Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection

Abstract

Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized.

Introduction

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease or paratuberculosis, which is a chronic debilitating disease in ruminants that is characterized by incurable enteritis and persistent diarrhea [1]. The disease is distributed worldwide and causes significant economic losses to the livestock industry because of premature culling and production losses [2, 3]. In the United States, MAP-positive herds experience economic losses of almost US $100 per cow and a disease cost of US $200 to 250 million annually [4]. At the herd level, it has been estimated that more than 50% of dairy cattle farms were infected with MAP in most major dairy-producing countries [4, 5]. Moreover, the most recent herd level prevalence estimates are as high as 90% in the U.S. dairy cattle industry [6]. These findings indicate that an infection rate of MAP is increasing and there is a need to establish an efficient program for control of this pathogen.

Clinical signs of the disease, such as diarrhea, loss of milk production and weight loss, are usually absent until two or more years after initial infection [7]. Stages of the MAP infection can be divided into four categories according to severity of clinical signs, the potential for shedding organisms, and the possibility of detection using current diagnostic methods [3]. The first stage, silent infection, is generally observed in young animals less than two years of age. These animals have no signs of infection clinically or microbiologically. Furthermore, there are no cost-effective diagnostic methods to detect animals in this stage [8]. The second stage is subclinical infection. Although animals in this stage still have no clinical signs of infection, they may be detected through cost-effective diagnostic tests such as serum enzyme-linked immunosorbent assay (ELISA) and fecal culture [9]. However, many of the animals in this stage are not detected by such tests because the animals shed organisms in an intermittent manner [10], and antibodies against MAP are usually produced when they are close to the next stage of disease (clinical stage) [11]. These undetected subclinical fecal shedders become a source of infection that consistently contaminate the environment. Therefore, many attempts have been made to detect these animals based on immunological knowledge. One of the major attempts is to identify MAP specific antigens that can be used in the interferon γ (IFN-γ) assay to measure Th1-mediated immune response elicited by animals in the early stage of infection [12, 13]. Another attempt is to identify biomarkers of the MAP infected animals by analysis of transcriptional changes that show early responses to infection. Accordingly, host transcriptional profiles during the early stage of infection in mouse RAW264.7 cells, MAP infected mouse models, and naturally infected cattle have been analyzed [14, 15, 16].

There are three major approaches to reduce or eradicate the Johne's disease, efficient management to decrease transmission, testing and culling, and vaccination [17]. Management using testing and culling practices are used in most countries [18]. Although the incidence of Johne's disease can be reduced by efficient management, eradication can only be accomplished when all the infected animals are detected and culled [19]. Although diagnostic tests for Johne's disease are improving, it is still not possible to detect all infected animals. For these reasons, testing and culling strategies using the present diagnostic methods are ineffective for eradication of the disease except when targeting only-high shedding animals [17, 20]. Under these circumstances, vaccination can be the best control strategy unless animals can be detected during early infection. This is because vaccination can reduce the incidence of MAP shedding and manifestation of clinical signs, which is more cost-effective than testing and culling [21]. However, vaccination is probably the least accepted strategy because of several drawbacks, which are discussed in the next section of this review.

MAP can infect a wide range of animals, therefore, it is important to determine if wildlife can act as a maintenance host or act as spillover host because the infection can persist via intraspecies transmission alone in maintenance hosts. In fact, one of the difficulties of eradication of bovine tuberculosis is blocking contacts with livestock from wildlife such as badgers, brush-tailed possums, and white-tail deer [22]. Some species susceptible to paratuberculosis such as farmed deer could maintain the infection when they are in high-density populations [23]. In South Korea, there have been several reports that MAP has infected wild boar, sika deer, and mouflon [24, 25, 26]. In this review, the general characteristics of Johne's disease with respect to the pathogenesis and immune response to MAP, as well as recent advances in development of vaccines were briefly examined.

