Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer.

Although the causative agent of Johne's disease, Mycobacterium avium subsp. paratuberculosis, is well known, the etiology of disease and the immune responses generated in response to infection are still poorly understood. Knowledge of definitive markers of protective immunity, infection, and the establishment of chronic granulomatous Johne's disease is necessary to advance vaccine and diagnostic development. We sought to profile the immune responses occurring within jejunal lymph nodes of experimentally challenged red deer (Cervus elaphus). Quantitative PCR was utilized to measure a range of cytokines, signaling molecules, and transcription factors involved in Th1, Th2, Treg, and Th17 immune responses. Significant differences in gene expression were observed between control, minimally diseased, and severely diseased animals, with severely diseased animals showing elevated proinflammatory transcripts and reduced anti-inflammatory transcripts. We identified a proinflammatory cytokine milieu of gamma interferon, interleukin-1α (IL-1α), and IL-17, which may contribute to the immunopathology observed during clinical Johne's disease and suggest that Th2 and Treg immune responses may play an important role in controlling the development of immunopathology in infected animals.

Histopathology of grossly normal mesenteric lymph nodes of New Zealand farmed red deer (Cervus elaphus) including identification of lipopigment.

This article describes the histopathology of grossly normal mesenteric lymph nodes (MLNs) of New Zealand farmed red deer (Cervus elaphus). Eighty MLNs were sourced from 10 deer from 5 North Island herds and 5 South Island herds classified as low risk and high risk of Mycobacterium avium subspecies paratuberculosis (MAP) infection, respectively. Fixed sections were stained with hematoxylin and eosin; Ziehl-Neelsen; and, selectively, periodic acid-Schiff, Perl's, and Sudan black. Positive Ziehl-Neelsen stain, follicular hyperplasia, capsular eosinophil infiltration, focal granulomas, foci of macrophages containing lipopigment, parasitic granulomas, and calcified foci are described and severity graded where appropriate. Animal age, sex, and herd of origin are variably associated with the presence of one or more features. Trabecular fibrosis and dilated edema-filled sinusoids are described. These observations allow differentiation between likely nonpathologic histologic features in deer MLNs and features possibly attributable to infection with a pathogen such as MAP.

Knowledge gaps that hamper prevention and control of Mycobacterium avium subspecies paratuberculosis infection.

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps.

Tuberculosis in ruminants: characteristics of intra-tonsilar Mycobacterium bovis infection models in cattle and deer.

Mycobacterium bovis infection produces tubercular lymphadenitis in the head lymphatics of cattle and deer, in addition to pulmonary disease. A low-dose intra-tonsilar infection model that establishes tuberculosis (Tb) lymphadenitis in cattle and deer is characterised in this study. Intra-tonsilar infection of red deer (500 cfus of M. bovis) was monitored longitudinally at 6-week intervals over a period of 23 weeks. Lesion characteristics, bacteriological and immunological parameters were assessed, and compared against those observed in cattle at 20 weeks post-infection, where the latter were infected with 500 or 5000 cfus of M. bovis. Intra-tonsilar inoculation of M. bovis established infection in >90% of deer and cattle, with lesion frequencies at the draining sentinel lymphatic site (left medial retropharyngeal node) of 68-86% and tissue bacterial burdens >3.5 logs/g of tissue, the tonsil being a major site of M. bovis persistence in deer only. Mineralisation occurred at lesion sites in both species in the later stages (18-23 weeks) of infection, with extensive coarse mineralisation observed mainly in cattle. The severity of infection or disease in cattle that received the higher or lower dose of M. bovis did not differ markedly. Pathogen-induced cellular immune response (lymphocyte transformation) and humoral responses (IgG and IgG(1) anti-mycobacterial antibodies) were recorded in both species, and the magnitude of these was noticeably amplified by skin tuberculin testing. IgG(1) antibodies were detectable within 6 weeks post-inoculation in deer and could be associated with early detection of lymphadenitis. Deer and cattle show similar levels of susceptibility to M. bovis infection.

An experimental intratonsilar infection model for bovine tuberculosis in African buffaloes, Syncerus caffer.

An infection model for Mycobacterium bovis in African buffaloes, Syncerus caffer, was developed, using the intratonsilar route of inoculation. Two groups of 11 buffaloes each, aged approximately 18 months, were infected with either 3.2 x 10(2) cfu (low dose) or 3 x 10(4) cfu (high dose) of M. bovis strain isolated from a buffalo. A control group of six buffaloes received saline via the same route. The infection status was monitored in vivo using the comparative intradermal tuberculin test, and in vitro by the modified interferon-gamma assay. All buffaloes were euthanazed 22 weeks post infection and lesion development was assessed by macroscopic examination, culture and histopathology. It was found that the high dose caused macroscopic lesions in nine out of 11 buffaloes. Mycobacterium bovis was isolated from all buffaloes in the high-dose group and from six out of 11 in the low-dose group.

Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling.

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement. METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA. RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ∼10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69-70% mapped uniquely to version 3.1 of the ovine genome, and 48-49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study. CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.

SOLiD SAGE sequencing shows differential gene expression in jejunal lymph node samples of resistant and susceptible red deer (Cervus elaphus) challenged with Mycobacterium avium subsp. paratuberculosis.

This study compared in vivo lymph node gene expression levels between six young red deer that were either relatively resistant (R) or susceptible (S) to paratuberculosis following experimental challenge with Mycobacterium avium subsp. paratuberculosis. Intestinal lymph nodes were biopsied at 4, 12 and 50 weeks post challenge (pc) and parallel changes in histopathology, immunology and bacterial load monitored. SOLiD SAGE (serial analysis of gene expression) next generation sequencing of biopsied lymph node samples generated a total of 373 million transcript tags 26-28bp in length after filtering. A total of 36,632 unique transcripts were identified and 14,325 of these were able to be annotated. The copy number of each transcript was counted, averaged and compared for R and S animals (R-S). P values and False Discovery Rates (FDR) were calculated for each transcript. Genes differentially upregulated ≥2 fold (FDR<0.5) totalled 9, 40 and 32 in R animals (+ values) and 23, 164 and 47 in S animals (- values) at weeks 4, 12, and 50pc, respectively. Transcripts displaying greatest differential expression between R and S animals at each time point were IFIT2 (189 fold) and S100A8 (-32.7 fold) at week 4, LRR1 (52.7 fold), SERPINF2 (-214.6 fold) at week 12 and CEACAM8 (84.6 fold), and STK31 (-129.5 fold) at week 50, respectively. All 9 genes significantly upregulated at week 4 in R animals relate specifically to host defence and all involve Type I interferon stimulated genes. By contrast genes upregulated in S animals at week 4, relate predominantly to inflammation, but also involve adaptive immune responses, mitochondrial function and apoptosis regulation. At week 12, the genes differentially upregulated in R animals are linked predominantly to regulation of adaptive immunity and mucosal immunity, while many of the genes in S animals are associated with pro-inflammatory interleukins involved with innate and adaptive immunity. These correlated with greater lesion severity and higher MAP numbers in lymph nodes of S animals. By week 50 the number of upregulated genes declined in both groups. A number of genes upregulated in R animals appear to be associated with host resistance and regulation of adaptive immunity, especially CEACAM8. Genes upregulated in S animals involve antigen presentation (ENDOD1) and gut associated immune pathology (HSH2D). In conclusion, gene expression in jejunal lymph nodes of resistant and susceptible deer infer that the resistant phenotype is associated with pathways of adaptive immunity, while susceptibility is linked with upregulated non-protective pro-inflammatory responses, following experimental MAP infection.

Animal models of protective immunity in tuberculosis to evaluate candidate vaccines.

While the etiology of tuberculosis is well understood, the nature of the protective immune response to the causative mycobacteria has remained a mystery. There is an urgent need to define protective immunity critically, and to develop alternative animal models to evaluate the efficacy of new-generation vaccines against tuberculosis in a cost-effective way.

Optimal models to evaluate the protective efficacy of tuberculosis vaccines.

BCG has been used widely as a vaccine to prevent tuberculosis (TB) for 80 years, yet there is still considerable controversy about its efficacy. Many experimental variables have obscured the true efficacy of BCG. The absence of appropriate animal models for the study of protective efficacy and the lack of in vitro correlates of protective immunity have impeded progress. Laboratory animal studies, which have contributed to understanding the pathogenesis, heritability of resistance and immunology of TB, have failed to identify the immunological pathways necessary for protective immunity. In recent years, cattle and deer, which are naturally susceptible to TB, have been used to study protective immunity in vaccinated animals, challenged with virulent bacteria. A deer TB infection model has been developed that can measure protection against TB infection or the development of disease. Data from this model show that, providing live BCG is administered in a short interval prime-boost protocol, significant protection against infection and disease can be obtained. Single dose vaccine provides suboptimal protection that attenuates pathology but does not prevent infection. Low dose BCG vaccine (10(4)cfu), administered in a prime-boost protocol, sufficient to prevent infection, does not cause conversion to delayed type hypersensitivity or produce unacceptable side-effects. Immune memory for protection against infection persists at optimal levels for at least 12 months post vaccination. Used optimally, BCG produces good levels of protection against TB and improved protocols or its use should be explored, before attempts are made to replace it with new-generation vaccines. It is now possible to integrate the fundamental information obtained from laboratory animals with studies of functional immune protection in target host species. Justification for the use of TB vaccines for domestic livestock under field conditions, must be underpinned by scientific evidence that they provide acceptable levels of protection long term.

