M ini Review Farmed deer: A veterinary model for chronic mycobacterial diseases that is accessible, appropriate and cost‑effective Frank Griffin
Department of Microbiology and Immunology, Disease Research Laboratory, University of Otago, Dunedin, New Zealand Address for correspondence: Dr. Frank Griffin, E‑mail: frank.griffin@otago. ac.nz
Received : 20‑11‑13 Review completed : 20‑11‑13 Accepted : 20‑11‑13
ABSTRACT Although most studies in immunology have used inbred mice as the experimental model to study fundamental immune mechanisms they have been proven to be limited in their ability to chart complex functional immune pathways, such as are seen in outbred populations of humans or animals. Translation of the findings from inbred mouse studies into practical solutions in therapeutics or the clinic has been remarkably unproductive compared with many other areas of clinical practice in human and veterinary medicine. Access to an unlimited array of mouse strains and an increasing number of genetically modified strains continues to sustain their paramount position in immunology research. Since the mouse studies have provided little more than the dictionary and glossary of immunology, another approach will be required to write the classic exposition of functional immunity. Domestic animals such as ruminants and swine present worthwhile alternatives as models for immunological research into infectious diseases, which may be more informative and cost effective. The original constraint on large animal research through a lack of reagents has been superseded by new molecular technologies and robotics that allow research to progress from gene discovery to systems biology, seamlessly. The current review attempts to highlight how exotic animals such as deer can leverage off the knowledge of ruminant genomics to provide cost‑effective models for research into complex, chronic infections. The unique opportunity they provide relates to their diversity and polymorphic genotypes and the integrity of their phenotype for a range of infectious diseases. KEY WORDS: Deer, immune markers, infectious diseases, vaccine development
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esearch into many facets of experimental biology has been dominated by inbred mice since they were first derived >100 years ago.[1] Inbred mice have made a vital contribution to our understanding of transplantion biology[2] and immunology, by disclosing the multiple cell subpopulations that contribute to the diverse immune pathways and the regulatory and effector molecules that influence the highly plastic immune system of higher organisms. The ability to transfer syngeneic cells from inbred donors to histocompatible recipients has made an important contribution to understanding Access this article online Quick Response Code:
Website: www.jpbsonline.org DOI: 10.4103/0975-7406.124302
the cellular molecular basis of many areas of immunological research and has underwritten most of the theoretical dogma of modern immunology. Research findings from inbred mice continue to dominate published outputs (>80%) in fundamental immunological research involving infection, allergy, autoimmunity and cancer. Nonetheless, while inbred mice have been valuable in illuminating the rich tapestry of immune circuitry they have provided more limited insights into the underlying mechanisms involved in complex, naturally occurring diseases. These include infection,[3] malignancy,[4] metabolic[5] and neurological disorders,[6] where inbred mice have not elucidated the mechanisms of disease or facilitated therapeutic interventions or have allowed translation of fundamental discoveries into applied solutions. Two important human diseases; malaria and tuberculosis (TB), will be used to highlight the limitations of inbred murine models to advance knowledge into infectious diseases that affect outbred human or domestic livestock populations.
How to cite this article: Griffin F. Farmed deer: A veterinary model for chronic mycobacterial diseases that is accessible, appropriate and cost-effective. J Pharm Bioall Sci 2014;6:10-5.
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Griffin: Outbred animal models for infectious diseases
Malaria A striking example of the limitations of inbred murine models is seen from multiple murine studies designed to evaluate novel therapeutics for cerebral malaria in humans.[7] In a large series (48) of therapeutic interventions evaluated over the past two decades in mice, 44 (92%) were considered to prevent or ameliorate the cerebral disease, preventing or reducing disease severity by 50‑100%. In stark contrast, only 1/11 human adjunctive intervention therapies trialed over the same timeframe provided a mortality advantage. Explanations that have been advanced to account for this dichotomy include the diverse phenotypes of the pathogen studied (Plasmodium falciparum [human] vs. Plasmodium berghei ANKA [mice]), the host genotype (Polymorphic genetics ‑ humans vs. monomorphic genetics ‑ mice), phenotypic differences, driven by closed (murine) versus open (human) environments, immunopathology (Intravascular coagulation [human] vs. inflammation [murine]), or the intervention strategy (prophylactic [mice] vs. therapeutic [human]). Irrespective of the point of difference,[7] inbred mouse model studies appear to have serious limitations in informing new approaches to solve the human malaria conundrum, by developing effective prophylactic vaccines to prevent infection or therapeutics to treat affected individuals. An evaluation of animal models for malaria,[8] involving 22 international experts failed to reconcile the points of differences between the expectations of human researchers and the outputs from murine malaria models.