Pathogenesis and Immune Responses to MAP

MAP infection is initiated by ingestion of fecal material orally contaminated by MAP (fecal-oral route). Following ingestion, MAP can pass through the M cell, which is specialized for uptake of particles that mainly bind to bacteria and transport them into the submucosal layer. After crossing the epithelial layer, MAP is phagocytosed by submucosal macrophages [27]. Like other mycobacterium, MAP is able to survive and replicate in non-activated macrophages by inhibiting phagosome-lysosome maturation [28, 29]. MAP eventually causes the cell death of infected cells, after which liberated MAP can be phagocytosed by freshly accumulated macrophages and dendritic cells that are activated by cytokines such as tumor necrosis factor α and IFN-γ. IFN-γ, which plays an important role in activation of macrophages and T cells, is produced by infected cells, local γδ T cells, and natural killer cells [30, 31]. Activated macrophages and dendritic cells produce interleukin (IL) 12 and present antigen to naïve CD4+ T cells through MHC class II molecules. IL-12 triggers the differentiation of naïve CD4+ T cells into T helper 1 (Th1) cells. Th1 cells produce cytokines such as IL-2 and IFN-γ, which play roles in promotion of expansion of antigen specific Th1 cells and maturation of macrophages. These antigen specific responses change from innate immunity to cell mediated adaptive immunity.

In the later stages of infection, increasing antibodies are frequently observed with increasing bacterial shedding [32]. Therefore, it has been accepted that switching from Th1 immune responses to Th2 responses is the cause of disease progression [33, 34, 35]. Many researchers investigated the immune regulatory mechanisms of host animals to identify causes of Th1-Th2 switches. An increase of IL-10 production by regulatory T cells (Tregs) or macrophages, which induces the down regulation of Th1 responses and stimulation of antibody production, is considered to cause this switch [36, 37, 38]. Along with Tregs, γδ T cells have been known to play a role in immune regulation. The progressive decrease of CD4+ T cell population in local immune response has been shown to be accompanied by increasing γδ T cell population [32]. The cytokine mRNA profiles of γδ T cells demonstrated that subsets of bovine γδ T cells encode IL-10 and transforming growth factor β, suggesting a potential regulatory role of γδ T cells [39, 40]. Recently, however, a typical pattern of disease progression that can be explained by the Th1-Th2 switch was observed in only 40% of MAP-infected sheep with other cases showing simultaneous responses of both cellular and humoral immunity or cellular immunity only [41]. A recent study using mathematical modeling suggested that Th1-Th2 switch may be a result of disease progression (increasing of extracellular bacteria) rather than cause [42]. A similar study that used a mathematical model to analyze the correlation between Th1, Th2 expression and bacterial shedding revealed a positive correlation between the amount of bacteria and humoral immune response was observed. However, there was no evidence of competition or synergy between Th1 and Th2 immunity [43]. This study also suggested that MAP-specific cellular immune responses were predicted to increase shedding, whereas in some animals it was predicted to suppress the shedding. As a result, it can be inferred that adaptive immune responses play a limited role in disease protection. However, these mathematical modeling studies have some vulnerable points because they cannot consider all of the parameters. Therefore, these results or hypotheses should be confirmed by both in vitro and in vivo studies. Another modeling study suggested that long-term subclinical infection may related to innate immunity rather than adaptive immunity [44]. The results using structural models (villus model, granulomatous model) of local infection showed that the long subclinical phase was due to structural organization of the granulomatous lesion. Moreover, the authors predicted that intermittent shedding was due to changes in the recruiting efficiency of macrophages influenced by external factors such as hormonal changes (ex., pregnancy, lactation).