Systemic but not intra-intestinal vaccination with BCG reduces the severity of tuberculosis infection in ferrets (Mustela furo).

SETTING: Ferrets are important wildlife vectors of bovine tuberculosis (Mycobacterium bovis) in New Zealand. By reducing the severity and/or incidence of tuberculosis (TB) in wild ferret populations, vaccination may limit disease transmission to livestock. OBJECTIVE: To investigate whether vaccination of ferrets with attenuated M. bovis BCG via systemic or intraintestinal routes can reduce the severity of TB resulting from oral M. bovis challenge. DESIGN: Groups of captive ferrets were vaccinated with live BCG via sub-cutaneous injection or intra-duodenal inoculation, twice, 4 weeks apart. Vaccinated and non-vaccinated (control) ferrets were subsequently challenged orally with virulent M. bovis to simulate the natural route of infection. Peripheral blood lymphocyte reactivity was longitudinally monitored, and the outcome of challenge was determined 20 weeks later by autopsy, histology and bacteriological culture. RESULT: Both vaccination routes induced tuberculin-specific lymphocyte reactivity; however, only the subcutaneous route was effective in reducing disease. Subcutaneous vaccinated ferrets had a lower severity of infection than non-vaccinated control animals, as indicated by significant reductions in viable bacterial burdens and prevention of gross lesions in mesenteric lymph nodes (the primary site of infection), and a lower incidence of bacterial translocation to thoracic lymph nodes. However, sub-cutaneous vaccination did not reduce the incidence of mesenteric lymph node infection. CONCLUSIONS: Systemic vaccination with BCG can reduce the severity of TB resulting from oral challenge with virulent M. bovis; however, delivery of viable BCG to the upper intestinal tract may not protect ferrets against TB.

Partial protection against oral challenge with Mycobacterium bovis in ferrets (Mustela furo) following oral vaccination with BCG.

SETTING: Ferrets (Mustela furo) are important wildlife vectors of bovine tuberculosis (TB) in New Zealand. Protective vaccination of ferrets may limit the potential of transmission to livestock. OBJECTIVE: To determine whether orally-delivered Mycobacterium bovis BCG can confer protection against oral challenge with virulent M. bovis. DESIGN: Ten ferrets were vaccinated by feeding measured doses of live BCG, and subsequently challenged with virulent M. bovis via the oral route. Ten non-vaccinated (control) ferrets were similarly challenged. Live body weights and lymphocyte reactivity were monitored longitudinally, and ferrets were killed 20 weeks following challenge. Necropsy, histological examination and bacterial culture of alimentary tract lymphatic tissues were undertaken. RESULTS: There was a significant reduction in the incidence of gross tuberculous lesions among vaccinated ferrets compared to control animals, and fewer vaccinated ferrets had histologically-detectable acid-fast organisms in mesenteric lymph node (LN) tissues. There were significantly fewer vaccinated ferrets with culture-positive retropharyngeal LNs, and the mean bacterial burden was significantly lower for retropharyngeal LNs isolated from vaccinated animals than from controls. CONCLUSION: These results demonstrate that oral BCG vaccination of ferrets can confer partial protection against M. bovis, and suggest that systemic immune responses may be less important in mediating this degree of protection than local immunity.

Intracellular survival of virulent Mycobacterium bovis and M. bovis BCG in ferret macrophages.

The intracellular survival of virulent Mycobacterium bovis and avirulent M. bovis BCG in ferret alveolar macrophages was investigated. In addition, the effects of endogenous and exogenous modulators of macrophage oxidative function on bacterial survival and growth in vitro were determined. Ferret macrophages limited the initial growth of BCG, while virulent M. bovis replicated within macrophages. Intracellular bacterial survival was unaffected by the addition of specific inhibitors of macrophage oxidative function. A T-cell supernatant (TCS), derived from mitogen-stimulated lymphocyte cultures, activated ferret macrophages for heightened oxidative burst performance. However, macrophages activated by TCS, bacterial LPS or a combination of both, failed to control infection, and actually enhanced the intracellular survival of M. bovis. These results are discussed in relation to the role of macrophages in mediating tuberculosis-related pathogenesis, with respect to the fact that ferrets are important wildlife vectors of bovine tuberculosis in New Zealand.