TB A huge research investment has been made in the past two decades in an effort to develop new vaccines that provide prophylactic or therapeutic protection against human TB caused by Mycobacterium tuberculosis. This is because the existing human Bacille Calmette‑Guerin (BCG) vaccine is considered to provide suboptimal protection against TB in humans, domestic livestock and wildlife, all of which are affected by naturally occurring disease. BCG was derived by Calmette‑Guerin in 1908 from a primary isolate of Mycobacterium bovis; isolated from a tuberculous cow, that was subsequently passaged in the laboratory >120 times to produce the safe live attenuated BCG vaccine. This was first introduced as a prophylactic vaccine for human TB in 1921 and since then it has been used in more than 1 × 109 people world‑wide, without significant side‑effects or adverse reactions. Although it has not resulted in the eradication of TB world‑wide it has made a dramatic impact on severe disease (tubercular meningitis and miliary TB) in generations of children that have been vaccinated with BCG over the past 90 years. Nonetheless, it is far from optimal as a single dose vaccine for adults, where its widespread use has failed to control pulmonary disease or prevent spread of infection, especially in developing countries.[9] Since 1990 in excess of US$200 million has been invested in research world‑wide to develop improved vaccination protocols to improve the efficacy of human and animal TB vaccines. New candidate vaccines that have been developed include Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
recombinant BCG, M. tuberculosis auxotrophs, recombinant virus and recombinant mycobacterial peptides that are first screened in inbred mice to establish immunogenicity and “proof‑of‑efficacy”. Tests carried out on more than 200 candidate vaccines has failed to show one single candidate vaccine that has superior efficacy to BCG.[10] This has refocused the direction of research such that the current goal is not to replace BCG, but to develop a complementary vaccine that can be used to boost levels of protective immunity following primary vaccination with BCG.[10] There are currently up to 12 candidate TB vaccines in the pipeline in Phases I, II or III clinical trials. Testing these in human clinical trials is extremely expensive and fraught with challenges. The lead candidate vaccine (modified vaccinia virus ANKA 85A [MVA85A]), first tested in inbred mice[11] is a recombinant MVA that contained the 85A gene, which codes for a major cell wall antigen found in M. tuberculosis, M. bovis and BCG. It was seen to produce significantly increased levels of protection when used to boost inbred mice, previously vaccinated with BCG. Considering that this study provided the foundation data to justify further trials with the MVA85A vaccine in primates and later in humans, it is surprising that researchers dismissed the finding that prime‑boost with homologous BCG gave equivalent levels of protection to that seen with prime BCG followed by boosting with MVA85A. Superior protection against experimental infection with virulent M. tuberculosis, instilled into the lungs of vaccinated mice, was seen when the vaccine was administered intranasally rather than subcutaneously. Again two doses of BCG gave equivalent levels of protection as BCG‑MVA85A. Subsequent vaccine efficacy studies[12] in non‑human primates also showed that the vaccine was safe and stimulated protection against experimental challenge with virulent M. tuberculosis. A recent safety/efficacy trial on infants vaccinated with BCG and boosted with MVA85A showed that while the recombinant vaccine was safe for use in infants it produced no demonstrable protective efficacy against TB.[13]
Outbred Animal Models for Infection While inbred mice may provide spurious information for the study of TB it must be recognized that in selected areas of research that has involved murine virus infection, inbred mouse strains have provided outstanding fundamental information that has influenced the central dogma of immunology. The study of lymphocytic choriomeningitis virus in inbred strains of mice unraveled the importance of the critical role of the major histocompatibility complex markers for the recognition of virus infected cells by T cells (Tc).[14] This has become a critical paradigm that has facilitated the discovery of cytotoxic Tc, essential for immune protection against virus infection. Nonetheless, the widespread use of inbred mice in other areas of infectious disease research has provided limited metrics for immune function that are necessary to develop improved immunodiagnostics for infectious diseases, immune markers for protection to aid development of new vaccines or a better understanding of heritability of resistance or susceptibility to important infectious diseases that affect humans and domestic livestock. A number of researchers[15,16] have expressed concern about the undue reliance on the inbred 11
Griffin: Outbred animal models for infectious diseases
mouse for immunology studies. Others suggest new approaches in human clinical immunology[17] or the use of experimental animal model including ruminants (cattle, sheep or goats)[18] or swine[19] will more accurately reflect functional pathways of protective immunity and pathology, that occur naturally in response to infection or vaccination.
Table 1: Advantages and limitations of animal models for infectious disease research
The contrasting features of experimental studies involving inbred mice and outbred animal models are shown in Table 1, highlighting the advantages of two model systems. Based on the relative weighting it would appear that inbred mice have significant advantages based on cost, easy of handling, availability of reagents, technology platforms and genomic information. In reality, the fact is that inbred mice, with a homozygous genotype are intrinsically restricted and unrepresentative and rarely express a phenotype for disease that equates with that found in wild type mice, outbred animals or humans. In disease research host phenotype is paramount, where the experimental model must mimic the patterns of disease that occur naturally in the host, which in part explains why inbred mice are so limited as models for TB. Mice appear relatively resistant and do not develop TB naturally and when experimentally infected with M. tuberculosis or M. bovis they produce pathology that is completely different from that seen in TB animals or people. By contrast domestic animals produce TB pathology that is indistinguishable from humans and show a spectrum of disease severity that equates with the relative susceptibility or resistance of individuals within an outbred population. When developing experimental animal models due cognizance must be given to the protocols for both the establishment of infection and vaccination, in order to accurately monitor infection or disease outcomes to monitor the efficacy of vaccines or effect of therapeutics. Ideally, infection should be established by a natural route using the minimum dose of the pathogen necessary to produce a predictable response. The resulting experimental challenge should produce patterns of infection and disease that are similar in their range and severity to that seen to occur naturally. While conducting vaccine efficacy studies it is necessary to consider additional levels of stringency to control for all the variables that may influence the metrics for vaccine efficacy. Ideally these should reflect phenotypic modifiers such as intercurrent infection, climatic and environmental variables that may influence host phenotype and impact on vaccine efficacy. It is self‑evident that a well‑fed, pathogen‑free inbred mouse held under sterile housing conditions in a biosafety level 3 facility, does not faithfully reflect the environment of an infant in an underdeveloped country or a lamb, calf or piglet on a conventional farm. When using animal models for efficacy testing of new TB vaccine the variables which must be taken into account are listed in Table 2.