Paratuberculosis Vaccines

Live attenuated vaccines

Recently, many researchers have been interested in development of live attenuated vaccines against MAP. These types of vaccines can elicit protective mucosal and systemic immune responses because the diverse antigens included in this vaccine can stimulate both innate and adaptive immunity [45, 46]. Another advantage of this vaccine is that manufacture of live attenuated vaccine is cost effective and easies than that of other vaccines such as subunit vaccines [47]. Many vaccine candidates have been produced by mutagenesis to attenuate the virulence of MAP. Mutants of MAP have been made by phage-mediated techniques, transposon mutagenesis and allelic exchange mutagenesis [48, 49, 50]. Many transposon mutant libraries have been created to identify virulence mechanisms thereby finding vaccine candidates [49, 51, 52]. Direct mutagenesis using allelic exchange techniques has also been tried by deletion of genes already known to be pathogenic or essential for intracellular survival in M. tuberculosis or M. bovis [46, 47, 50, 51, 53]. The ΔrelA, Δlsr2, and ΔpknG mutants were generated by Park et al. [50] and each gene was known to be related to virulence factors in M. tuberculosis and M. bovis [54, 55, 56]. Two of these candidates, ΔrelA and ΔpknG were evaluated for virulence attenuation and efficacy as vaccine candidates using macrophages and ileal cannulation models of natural hosts (cattle) and goats [57]. The result showed that ΔrelA mutant was a better vaccine candidate than ΔpknG mutant based on virulence attenuation and inhibition of MAP challenge in baby goats. The WAg906 (ΔMAP1566), WAg913, and WAg915 (ΔppiA) mutants were evaluated by Scandurra et al. [51]. WAg906 and WAg913 were made by transposon mutagenesis using MAP 989 strain, and WAg915 was made by allelic exchange of the ppiA gene [51, 58]. These live attenuated vaccine candidates were evaluated using monocyte derived macrophages (MDM) apoptosis, IL-10 production and animal models (mouse and goat) [51]. The results revealed that WAg906 mutant was the most attenuated strain. Mouse vaccinated with WAg915 mutant reduced bacterial loads in the spleen and liver after challenge with MAP. The ΔleuD, Δmpt64, and ΔsecA2 mutants were constructed by allelic exchange of these three genes to develop effective live attenuated vaccine [53]. These mutant candidates were selected based on previous studies. The auxotroph leuD mutant of M. bovis-BCG strain showed lower survival rates [59, 60], and M. bovis leuD mutant induced significant protective immune responses against a virulent M. bovis strain in cattle [61]. The mpt64 gene is related to apoptosis of multinucleated giant cells [62, 63], while the secA2 mutant of M. tuberculosis enhanced apoptosis of infected macrophages [64]. Testing using the mouse model revealed that the most obviously attenuated mutant was ΔleuD mutant strain. In addition, ΔleuD mutant induced a significant reduction of inflammation and bacterial load compared with the non-vaccinated group. The ΔsigL and ΔsigH mutants that are knocked out of the sigma factor gene were selected as live attenuated vaccine candidates because the sigma factors involved in part of the global virulence regulation provide resistance to the host bactericidal activities [46, 65]. The ΔsigL and ΔsigH mutants showed attenuated virulence in mice, and these mutants elicited significant protective immune responses against MAP infection in mouse models [46, 47].

Recently, a three phase vaccine candidate evaluation strategy was established by Johne's Disease Integrated Program (JDIP) research consortium to improve the efficiency of the efficacy test on the MAP live attenuated vaccines [66]. Phase I is a screening test using the MDM model, phase II is a challenge test using the mouse model, and phase III is an evaluation of protective effects using a goat model. The phase I test was conducted by Lamont et al. [67] to evaluate many live attenuated vaccine candidates constructed until 2014.

Subunit vaccines

Subunit vaccines have been developed to overcome the drawbacks of whole-cell based vaccines. Whole-cell based vaccines interfere with the diagnosis of both tuberculosis and paratuberculosis in vaccinated animals. However, subunit vaccines using well defined recombinant MAP proteins or DNA encoding immunogenic antigens can overcome the interference issues [68]. Many attempts have been made to identify MAP antigens to develop subunit vaccines using genomic or proteomic analysis. Because the production of IFN-γ induced by Th1-mediated immune responses is crucial to reducing the number of bacteria in the early stages of MAP infection, identifying antigens that induce strong Th1 responses is essential to the development of subunit vaccines [68]. Finding an antigen is also related to development of immunodiagnostic method as well as development of subunit vaccines. Several proteins have been identified as vaccine candidates. Several antigens were tested for their potential for use as a vaccine candidates: heat shock protein 70 (Hsp70) [69], antigen 85 complex proteins (Ag85A, Ag85B, and Ag85C) [70], lipoproteins (LprG and MAP0261c) [71, 72], PPE family proteins (MAP1518 and MAP3184) [73], superoxide dismutase [70], and alkyl hydroperoxide reductases (AhpC, AhpD) [74]. Among many antigens, the protein Hsp70 has been widely studied as a subunit vaccine candidate. Cattle vaccinated with Hsp70 containing an adjuvant, dimethyl dodecyl ammonium bromide, showed reduced bacterial load compared with a non-vaccinated group in animals experimentally challenged with MAP [75]. Furthermore, the cross-reactivity with serologic test of paratuberculosis was not observed when a preabsorption step with Hsp70 was included, and Hsp70 vaccination did not interfere with the skin test of tuberculosis, despite Hsp70 being a major component of mycobacterial tuberculin [76, 77]. However, recent study suggested that the protective effects of Hsp70 protein are due to B-cell activation and therefore the production of Hsp70-specific IgG1, instead of Th1-mediated immune response producing IFN-γ [78]. To date, researchers have only focused on cell-mediated immune responses to identify candidate vaccines. However, further studies are needed to understand protective mechanisms to MAP of host animals in greater detail, including humoral immune responses in the early stages of infection.