Oxidative responses in ferret macrophages.

Although the basic function of T and B lymphocytes in ferrets has been known for some time, the function of mononuclear phagocytes has not been described in this species. The present study has characterised basic oxidative responses in ferret macrophages, and has investigated the effects of endogenous and exogenous modulators of macrophage function on oxidative capacity in vitro. Macrophages derived from the blood or lungs of ferrets were shown capable of generating the reactive oxygen intermediate (ROI) molecules superoxide and hydrogen peroxide, and secreting a lysosomal enzyme (acid phosphatase), in response to appropriate stimuli. A T cell supernatant (derived from mitogen-stimulated peripheral blood lymphocytes) was able to activate both blood- and lung-derived macrophages for enhanced ROI production, while specific ROI inhibitors (superoxide dismutase and catalase) were able to partially ablate ROI activity. The accumulation of nitrite in culture supernatants, as an indicator for the production of reactive nitrogen intermediates, could not be demonstrated by ferret macrophages derived from either tissue source. In contrast to the enhancing effects of TCS on the oxidative function of blood-derived macrophages, exposure to bacterial LPS caused marked suppression of ROI and lysosomal enzyme production by these cells. Finally, the generation of superoxide anion, following phagocytosis of live or heat-killed Mycobacterium bovis or zymosan, indicated that ROI production in response to phagocytic stimulation was relatively weak in ferret blood-derived macrophages. These results are discussed in relation to the study of immune function in a novel species, and with particular reference to research into tuberculosis (Tb), since ferrets are important wildlife vectors of bovine Tb in New Zealand.

Efficacy trial of an irradiated cattle lungworm vaccine in red deer (Cervus elaphus).

Lungworm (Dictyocaulus sp.) is the parasite of most concern to the New Zealand deer industry. Although lungworm can be controlled by anthelmintics there is an increasing concern over excessive drenching programmes and reliance on chemicals for parasite control. A live irradiated larval vaccine developed for cattle has been available in Europe for the past 40 years but has never been evaluated in red deer in New Zealand. Four groups of red deer and two of cattle were hand reared from birth in parasite-free conditions. The cattle acted as a control group to ensure that the vaccine was still efficacious on arrival in New Zealand. Two groups of deer were vaccinated, and all four groups were challenged with either D. viviparus or deer origin Dictyocaulus, tentatively identified as D. eckerti. The vaccine provided excellent protection to cattle under New Zealand conditions, there was no larval output in the vaccinated cattle and no adults were found in their lungs at necropsy. In red deer, patency was delayed in the vaccinated groups regardless of challenge species and larval output was lower but was not prevented. Adult lungworms were found in the lungs of all deer at necropsy but fewer were recorded in the vaccinated deer. Although Huskvac provided a degree of protection for red deer it was not effective enough to recommend its use.

Interpreting comparative experiments on parasite burdens in animals using randomization and canonical variates.

Comparative experiments on parasite burdens present difficulties for modelling and interpretation: data tend to have highly skewed distributions, some standard methods of testing for effects have low power, and there is a need to make allowance for dependencies among the various measurements. Randomization testing and canonical variates analysis are advocated as a means of minimising these problems.

Oral infection of ferrets with virulent Mycobacterium bovis or Mycobacterium avium: susceptibility, pathogenesis and immune response.

Ferrets are important wildlife reservoirs of tuberculosis in New Zealand, where they acquire infection primarily through scavenging infected carrion. In the present study, groups of laboratory-reared ferrets were infected orally with 5 x 10(6)colony-forming units of Mycobacterium bovis or Mycobacterium avium. Body weight and tuberculin-specific immune reactivity were monitored at intervals (pre-infection, and 4 and 20 weeks post-infection) and animals were killed at 20 weeks post-infection for post-mortem, histopathological and bacteriological examinations. Weight loss was significantly greater in M. bovis -infected than in M. avium -infected ferrets. M. bovis, unlike M. avium, sometimes produced gross necrotic lesions in the mesenteric lymph nodes. M. bovis invariably produced microscopical foci of mycobacterial infection or tissue necrosis typical of tuberculosis, whereas M. avium did so in only one of nine animals. Mycobacteria were recovered from the lymphatic tissues of all M. bovis -infected ferrets but from only five of nine M. avium -infected animals; and the mean bacterial burdens of the lymph nodes of the head and intestinal regions were > 10-fold and > 100-fold greater, respectively, for M. bovis -infected than for M. avium -infected animals. M. bovis, unlike M. avium, evoked tuberculin-specific peripheral blood lymphocyte reactivity and serum antibody responses.