Experimental Studies in Farmed Deer During the past 20 years, we have explored a number of strategies for the control of mycobacterial disease in livestock
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Feature
Inbred mice
Large animal
Cost Containment/husbandry Breeding
Relatively low Relatively cheap Prolific/cheap
Genetic modification Availability of reagents Genetic diversity
Almost unlimited Excellent Extremely low (monomorphic) Poor Restricted (single) Extensive Fully annotated
High Expensive Prolific (embryo transfer) expensive Restricted Relatively poor High (polymorphic)
Integrity of phenotype Sampling Supporting literature Genome database
Purpose designed equipment Available Adoptive cell transfer Histocompatible
Excellent Unlimited (sequential) Relatively limited Most often not available Non‑existent Incompatible‑graft rejection
Table 2: Variables that impact on TB vaccine efficacy studies Choice of animal model Type of vaccine Prime versus boost Route of vaccination/ challenge Interval from vaccination to challenge Putative markers for protection Phenotype for protection Phenotypic modifiers
Mouse, guinea pig, rabbit Non‑human primate, livestock BCG, rBCG, rVector, peptide, DNA Interval Subcutaneous, intradermal, oral, intranasal Weeks versus months Immune cell phenotype, cytokines, mRNA Bacterial numbers, bacterial spread, nature of pathology, clinical manifestations, mortality Environmental infection, climate, nutrition
TB: Tuberculosis, BCG: Bacille Calmette‑Guerin, rBCG: Recombinant BCG, rVector: Recombinant vector
and consider that these findings may be translated to other species for the control of mycobacterial diseases such as TB in humans and other ruminant species. Our studies have involved TB, caused by infection of cattle and deer with M. bovis and M. paratuberculosis, which causes Johne’s disease (JD); a chronic enteritis in cattle, deer and sheep. As the majority of our research has been carried out with farmed deer this review will outline the chronology of the research findings in deer. It will cover the evolution of the research to develop laboratory‑based diagnostics, for use in test and cull programs, to research into vaccination and the discovery of heritable resistance to infection. The study of this exotic farmed species highlights the challenges associated with the intensified farming of a new species, but also identifies the opportunities to study novel animal species now that we have access to new molecular immunology platforms available to study infection in outbred populations. Deer also present unique opportunities to study how individual genotypes influence disease phenotypes as the process of domestication of these wild animals for intensive introduces stressors that can amplify the disease phenotypes. Within a decade of the first intensive management of wild captured deer (Circa 1970), TB infection was identified as a disease that had the potential to compromise successful Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
Griffin: Outbred animal models for infectious diseases
domestication of deer. In 1985, our laboratory was commissioned by the New Zealand Deer Industry to develop a laboratory test for TB diagnosis in deer to complement the existing intradermal cervical skin test (CT). The CT had been used for >50 years to diagnose TB infection in humans and domestic livestock (mainly cattle). The deer industry was concerned about the performance of CT for TB diagnosis in deer, because it produced False (+) reactions in deer exposed to non‑pathogenic environmental mycobacteria and False (−) reactions in a proportion of TB animals. Our laboratory developed a composite blood test for TB (BTB)[20] which measured antibody, T‑cell transformation and inflammatory markers, simultaneously. This test improved the precision of TB diagnosis in deer allowing for the salvage of False (+) CT reactors and culling of BTB (+)/ CT (−) non‑reactors. The value of the laboratory‑based tests is evidenced by the striking progress in TB control in farmed deer over the past 3 decades, where infected herd numbers decreased from >200 herds in the 1980s to >5 in 2012. This is impressive considering the ongoing threat of TB infection in livestock in New Zealand posed by wildlife reservoirs of TB infection (e.g., possums/ferrets). Retrospective post‑mortem analysis of thousands of deer from which BTB tests had been carried out prior to slaughter, showed clearly that different components of the BTB could be used to predict whether individual animals were immune, infected or diseased, after exposure to pathogenic mycobacteria (M. bovis).[21,22] The ability of these tests to predict that an individual animal could become immune after exposure to virulent M. bovis prompted us to hypothesize that a proportion of animals were resistant to infection and could develop a protective immune response. In order to explore if protective immunity and disease resistance was attainable it was necessary to develop an experimental infection model and hence that we could trace the course of infection from exposure to virulent M. bovis, through to development of immunity, infection or disease. In a comparative study involving multiple routes of exposure (intratracheal, intratonsil, intranasal and subcutaneous) to virulent M. bovis the intratonsilar route of exposure was shown to be superior to all others in that it produced disease that was indistinguishable from TB in naturally infected animals.[23] All other routes of exposure produced aberrant or inconsistent responses that did not resemble TB in deer. Intratonsilar challenge with 100 colony forming units of virulent M. bovis, instilled with a blunt needle into the crypt of the tonsil, produced a spectrum of reactivity in different animals that ranged from, infection without any pathology, through to single lesions or generalized lesions involving lymphatic or bacteremic spread to multiple organs. The histopathology of the lesions in experimentally infected animals was indistinguishable from that seen in naturally infected animals, where lesions were also found at equivalent sites throughout the body. The validity of the intratonsilar infection model has subsequently been confirmed in Cape buffalo[24] and cattle.[25] The intratonsilar infection model in deer is sufficiently sensitive that it can be used to measure levels of infection as reflected by the recovery of live bacteria from infected tissues, or patterns of disease, as defined by the severity of the histopathological changes. Data from the vaccine studies[26] using BCG vaccines showed that a Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
single dose of live BCG given to 3 month old deer produced protection against disease but did not protect against infection. Protection against infection is considered to be the “holy grail” in TB vaccinology. By contrast prime‑boost live BCG vaccination protected against animals against disease and infection. Killed BCG vaccines were ineffective and the protective effect of live BCG ×2 was ablated in animals treated with steroids. Subsequent studies[27] showed that the interval between prime and booster inoculations was important, where animals boosted at a 8 or 16 week interval generated protective immunity, while animals boosted at a 43 week interval were not protected. This corroborates findings from the Karonga study[28] on human BCG vaccination, where boosting children years after their primary BCG vaccination did not produce a demonstrable increase in protection. A recent booster BCG vaccine study in Brazilian children[29] showed that long term boosting does provide a measurable increase in vaccine protection in some regions, where environmental mycobacteria do not appear to interfere with protective immunity generated following prime BCG vaccination. The age of animals at primary vaccination may also be important as the prime‑boost effect we see in 3 month old deer was not seen in vaccinated neonatal (1 day old) calves, where a single dose of BCG provided optimal protection.[30] Not only has the deer infection model been valuable in evaluating vaccine mediated protection but it also has provided insights into heritable resistance and susceptibility to TB in deer. Experimental infection of a group of 75 stags was used to select small groups (3) of animals which displayed an extreme phenotype for either resistance (R) or susceptibility (S) to TB challenge.[31] Semen from R and S animals was used in an artificial insemination program to breed offspring which were subsequently challenged with M. bovis by the intratonsilar route. The results show that resistance or susceptibility to TB is highly heritable (0.48 ± 0.096). Subsequent field studies confirmed that heritability and selection for resistance to TB was evident on a deer farm that experienced a catastrophic outbreak of TB.[32] The establishment of the diagnostic platform for TB in deer in the 1980s and the subsequent development of experimental TB infection models have been extended into an equivalent program for control of M. paratuberculosis infection, which is the causative agent for JD in deer.[33] In the past decade our laboratory has developed and validated an immunoassay for JD (Paralisa TM) in deer [34] and developed a sensitive molecular (quantitative polymerase chain reaction) assay to monitor microbial pathogens excreted in feces or present in intestinal tissues.[35] Extensive histopathological assessments[36] are also required to confirm the infection or disease status of each animal that has been infected with M. paratuberculosis as each may respond differently to an equivalent exposure to this mycobacterial pathogen. We have also developed a M. paratuberculosis experimental infection model for deer,[37] which replicates many of the key features originally incorporated into the TB infection model. Because JD is an enteric infection it is possible to establish infection and disease at predictable levels in young deer by oral gavage with virulent M. paratuberculosis. We have also been fortunate to 13
Griffin: Outbred animal models for infectious diseases
identify different breeds of farmed deer that express extreme phenotypes for resistance and susceptibility to infection.[38] Breeding from stags with a R or S phenotype shows high levels of heritability for the paternal phenotype, where up to 80% of progeny express the same phenotype as the sire. This facilitates cost‑effective breeding of animals with a prescribed phenotype for disease resistance or susceptibility. These are especially valuable because animals with a susceptible phenotype provide an immunological signature for the infected or diseased phenotype. By contrast, animals with a resistant phenotype can be used to target protective immune pathways such as would be required to produce protective immunity following vaccination. The challenge has been to extend the historical concept of immune diagnostics beyond the limited scope in diagnosing infection or disease to include the spectrum of reactivity that occurs following infection and provide a metric that is predictive for different phenotypic responses in the host. These include: Exposed-Immune-Infected-Diseased.
