection that over 24 weeks was non-progressive with limited dissemination of infection from the thoracic cavity, mainly to the hepatic and mesenteric lymph nodes. However, in one of the badgers examined at 18 weeks pi and one at 24 weeks pi, infection was widely disseminated. The disease induced by the endobronchial inoculation displayed the characteristics of disease observed in naturally infected badgers.     

Description of lesions in cattle in a natural outbreak of Babesia bovis infection in Brazil

This paper describes the pathologic features of a natural infection by Babesia bovis (B. argentina) in Brazil. Microscopic examination of cerebrum, midbrain, cerebellum, liver, kidney, heart and spleen of five fatal cases revealed variable degrees of congestion, particularly in the brain, liver and kidney. The packing of erythrocytes, the majority of which were parasitized, was most marked in the capillaries of brain, kidney and less in liver. Lymphocytic glomerulonephritis was observed. Variable degrees of fatty degeneration were noted in the liver, distention of hepatic canaliculi and biliary retention was marked. A strong activation of the mononuclear phagocytic system was evident in all the subjects studied.     

Cytokine expression in colostrum-deprived pigs immunized and challenged with Haemophilus parasuis

The expression of several cytokines in spleen, pharyngeal lymph nodes, lung and brain after different immunization procedures and a challenge with 5 × 10⁹ CFU of Haemophilus parasuis was compared. Five groups of colostrum-deprived pigs were used: vaccinated with (I) a bacterin, (II) an outer-membrane-protein-vaccine, (III) a recombinant transferring-binding protein B, (IV) exposed to a total dose of 10⁵ CFU, and (V) not previously immunized. All pigs in groups III and V died, while all animals in group I, most of group IV and half of group II survived until the end of the experiment. IL-1α was found in significantly higher levels (p < 0.05) in spleen, lymph nodes and brain of dead pigs, which could be explained by the major severity of lesions in these animals. However, IL-4, IL-10, TNF-α and IFN-γ were expressed in significantly higher levels by survivors (for all the four cytokines in lymph nodes; for IL-4, IL-10 and TNF-α in spleen; for IL-4, TNF-α and IFN-γ in lung, and only for TNF-α in brain), thus suggesting a role of these four cytokines in the adaptive response, which might contribute to protection against H. parasuis infection.     

AN ABATTOIR SURVEY OF TUBERCULOSIS IN FERAL BUFFALOES

Tuberculosis lesions were found in 193 (1.7%) of 11,322 buffaloes examined during routine post-mortem inspection at 2 export abattoirs. The prevalence of tuberculosis in buffaloes supplied from 17 separate farms ranged from 0.3% to 8.22%, with the highest levels occurring on the coastal plains. Lesions were confined to one major body region in 50 of 72 randomly chosen cases of tuberculosis and to 2 or more regions in 22 cases. Thoracic lesions occurred in 65 of the 72 cases, abdominal lesions in 19, head lesions in 18 and carcase lesions in 9. In the thoracic cavity, lesions occurred most frequently in mediastinal and bronchial lymph nodes. In the head region the retropharyngeal lymph node was most frequently involved, in the abdominal cavity, the liver, and in the carcase, the deep inguinal lymph node. Tuberculosis lesions in buffaloes had a lardaceous consistency and were paler in colour and less calcified than those normally exhibited by cattle. Mycobacteria were isolated from 30 of 31 lesion samples submitted for bacteriological examination. Of the isolates, 25 were identified as Mycobacterium bovis, 3 as M. avium-intracellulare-scrofulaceum complex, one as M. fortuitum and one as M. flavescens. The M. bovis isolates from buffaloes showed minor cultural differences to those normally characteristic of bovine isolates.     

Occurrence of Mycobacterium spp. and other pathogens in lymph nodes of slaughtered swine and wild boars (Sus scrofa)

Mycobacterium spp. and other pathogens were investigated in 258 swine lymph nodes (129 with and 129 without apparent lesions), and 120 lymph nodes (60 with and 60 without lesions) from wild boars (Sus scrofa). A total of lymph nodes from swine and wild boars were collected of different animals. Submaxillar and mesenteric lymph nodes were submitted to microbiological examination and colonies suggestive of Mycobacterium spp. (alcohol-acid bacilli) were submitted to PCR Restriction Assay (PRA). In swine with lymphadenitis, Mycobacterium spp. (24.1%) and Rhodococcus equi (13.2%) were the most prevalent microorganisms, while in lymph nodes without lesions were identified a complex of microorganisms, including of environmental mycobacteria. In wild boars with lymphadenitis, ß-haemolytic Streptococcus (10.0%), Mycobacterium spp (8.4%) and R. equi (6.6%) were the most frequent. Among mycobacterias were identified predominantly Mycobacterium avium subspecies type 1 (48.3%) and M. avium subspecies type 2 (16.1%), followed by Mycobacterium intracellulare, Mycobacterium szulgai,Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium simiae, Mycobacterium nonchromogenicum and Mycobacterium intracellulare type 2.