Superovulation and embryo recovery in Red deer (Cervus elaphus ) hinds.

In two experiments, Red deer hinds were synchronized with intravaginal progesterone and were given 4 d of treatment (3 d before progesterone withdrawal and 1 d after) with an ovine follicle stimulating hormone (FSH) preparation which had a claimed low level of luteinizing hormone (LH) contamination. In Experiment 1, 12 hinds received one of four FSH levels by osmotic minipump. Hinds were run with fertile stags, and laparotomy and embryo recovery were performed 9 d after progesterone withdrawal. The ovulation rates (mean of three hinds per dosage) were 1.0, 2.0, 4.3 and 15.3 (number of corpora lutea counted) for estimated daily dosages rates of 0.036, 0.071, 0.11 and 0.14 units FSH preparation/day; the response to the increasing dosage was exponential (P<0.01). The recovery rate of ova on flushing was 38% (24 63 ), with all recovered ova being fertilized and of transferable quality. In Experiment 2, performed later in the breeding season, eight hinds received 0.14 units FSH/day either by minipump or by intramuscular injection. The mean ovulation rates were 3.0 and 11.0 (a significant difference, P<0.01), respectively, with a recovery rate of 72% (34 47 ), and with only 18 34 ova considered to be of transferable quality. The recovery rate in Experiment 2 was significantly higher than that in Experiment 1 (P<0.001). Overall, the results were better than those previously recorded for red deer, perhaps a function of both the FSH preparation used and an improved progesterone profile in estrus synchronization.

Effect of physical restraint and xylazine sedation on haematological values in red deer (Cervus elaphus).

Blood samples were taken from healthy red deer (Cervus elaphus) using physical restraint or xylazine sedation. Red and white cell and platelet parameters were measured using a Technicon H6000/C analyser. Lower circulating red cell mass, lymphocyte and platelet counts were found in sedated deer. Mean red cell volume was significantly lower, probably due to the preferential sequestration of young red cells in the spleen. While the main cause of the observed differences is likely to be splenic contraction during physical restraint, the lower plasma viscosity and fibrinogen are suggestive of a haemodynamic component.

Tuberculosis in deer: perceptions, problems and progress.

Since the emergence of deer farming as an alternative farming enterprise over the past 30 years, there has been an increasing awareness of the potential threat posed by tuberculosis (TB) to domesticated deer. TB, caused by Mycobacterium bovis, has been found in deer in every country involved with deer farming. Different types of TB control policies, which vary from whole-herd depopulation to selective testing and slaughter of reactor animals, have been implemented. Extensive research has been carried out, incorporating modern microbiological and immunological concepts and advanced molecular methodologies, to find new solutions for the eradication of TB from domesticated deer. This work has resulted in valuable new insights into the aetiology, transmission, pathogenesis, diagnosis, prevention and heritability of resistance to M. bovis infection in ruminants. This knowledge has complemented the existing literature database on bovine and human TB and will provide new strategies for improved diagnosis, vaccination and selective breeding to control TB, which should be relevant for human, domestic livestock and wildlife populations.

Mycobacterium bovis in free-living and captive wildlife, including farmed deer.

Mycobacterium bovis has been isolated from a wide range of wildlife species, in addition to domestic animals. This review examines the role played by various species in the maintenance of M. bovis in wildlife communities and the spread to domestic animals. Badgers (Meles meles), brushtail possums (Trichosurus vulpecula), deer (Odocoileus virginianus), bison (Bison bison) and African buffalo (Syncerus caffer) are examples of wildlife that are maintenance hosts of M. bovis. The importance of these hosts has been highlighted by the growing realisation that these animals can represent the principal source of infection for both domestic animals and protected wildlife species. The range of methods for controlling M. bovis in wildlife is limited. While population control has been used in some countries, this approach is not applicable in many situations where protected wildlife species are concerned. Vaccination is a potential alternative control method, although as yet, no practical, effective system has been developed for vaccinating wildlife against bovine tuberculosis. Tuberculosis caused by M. bovis has also been a problem in captive wildlife and in recently domesticated animals such as farmed deer. Control of M. bovis in this group of animals is dependent on the judicious use of diagnostic tests and the application of sound disease control principles. The advances in the development of bovine tuberculosis vaccines for cattle and farmed deer may offer valuable insights into the use of vaccination for the control of tuberculosis in a range of captive wildlife species.