DNA vaccination against mycobacteria showed very effective protective immune responses in small rodents [79, 80]. Moreover, DNA vaccines have advantages of storage and delivery because they are very stable. Several candidates were evaluated for their ability to induce protective immune responses; however, they were only evaluated in mouse models. Recently, combination of MAP-specific antigens and viral vectors was attempted to increase the ability of antigenic effects of DNA vaccines [81, 82]. The advantage of viral vectored vaccines is to provide high delivery of antigens to antigen presenting cells, thereby increasing antigen specific CD4+ and CD8+ immune responses [83, 84, 85].

Commercially available vaccines

The first MAP vaccine, which was developed in 1926 by Vallee and Rinjard, consisted of a live non-virulent MAP and oil based adjuvants. Since then, a number of whole-cell based vaccines, live attenuated vaccines and inactivated vaccines were developed to prevent bovine and ovine Johne's disease. Currently, three commercial vaccines are all based on inactivated whole bacteria, Mycopar, Gudair, and Silirum, of which only Mycopar is approved for use in the United States [17]. Mycopar is manufactured by Boehringer Ingelheim Vetmedica Inc. using MAP strain 18 for use in cattle. Interestingly, strain 18 is not a MAP, although it is a member of the family of Mycobacterium avium species [86]. Gudair is manufactured by CZ Veterinaria in Spain for use in sheep and goats using heat inactivated MAP F316 strain adjuvanted with mineral oil. Gudair vaccination is encouraged in Australia for controlling ovine Johne's disease [87]. An Australian study revealed that vaccination could reduce the prevalence of MAP shedding with their longitudinal study [88]. However, another cross-sectional study reported that shedding of MAP persisted in the majority of flocks, despite vaccination of lambs [89]. Silirum consists of MAP F316, similar to Gudair. This vaccine is manufactured by Zoetis to prevent bovine Johne's disease. An efficacy test with a randomized control of Johne's disease in young farmed deer in New Zealand revealed that vaccination of Silirum reduced the prevalence of clinical disease [90]. Meta-analysis of the efficacy of MAP vaccination, especially its production, epidemiological effects, and pathogenic effects, was conducted by Bastida and Juste in 2011 [17] using previously published papers. From this meta-analysis, it was concluded that vaccination against MAP is a useful strategy for reducing contamination by this pathogen, production losses and pathologic effects. Despite the many advantages of vaccination, it has not been encouraged in cattle in most of countries because of several drawbacks mentioned in the introduction section. One major drawback of whole-cell based vaccination is interference with diagnostic tests currently used in bovine tuberculosis and paratuberculosis [91, 92]. These vaccines have the potential to produce false positive animals in serological tests for paratuberculosis such as ELISA because the commercial ELISA kit consisted of crude MAP antigens, which hinder differentiation of infected animals from vaccinated animals [93]. The caudal fold skin test using M. bovis purified protein derivatives (PPD-B) is most widely used field screening tool for diagnosis of bovine tuberculosis [94]. However, in the IFN-γ assay, stimulation with PPD-B produced robust responses similar to PPD-J (MAP purified protein derivatives) in MAP vaccinated animals [92, 95]. Because of this cross-reaction with other mycobacteria such as M. avium subspecies, comparative cervical test has been used as a complementary test to discriminate M. bovis infection from other mycobacterial infections by comparing the reactivity of each antigen using PPD-B and PPD-A (M. avium purified protein derivatives). However, this strategy may also cause problems with diagnostic sensitivity owing to the higher PPD-A reactivity because MAP vaccination can reduce the differences between PPD-B and PPD-A in M. bovis infected animals [93]. Therefore, many countries that are running M. bovis eradication programs do not use vaccination policies. However, these problems can be overcome by development of new diagnostic methods or vaccines. Emerging serologic tests using M. bovis specific antigens such as ESAT-6, CFP-10, and MPB83 did not produce positive results in MAP vaccinated animals [95]. Another drawback of whole cell based vaccines is the substantial tissue damage at the injection site and accidental self-inoculation, which may cause serious side-effects [96]. However, there is a vaccine adjuvanted with highly refined mineral oils such as Silirum to reduce the formation of granuloma at the site of injection [68].