3. 4.
5. 6. 7. 8. 9. 10. 11.
12.
Phenotype: Resistant(R)-Intermediate(I)-Susceptible(S). While the composite BTB diagnostic test developed for TB in deer[20] has provided some initial insights into different phenotypic responses to TB, the small range of biomarkers markers available (inflammatory/antibody/T‑cell) do not provide sufficient clarity to distinguish different phases of infection. Considering the complexity of the host response it is likely that a new systems based approach will be required to chart the relevant diagnostic markers for diagnosis of mycobacterial infections/disease, protective immunity to vaccines or to identify susceptible or resistant phenotypes. This approach incorporates monitoring gene expression levels for an array of immunological markers involving both the adaptive (T‑cell)[39] and the innate (Macrophage –Mf)[40,41] pathways of immunity. The initial findings suggest that it will be possible to assemble an array of markers that can be correlated with different animal phenotypes that distinguish infection, latency, disease or immunity. It appears that susceptibility is associated with dysfunctionality of the innate system, while resistance relates to immune competence of the adaptive T‑cell responses. Fortuitously, access to new molecular technologies circumvents the need for customized reagents when studying complex immune responses to infection. Based on published genomic data from other ruminants (cattle/sheep) it is possible to design primers for any annotated gene sequence in the database and monitor equivalent gene expression in more exotic animals such as deer. The use of transcriptomics to monitor gene expression and parallel sequencing can be used to identify candidate genes that contribute to functional immunity in experimentally infected animals. A similar approach involving the monitoring of biomarkers has been proposed for diagnosis of human TB.[42]
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Griffin: Outbred animal models for infectious diseases 28. Randomised controlled trial of single BCG, repeated BCG, or combined BCG and killed Mycobacterium leprae vaccine for prevention of leprosy and tuberculosis in Malawi. Karonga Prevention Trial Group. Lancet 1996;348:17‑24. 29. Barreto ML, Pereira SM, Pilger D, Cruz AA, Cunha SS, Sant’Anna C, et al. Evidence of an effect of BCG revaccination on incidence of tuberculosis in school‑aged children in Brazil: Second report of the BCG‑REVAC cluster‑randomised trial. Vaccine 2011;29:4875‑7. 30. Buddle BM, Wedlock DN, Parlane NA, Corner LA, De Lisle GW, Skinner MA. Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination. Infect Immun 2003;71:6411‑9. 31. Mackintosh CG, Qureshi T, Waldrup K, Labes RE, Dodds KG, Griffin JF. Genetic resistance to experimental infection with Mycobacterium bovis in red deer (Cervus elaphus). Infect Immun 2000;68:1620‑5. 32. Griffin JF, Chinn DN, Rodgers CR. Diagnostic strategies and outcomes on three New Zealand deer farms with severe outbreaks of bovine tuberculosis. Tuberculosis (Edinb) 2004;84:293‑302. 33. Griffin JF, Spittle E, Rodgers CR, Liggett S, Cooper M, Bakker D, et al. Immunoglobulin G1 enzyme‑linked immunosorbent assay for diagnosis of Johne’s Disease in red deer (Cervus elaphus). Clin Diagn Lab Immunol 2005;12:1401‑9. 34. O’Brien R, Mackintosh CG, Bakker D, Kopecna M, Pavlik I, Griffin JF. Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp. paratuberculosis infection in the red deer (Cervus elaphus). Infect Immun 2006;74:3530‑7. 35. O’Brien R, Hughes A, Liggett S, Griffin F. Composite testing for ante‑mortem diagnosis of Johne’s disease in farmed New Zealand deer: Correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR. BMC Vet Res 2013;9:72. 36. Clark RG, Griffin JF, Mackintosh CG. Johne’s disease caused by
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Source of Support: Nil, Conflict of Interest: None declared.
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Department of Microbiology and Immunology, Disease Research Laboratory, University of Otago, Dunedin, New Zealand Address for correspondence: Dr. Frank Griffin, E‑mail: frank.griffin@otago. ac.nz
Received : 20‑11‑13 Review completed : 20‑11‑13 Accepted : 20‑11‑13
ABSTRACT Although most studies in immunology have used inbred mice as the experimental model to study fundamental immune mechanisms they have been proven to be limited in their ability to chart complex functional immune pathways, such as are seen in outbred populations of humans or animals. Translation of the findings from inbred mouse studies into practical solutions in therapeutics or the clinic has been remarkably unproductive compared with many other areas of clinical practice in human and veterinary medicine. Access to an unlimited array of mouse strains and an increasing number of genetically modified strains continues to sustain their paramount position in immunology research. Since the mouse studies have provided little more than the dictionary and glossary of immunology, another approach will be required to write the classic exposition of functional immunity. Domestic animals such as ruminants and swine present worthwhile alternatives as models for immunological research into infectious diseases, which may be more informative and cost effective. The original constraint on large animal research through a lack of reagents has been superseded by new molecular technologies and robotics that allow research to progress from gene discovery to systems biology, seamlessly. The current review attempts to highlight how exotic animals such as deer can leverage off the knowledge of ruminant genomics to provide cost‑effective models for research into complex, chronic infections. The unique opportunity they provide relates to their diversity and polymorphic genotypes and the integrity of their phenotype for a range of infectious diseases. KEY WORDS: Deer, immune markers, infectious diseases, vaccine development
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esearch into many facets of experimental biology has been dominated by inbred mice since they were first derived >100 years ago.[1] Inbred mice have made a vital contribution to our understanding of transplantion biology[2] and immunology, by disclosing the multiple cell subpopulations that contribute to the diverse immune pathways and the regulatory and effector molecules that influence the highly plastic immune system of higher organisms. The ability to transfer syngeneic cells from inbred donors to histocompatible recipients has made an important contribution to understanding Access this article online Quick Response Code:
Website: www.jpbsonline.org DOI: 10.4103/0975-7406.124302
the cellular molecular basis of many areas of immunological research and has underwritten most of the theoretical dogma of modern immunology. Research findings from inbred mice continue to dominate published outputs (>80%) in fundamental immunological research involving infection, allergy, autoimmunity and cancer. Nonetheless, while inbred mice have been valuable in illuminating the rich tapestry of immune circuitry they have provided more limited insights into the underlying mechanisms involved in complex, naturally occurring diseases. These include infection,[3] malignancy,[4] metabolic[5] and neurological disorders,[6] where inbred mice have not elucidated the mechanisms of disease or facilitated therapeutic interventions or have allowed translation of fundamental discoveries into applied solutions. Two important human diseases; malaria and tuberculosis (TB), will be used to highlight the limitations of inbred murine models to advance knowledge into infectious diseases that affect outbred human or domestic livestock populations.
How to cite this article: Griffin F. Farmed deer: A veterinary model for chronic mycobacterial diseases that is accessible, appropriate and cost-effective. J Pharm Bioall Sci 2014;6:10-5.
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Malaria A striking example of the limitations of inbred murine models is seen from multiple murine studies designed to evaluate novel therapeutics for cerebral malaria in humans.[7] In a large series (48) of therapeutic interventions evaluated over the past two decades in mice, 44 (92%) were considered to prevent or ameliorate the cerebral disease, preventing or reducing disease severity by 50‑100%. In stark contrast, only 1/11 human adjunctive intervention therapies trialed over the same timeframe provided a mortality advantage. Explanations that have been advanced to account for this dichotomy include the diverse phenotypes of the pathogen studied (Plasmodium falciparum [human] vs. Plasmodium berghei ANKA [mice]), the host genotype (Polymorphic genetics ‑ humans vs. monomorphic genetics ‑ mice), phenotypic differences, driven by closed (murine) versus open (human) environments, immunopathology (Intravascular coagulation [human] vs. inflammation [murine]), or the intervention strategy (prophylactic [mice] vs. therapeutic [human]). Irrespective of the point of difference,[7] inbred mouse model studies appear to have serious limitations in informing new approaches to solve the human malaria conundrum, by developing effective prophylactic vaccines to prevent infection or therapeutics to treat affected individuals. An evaluation of animal models for malaria,[8] involving 22 international experts failed to reconcile the points of differences between the expectations of human researchers and the outputs from murine malaria models.
TB A huge research investment has been made in the past two decades in an effort to develop new vaccines that provide prophylactic or therapeutic protection against human TB caused by Mycobacterium tuberculosis. This is because the existing human Bacille Calmette‑Guerin (BCG) vaccine is considered to provide suboptimal protection against TB in humans, domestic livestock and wildlife, all of which are affected by naturally occurring disease. BCG was derived by Calmette‑Guerin in 1908 from a primary isolate of Mycobacterium bovis; isolated from a tuberculous cow, that was subsequently passaged in the laboratory >120 times to produce the safe live attenuated BCG vaccine. This was first introduced as a prophylactic vaccine for human TB in 1921 and since then it has been used in more than 1 × 109 people world‑wide, without significant side‑effects or adverse reactions. Although it has not resulted in the eradication of TB world‑wide it has made a dramatic impact on severe disease (tubercular meningitis and miliary TB) in generations of children that have been vaccinated with BCG over the past 90 years. Nonetheless, it is far from optimal as a single dose vaccine for adults, where its widespread use has failed to control pulmonary disease or prevent spread of infection, especially in developing countries.[9] Since 1990 in excess of US$200 million has been invested in research world‑wide to develop improved vaccination protocols to improve the efficacy of human and animal TB vaccines. New candidate vaccines that have been developed include Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
recombinant BCG, M. tuberculosis auxotrophs, recombinant virus and recombinant mycobacterial peptides that are first screened in inbred mice to establish immunogenicity and “proof‑of‑efficacy”. Tests carried out on more than 200 candidate vaccines has failed to show one single candidate vaccine that has superior efficacy to BCG.[10] This has refocused the direction of research such that the current goal is not to replace BCG, but to develop a complementary vaccine that can be used to boost levels of protective immunity following primary vaccination with BCG.[10] There are currently up to 12 candidate TB vaccines in the pipeline in Phases I, II or III clinical trials. Testing these in human clinical trials is extremely expensive and fraught with challenges. The lead candidate vaccine (modified vaccinia virus ANKA 85A [MVA85A]), first tested in inbred mice[11] is a recombinant MVA that contained the 85A gene, which codes for a major cell wall antigen found in M. tuberculosis, M. bovis and BCG. It was seen to produce significantly increased levels of protection when used to boost inbred mice, previously vaccinated with BCG. Considering that this study provided the foundation data to justify further trials with the MVA85A vaccine in primates and later in humans, it is surprising that researchers dismissed the finding that prime‑boost with homologous BCG gave equivalent levels of protection to that seen with prime BCG followed by boosting with MVA85A. Superior protection against experimental infection with virulent M. tuberculosis, instilled into the lungs of vaccinated mice, was seen when the vaccine was administered intranasally rather than subcutaneously. Again two doses of BCG gave equivalent levels of protection as BCG‑MVA85A. Subsequent vaccine efficacy studies[12] in non‑human primates also showed that the vaccine was safe and stimulated protection against experimental challenge with virulent M. tuberculosis. A recent safety/efficacy trial on infants vaccinated with BCG and boosted with MVA85A showed that while the recombinant vaccine was safe for use in infants it produced no demonstrable protective efficacy against TB.[13]