Conclusion

Vaccines against paratuberculosis have been developed by diverse approaches. The most important factors to consider in vaccine studies are the mechanisms related to the host-pathogen interaction. Much more efforts are needed to understand exactly how bacteria can evade the host defense system, and these should focus on not only an adaptive immune system, but also innate immunity. Vaccines that can induce both immune responses may have improved protective effects. Despite some limitations, vaccines might still be an effective strategy to reduce or eradicate Johne's disease in livestock industries.

Notes

No potential conflict of interest relevant to this article was reported.

This work was carried out with the support of the "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00897001)" Rural Development Administration, the BK21 PLUS Program for Creative Veterinary Science Research and the Research Institute for Veterinary Science, Seoul National University, Republic of Korea.

References

1. Harris NB, Barletta RG. Mycobacterium avium subsp. paratuberculosis in Veterinary Medicine. Clin Microbiol Rev 2001;14:489–512.

2. Sweeney RW. Transmission of paratuberculosis. Vet Clin North Am Food Anim Pract 1996;12:305–312.

3. Whitlock RH, Buergelt C. Preclinical and clinical manifestations of paratuberculosis (including pathology). Vet Clin North Am Food Anim Pract 1996;12:345–356.

4. Ott SL, Wells SJ, Wagner BA. Herd-level economic losses associated with Johne's disease on US dairy operations. Prev Vet Med 1999;40:179–192.

5. Nielsen SS, Toft N. A review of prevalences of paratuberculosis in farmed animals in Europe. Prev Vet Med 2009;88:1–14.

6. Lombard JE, Gardner IA, Jafarzadeh SR, et al. Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis infection in United States dairy herds in 2007. Prev Vet Med 2013;108:234–238.

7. Irenge LM, Walravens K, Govaerts M, et al. Development and validation of a triplex real-time PCR for rapid detection and specific identification of M. avium subsp. paratuberculosis in faecal samples. Vet Microbiol 2009;136:166–172.

8. Tiwari A, VanLeeuwen JA, McKenna SL, Keefe GP, Barkema HW. Johne's disease in Canada Part I: clinical symptoms, pathophysiology, diagnosis, and prevalence in dairy herds. Can Vet J 2006;47:874–882.

9. Tiwari A, VanLeeuwen JA, Dohoo IR, Stryhn H, Keefe GP, Haddad JP. Effects of seropositivity for bovine leukemia virus, bovine viral diarrhoea virus, Mycobacterium avium subspecies paratuberculosis, and Neospora caninum on culling in dairy cattle in four Canadian provinces. Vet Microbiol 2005;109:147–158.

10. Merkal RS, Thurston JR. Comparison of Mycobacterium paratuberculosis and other mycobacteria, using standard cytochemical tests. Am J Vet Res 1966;27:519–521.

11. Kennedy DJ, Benedictus G. Control of Mycobacterium avium subsp. paratuberculosis infection in agricultural species. Rev Sci Tech 2001;20:151–179.

12. Mortier RA, Barkema HW, Wilson TA, Sajobi TT, Wolf R, De Buck J. Dose-dependent interferon-gamma release in dairy calves experimentally infected with Mycobacterium avium subspecies paratuberculosis. Vet Immunol Immunopathol 2014;161:205–210.

13. Stabel JR. Host responses to Mycobacterium avium subsp. paratuberculosis: a complex arsenal. Anim Health Res Rev 2006;7:61–70.

14. Cha SB, Yoo A, Park HT, Sung KY, Shin MK, Yoo HS. Analysis of transcriptional profiles to discover biomarker candidates in Mycobacterium avium subsp. paratuberculosis-infected macrophages, RAW 264.7. J Microbiol Biotechnol 2013;23:1167–1175.