Outbred Animal Models for Infection While inbred mice may provide spurious information for the study of TB it must be recognized that in selected areas of research that has involved murine virus infection, inbred mouse strains have provided outstanding fundamental information that has influenced the central dogma of immunology. The study of lymphocytic choriomeningitis virus in inbred strains of mice unraveled the importance of the critical role of the major histocompatibility complex markers for the recognition of virus infected cells by T cells (Tc).[14] This has become a critical paradigm that has facilitated the discovery of cytotoxic Tc, essential for immune protection against virus infection. Nonetheless, the widespread use of inbred mice in other areas of infectious disease research has provided limited metrics for immune function that are necessary to develop improved immunodiagnostics for infectious diseases, immune markers for protection to aid development of new vaccines or a better understanding of heritability of resistance or susceptibility to important infectious diseases that affect humans and domestic livestock. A number of researchers[15,16] have expressed concern about the undue reliance on the inbred 11
Griffin: Outbred animal models for infectious diseases
mouse for immunology studies. Others suggest new approaches in human clinical immunology[17] or the use of experimental animal model including ruminants (cattle, sheep or goats)[18] or swine[19] will more accurately reflect functional pathways of protective immunity and pathology, that occur naturally in response to infection or vaccination.
Table 1: Advantages and limitations of animal models for infectious disease research
The contrasting features of experimental studies involving inbred mice and outbred animal models are shown in Table 1, highlighting the advantages of two model systems. Based on the relative weighting it would appear that inbred mice have significant advantages based on cost, easy of handling, availability of reagents, technology platforms and genomic information. In reality, the fact is that inbred mice, with a homozygous genotype are intrinsically restricted and unrepresentative and rarely express a phenotype for disease that equates with that found in wild type mice, outbred animals or humans. In disease research host phenotype is paramount, where the experimental model must mimic the patterns of disease that occur naturally in the host, which in part explains why inbred mice are so limited as models for TB. Mice appear relatively resistant and do not develop TB naturally and when experimentally infected with M. tuberculosis or M. bovis they produce pathology that is completely different from that seen in TB animals or people. By contrast domestic animals produce TB pathology that is indistinguishable from humans and show a spectrum of disease severity that equates with the relative susceptibility or resistance of individuals within an outbred population. When developing experimental animal models due cognizance must be given to the protocols for both the establishment of infection and vaccination, in order to accurately monitor infection or disease outcomes to monitor the efficacy of vaccines or effect of therapeutics. Ideally, infection should be established by a natural route using the minimum dose of the pathogen necessary to produce a predictable response. The resulting experimental challenge should produce patterns of infection and disease that are similar in their range and severity to that seen to occur naturally. While conducting vaccine efficacy studies it is necessary to consider additional levels of stringency to control for all the variables that may influence the metrics for vaccine efficacy. Ideally these should reflect phenotypic modifiers such as intercurrent infection, climatic and environmental variables that may influence host phenotype and impact on vaccine efficacy. It is self‑evident that a well‑fed, pathogen‑free inbred mouse held under sterile housing conditions in a biosafety level 3 facility, does not faithfully reflect the environment of an infant in an underdeveloped country or a lamb, calf or piglet on a conventional farm. When using animal models for efficacy testing of new TB vaccine the variables which must be taken into account are listed in Table 2.
Experimental Studies in Farmed Deer During the past 20 years, we have explored a number of strategies for the control of mycobacterial disease in livestock
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Feature
Inbred mice
Large animal
Cost Containment/husbandry Breeding
Relatively low Relatively cheap Prolific/cheap
Genetic modification Availability of reagents Genetic diversity
Almost unlimited Excellent Extremely low (monomorphic) Poor Restricted (single) Extensive Fully annotated
High Expensive Prolific (embryo transfer) expensive Restricted Relatively poor High (polymorphic)
Integrity of phenotype Sampling Supporting literature Genome database
Purpose designed equipment Available Adoptive cell transfer Histocompatible
Excellent Unlimited (sequential) Relatively limited Most often not available Non‑existent Incompatible‑graft rejection
Table 2: Variables that impact on TB vaccine efficacy studies Choice of animal model Type of vaccine Prime versus boost Route of vaccination/ challenge Interval from vaccination to challenge Putative markers for protection Phenotype for protection Phenotypic modifiers
Mouse, guinea pig, rabbit Non‑human primate, livestock BCG, rBCG, rVector, peptide, DNA Interval Subcutaneous, intradermal, oral, intranasal Weeks versus months Immune cell phenotype, cytokines, mRNA Bacterial numbers, bacterial spread, nature of pathology, clinical manifestations, mortality Environmental infection, climate, nutrition
TB: Tuberculosis, BCG: Bacille Calmette‑Guerin, rBCG: Recombinant BCG, rVector: Recombinant vector
and consider that these findings may be translated to other species for the control of mycobacterial diseases such as TB in humans and other ruminant species. Our studies have involved TB, caused by infection of cattle and deer with M. bovis and M. paratuberculosis, which causes Johne’s disease (JD); a chronic enteritis in cattle, deer and sheep. As the majority of our research has been carried out with farmed deer this review will outline the chronology of the research findings in deer. It will cover the evolution of the research to develop laboratory‑based diagnostics, for use in test and cull programs, to research into vaccination and the discovery of heritable resistance to infection. The study of this exotic farmed species highlights the challenges associated with the intensified farming of a new species, but also identifies the opportunities to study novel animal species now that we have access to new molecular immunology platforms available to study infection in outbred populations. Deer also present unique opportunities to study how individual genotypes influence disease phenotypes as the process of domestication of these wild animals for intensive introduces stressors that can amplify the disease phenotypes. Within a decade of the first intensive management of wild captured deer (Circa 1970), TB infection was identified as a disease that had the potential to compromise successful Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
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domestication of deer. In 1985, our laboratory was commissioned by the New Zealand Deer Industry to develop a laboratory test for TB diagnosis in deer to complement the existing intradermal cervical skin test (CT). The CT had been used for >50 years to diagnose TB infection in humans and domestic livestock (mainly cattle). The deer industry was concerned about the performance of CT for TB diagnosis in deer, because it produced False (+) reactions in deer exposed to non‑pathogenic environmental mycobacteria and False (−) reactions in a proportion of TB animals. Our laboratory developed a composite blood test for TB (BTB)[20] which measured antibody, T‑cell transformation and inflammatory markers, simultaneously. This test improved the precision of TB diagnosis in deer allowing for the salvage of False (+) CT reactors and culling of BTB (+)/ CT (−) non‑reactors. The value of the laboratory‑based tests is evidenced by the striking progress in TB control in farmed deer over the past 3 decades, where infected herd numbers decreased from >200 herds in the 1980s to >5 in 2012. This is impressive considering the ongoing threat of TB infection in livestock in New Zealand posed by wildlife reservoirs of TB infection (e.g., possums/ferrets). Retrospective post‑mortem analysis of thousands of deer from which BTB tests had been carried out prior to slaughter, showed clearly that different components of the BTB could be used to predict whether individual animals were immune, infected or diseased, after exposure to pathogenic mycobacteria (M. bovis).[21,22] The ability of these tests to predict that an individual animal could become immune after exposure to virulent M. bovis prompted us to hypothesize that a proportion of animals were resistant to infection and could develop a protective immune response. In order to explore if protective immunity and disease resistance was attainable it was necessary to develop an experimental infection model and hence that we could trace the course of infection from exposure to virulent M. bovis, through to development of immunity, infection or disease. In a comparative study involving multiple routes of exposure (intratracheal, intratonsil, intranasal and subcutaneous) to virulent M. bovis the intratonsilar route of exposure was shown to be superior to all others in that it produced disease that was indistinguishable from TB in naturally infected animals.[23] All other routes of exposure produced aberrant or inconsistent responses that did not resemble TB in deer. Intratonsilar challenge with 100 colony forming units of virulent M. bovis, instilled with a blunt needle into the crypt of the tonsil, produced a spectrum of reactivity in different animals that ranged from, infection without any pathology, through to single lesions or generalized lesions involving lymphatic or bacteremic spread to multiple organs. The histopathology of the lesions in experimentally infected animals was indistinguishable from that seen in naturally infected animals, where lesions were also found at equivalent sites throughout the body. The validity of the intratonsilar infection model has subsequently been confirmed in Cape buffalo[24] and cattle.[25] The intratonsilar infection model in deer is sufficiently sensitive that it can be used to measure levels of infection as reflected by the recovery of live bacteria from infected tissues, or patterns of disease, as defined by the severity of the histopathological changes. Data from the vaccine studies[26] using BCG vaccines showed that a Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1
single dose of live BCG given to 3 month old deer produced protection against disease but did not protect against infection. Protection against infection is considered to be the “holy grail” in TB vaccinology. By contrast prime‑boost live BCG vaccination protected against animals against disease and infection. Killed BCG vaccines were ineffective and the protective effect of live BCG ×2 was ablated in animals treated with steroids. Subsequent studies[27] showed that the interval between prime and booster inoculations was important, where animals boosted at a 8 or 16 week interval generated protective immunity, while animals boosted at a 43 week interval were not protected. This corroborates findings from the Karonga study[28] on human BCG vaccination, where boosting children years after their primary BCG vaccination did not produce a demonstrable increase in protection. A recent booster BCG vaccine study in Brazilian children[29] showed that long term boosting does provide a measurable increase in vaccine protection in some regions, where environmental mycobacteria do not appear to interfere with protective immunity generated following prime BCG vaccination. The age of animals at primary vaccination may also be important as the prime‑boost effect we see in 3 month old deer was not seen in vaccinated neonatal (1 day old) calves, where a single dose of BCG provided optimal protection.[30] Not only has the deer infection model been valuable in evaluating vaccine mediated protection but it also has provided insights into heritable resistance and susceptibility to TB in deer. Experimental infection of a group of 75 stags was used to select small groups (3) of animals which displayed an extreme phenotype for either resistance (R) or susceptibility (S) to TB challenge.[31] Semen from R and S animals was used in an artificial insemination program to breed offspring which were subsequently challenged with M. bovis by the intratonsilar route. The results show that resistance or susceptibility to TB is highly heritable (0.48 ± 0.096). Subsequent field studies confirmed that heritability and selection for resistance to TB was evident on a deer farm that experienced a catastrophic outbreak of TB.[32] The establishment of the diagnostic platform for TB in deer in the 1980s and the subsequent development of experimental TB infection models have been extended into an equivalent program for control of M. paratuberculosis infection, which is the causative agent for JD in deer.[33] In the past decade our laboratory has developed and validated an immunoassay for JD (Paralisa TM) in deer [34] and developed a sensitive molecular (quantitative polymerase chain reaction) assay to monitor microbial pathogens excreted in feces or present in intestinal tissues.[35] Extensive histopathological assessments[36] are also required to confirm the infection or disease status of each animal that has been infected with M. paratuberculosis as each may respond differently to an equivalent exposure to this mycobacterial pathogen. We have also developed a M. paratuberculosis experimental infection model for deer,[37] which replicates many of the key features originally incorporated into the TB infection model. Because JD is an enteric infection it is possible to establish infection and disease at predictable levels in young deer by oral gavage with virulent M. paratuberculosis. We have also been fortunate to 13
Griffin: Outbred animal models for infectious diseases
identify different breeds of farmed deer that express extreme phenotypes for resistance and susceptibility to infection.[38] Breeding from stags with a R or S phenotype shows high levels of heritability for the paternal phenotype, where up to 80% of progeny express the same phenotype as the sire. This facilitates cost‑effective breeding of animals with a prescribed phenotype for disease resistance or susceptibility. These are especially valuable because animals with a susceptible phenotype provide an immunological signature for the infected or diseased phenotype. By contrast, animals with a resistant phenotype can be used to target protective immune pathways such as would be required to produce protective immunity following vaccination. The challenge has been to extend the historical concept of immune diagnostics beyond the limited scope in diagnosing infection or disease to include the spectrum of reactivity that occurs following infection and provide a metric that is predictive for different phenotypic responses in the host. These include: Exposed-Immune-Infected-Diseased.