15. Shin MK, Park H, Shin SW, et al. Host transcriptional profiles and immunopathologic response following Mycobacterium avium subsp. paratuberculosis infection in mice. PLoS One 2015;10:e0138770.

16. Shin MK, Park HT, Shin SW, et al. Whole-blood gene-expression profiles of cows infected with Mycobacterium avium subsp. paratuberculosis reveal changes in immune response and lipid metabolism. J Microbiol Biotechnol 2015;25:255–267.

17. Bastida F, Juste RA. Paratuberculosis control: a review with a focus on vaccination. J Immune Based Ther Vaccines 2011;9:8.

18. Geraghty T, Graham DA, Mullowney P, More SJ. A review of bovine Johne's disease control activities in 6 endemically infected countries. Prev Vet Med 2014;116:1–11.

19. Gumber S, Taylor DL, Whittington RJ. Evaluation of the immunogenicity of recombinant stress-associated proteins during Mycobacterium avium subsp. paratuberculosis infection: implications for pathogenesis and diagnosis. Vet Microbiol 2009;137:290–296.

20. Lu Z, Mitchell RM, Smith RL, et al. The importance of culling in Johne's disease control. J Theor Biol 2008;254:135–146.

21. Juste RA. Slow infection control by vaccination: paratuberculosis. Vet Immunol Immunopathol 2012;148:190–196.

22. Drewe JA, Pfeiffer DU, Kaneene JB. Epidemiology of Mycobacterium bovis. In: Thoen CO, Steele JH, Kaneene JB, editors. Zoonotic tuberculosis: Mycobacterium bovis and other pathogenic mycobacteria. 3rd ed. Hoboken, NJ: Wiley-Blackwell; 2014. pp. 63-77.

23. Balseiro A, Garcia Marin JF, Solano P, Garrido JM, Prieto JM. Histopathological classification of lesions observed in natural cases of paratuberculosis in free-ranging fallow deer (Dama dama). J Comp Pathol 2008;138:180–188.

24. Bae JH, Jean YH. Spontaneous paratuberculosis in a sika deer: a case report. Korean J Vet Res 1993;33:673–678.

25. Bae YC, Kim HY, Kim HJ, et al. Paratuberculosis in mouflon (Ovis musimon): a case report. Korean J Vet Res 2006;46:271–274.

26. Kim JM, Ku BK, Lee HN, et al. Mycobacterium avium paratuberculosis in wild boars in Korea. J Wildl Dis 2013;49:413–417.

27. Momotani E, Whipple DL, Thiermann AB, Cheville NF. Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves. Vet Pathol 1988;25:131–137.

28. Kuehnel MP, Goethe R, Habermann A, et al. Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria. Cell Microbiol 2001;3:551–566.

29. Sweeney RW. Pathogenesis of paratuberculosis. Vet Clin North Am Food Anim Pract 2011;27:537–546.

30. Boysen P, Storset AK. Bovine natural killer cells. Vet Immunol Immunopathol 2009;130:163–177.

31. Rogers AN, Vanburen DG, Hedblom EE, Tilahun ME, Telfer JC, Baldwin CL. Gammadelta T cell function varies with the expressed WC1 coreceptor. J Immunol 2005;174:3386–3393.

32. Koets A, Rutten V, Hoek A, et al. Progressive bovine paratuberculosis is associated with local loss of CD4(+) T cells, increased frequency of gamma delta T cells, and related changes in T-cell function. Infect Immun 2002;70:3856–3864.

33. Manning EJ, Collins MT. Mycobacterium avium subsp. paratuberculosis: pathogen, pathogenesis and diagnosis. Rev Sci Tech 2001;20:133–150.

34. Stabel JR. Transitions in immune responses to Mycobacterium paratuberculosis. Vet Microbiol 2000;77:465–473.

35. Sweeney RW, Jones DE, Habecker P, Scott P. Interferon-gamma and interleukin 4 gene expression in cows infected with Mycobacterium paratuberculosis. Am J Vet Res 1998;59:842–847.

36. Buza JJ, Hikono H, Mori Y, et al. Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis. Infect Immun 2004;72:2425–2428.

37. Coussens PM, Sipkovsky S, Murphy B, Roussey J, Colvin CJ. Regulatory T cells in cattle and their potential role in bovine paratuberculosis. Comp Immunol Microbiol Infect Dis 2012;35:233–239.