3. 4.
5. 6. 7. 8. 9. 10. 11.
12.
Phenotype: Resistant(R)-Intermediate(I)-Susceptible(S). While the composite BTB diagnostic test developed for TB in deer[20] has provided some initial insights into different phenotypic responses to TB, the small range of biomarkers markers available (inflammatory/antibody/T‑cell) do not provide sufficient clarity to distinguish different phases of infection. Considering the complexity of the host response it is likely that a new systems based approach will be required to chart the relevant diagnostic markers for diagnosis of mycobacterial infections/disease, protective immunity to vaccines or to identify susceptible or resistant phenotypes. This approach incorporates monitoring gene expression levels for an array of immunological markers involving both the adaptive (T‑cell)[39] and the innate (Macrophage –Mf)[40,41] pathways of immunity. The initial findings suggest that it will be possible to assemble an array of markers that can be correlated with different animal phenotypes that distinguish infection, latency, disease or immunity. It appears that susceptibility is associated with dysfunctionality of the innate system, while resistance relates to immune competence of the adaptive T‑cell responses. Fortuitously, access to new molecular technologies circumvents the need for customized reagents when studying complex immune responses to infection. Based on published genomic data from other ruminants (cattle/sheep) it is possible to design primers for any annotated gene sequence in the database and monitor equivalent gene expression in more exotic animals such as deer. The use of transcriptomics to monitor gene expression and parallel sequencing can be used to identify candidate genes that contribute to functional immunity in experimentally infected animals. A similar approach involving the monitoring of biomarkers has been proposed for diagnosis of human TB.[42]
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Griffin: Outbred animal models for infectious diseases 28. Randomised controlled trial of single BCG, repeated BCG, or combined BCG and killed Mycobacterium leprae vaccine for prevention of leprosy and tuberculosis in Malawi. Karonga Prevention Trial Group. Lancet 1996;348:17‑24. 29. Barreto ML, Pereira SM, Pilger D, Cruz AA, Cunha SS, Sant’Anna C, et al. Evidence of an effect of BCG revaccination on incidence of tuberculosis in school‑aged children in Brazil: Second report of the BCG‑REVAC cluster‑randomised trial. Vaccine 2011;29:4875‑7. 30. Buddle BM, Wedlock DN, Parlane NA, Corner LA, De Lisle GW, Skinner MA. Revaccination of neonatal calves with Mycobacterium bovis BCG reduces the level of protection against bovine tuberculosis induced by a single vaccination. Infect Immun 2003;71:6411‑9. 31. Mackintosh CG, Qureshi T, Waldrup K, Labes RE, Dodds KG, Griffin JF. Genetic resistance to experimental infection with Mycobacterium bovis in red deer (Cervus elaphus). Infect Immun 2000;68:1620‑5. 32. Griffin JF, Chinn DN, Rodgers CR. Diagnostic strategies and outcomes on three New Zealand deer farms with severe outbreaks of bovine tuberculosis. Tuberculosis (Edinb) 2004;84:293‑302. 33. Griffin JF, Spittle E, Rodgers CR, Liggett S, Cooper M, Bakker D, et al. Immunoglobulin G1 enzyme‑linked immunosorbent assay for diagnosis of Johne’s Disease in red deer (Cervus elaphus). Clin Diagn Lab Immunol 2005;12:1401‑9. 34. O’Brien R, Mackintosh CG, Bakker D, Kopecna M, Pavlik I, Griffin JF. Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp. paratuberculosis infection in the red deer (Cervus elaphus). Infect Immun 2006;74:3530‑7. 35. O’Brien R, Hughes A, Liggett S, Griffin F. Composite testing for ante‑mortem diagnosis of Johne’s disease in farmed New Zealand deer: Correlations between bacteriological culture, histopathology, serological reactivity and faecal shedding as determined by quantitative PCR. BMC Vet Res 2013;9:72. 36. Clark RG, Griffin JF, Mackintosh CG. Johne’s disease caused by
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Mycobacterium avium subsp. paratuberculosis infection in red deer (Cervus elaphus): An histopathological grading system, and comparison of paucibacillary and multibacillary disease. N Z Vet J 2010;58:90‑7. Mackintosh CG, Labes RE, Clark RG, de Lisle GW, Griffin JF. Experimental infections in young red deer (Cervus elaphus) with a bovine and an ovine strain of Mycobacterium avium subsp paratuberculosis. N Z Vet J 2007;55:23‑9. Mackintosh CG, Clark RG, Tolentino B, de Lisle GW, Liggett S, Griffin JF. Immunological and pathological responses of red deer resistant or susceptible genotypes, to experimental challenge with Mycobacterium avium subsp. paratuberculosis. Vet Immunol Immunopathol 2011;143:131‑42. Robinson MW, O’Brien R, Mackintosh CG, Clark RG, Griffin JF. Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer. Infect Immun 2011;79:2089‑97. Dobson B, Liggett S, O’Brien R, Griffin JF. Innate immune markers that distinguish red deer (Cervus elaphus) selected for resistant or susceptible genotypes for Johne’s disease. Vet Res 2013;44:5. Marfell BJ, O’Brien R, Griffin JF. Global gene expression profiling of monocyte‑derived macrophages from red deer (Cervus elaphus) genotypically resistant or susceptible to Mycobacterium avium subspecies paratuberculosis infection. Dev Comp Immunol 2013;40:210‑7. Wallis RS, Pai M, Menzies D, Doherty TM, Walzl G, Perkins MD, et al. Biomarkers and diagnostics for tuberculosis: Progress, needs, and translation into practice. Lancet 2010;375:1920‑37.
Source of Support: Nil, Conflict of Interest: None declared.
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