38. de Almeida DE, Colvin CJ, Coussens PM. Antigen-specific regulatory T cells in bovine paratuberculosis. Vet Immunol Immunopathol 2008;125:234–245.

39. Hsieh B, Schrenzel MD, Mulvania T, Lepper HD, DiMolfetto-Landon L, Ferrick DA. In vivo cytokine production in murine listeriosis: evidence for immunoregulation by gamma delta+ T cells. J Immunol 1996;156:232–237.

40. Park YH, Yoo HS, Yoon JW, Yang SJ, An JS, Davis WC. Phenotypic and functional analysis of bovine gammadelta lymphocytes. J Vet Sci 2000;1:39–48.

41. Begg DJ, de Silva K, Carter N, Plain KM, Purdie A, Whittington RJ. Does a Th1 over Th2 dominancy really exist in the early stages of Mycobacterium avium subspecies paratuberculosis infections? Immunobiology 2011;216:840–846.

42. Magombedze G, Eda S, Ganusov VV. Competition for antigen between Th1 and Th2 responses determines the timing of the immune response switch during Mycobaterium avium subspecies paratuberulosis infection in ruminants. PLoS Comput Biol 2014;10:e1003414.

43. Ganusov VV, Klinkenberg D, Bakker D, Koets AP. Evaluating contribution of the cellular and humoral immune responses to the control of shedding of Mycobacterium avium spp. paratuberculosis in cattle. Vet Res 2015;46:62.

44. Klinkenberg D, Koets A. The long subclinical phase of Mycobacterium avium ssp. paratuberculosis infections explained without adaptive immunity. Vet Res 2015;46:63.

45. Faisal SM, Chen JW, Yan F, et al. Evaluation of a Mycobacterium avium subsp. paratuberculosis leuD mutant as a vaccine candidate against challenge in a caprine model. Clin Vaccine Immunol 2013;20:572–581.

46. Ghosh P, Steinberg H, Talaat AM. Virulence and immunity orchestrated by the global gene regulator sigL in Mycobacterium avium subsp. paratuberculosis. Infect Immun 2014;82:3066–3075.

47. Ghosh P, Shippy DC, Talaat AM. Superior protection elicited by live-attenuated vaccines in the murine model of paratuberculosis. Vaccine 2015;33:7262–7270.

48. Foley-Thomas EM, Whipple DL, Bermudez LE, Barletta RG. Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology 1995;141(Pt 5):1173–1181.

49. Harris NB, Feng Z, Liu X, Cirillo SL, Cirillo JD, Barletta RG. Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol Lett 1999;175:21–26.

50. Park KT, Dahl JL, Bannantine JP, et al. Demonstration of allelic exchange in the slow-growing bacterium Mycobacterium avium subsp. paratuberculosis, and generation of mutants with deletions at the pknG, relA, and lsr2 loci. Appl Environ Microbiol 2008;74:1687–1695.

51. Scandurra GM, de Lisle GW, Cavaignac SM, Young M, Kawakami RP, Collins DM. Assessment of live candidate vaccines for paratuberculosis in animal models and macrophages. Infect Immun 2010;78:1383–1389.

52. Shin SJ, Wu CW, Steinberg H, Talaat AM. Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. Infect Immun 2006;74:3825–3833.

53. Chen JW, Faisal SM, Chandra S, et al. Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model. Vaccine 2012;30:3015–3025.

54. Colangeli R, Helb D, Vilcheze C, et al. Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis. PLoS Pathog 2007;3:e87.

55. Dahl JL, Kraus CN, Boshoff HI, et al. The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice. Proc Natl Acad Sci U S A 2003;100:10026–10031.

56. Walburger A, Koul A, Ferrari G, et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 2004;304:1800–1804.

57. Park KT, Allen AJ, Bannantine JP, et al. Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease. Vaccine 2011;29:4709–4719.

58. Cavaignac SM, White SJ, de Lisle GW, Collins DM. Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries. Arch Microbiol 2000;173:229–231.

59. Bange FC, Brown AM, Jacobs WR Jr. Leucine auxotrophy restricts growth of Mycobacterium bovis BCG in macrophages. Infect Immun 1996;64:1794–1799.

60. McAdam RA, Weisbrod TR, Martin J, et al. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infect Immun 1995;63:1004–1012.

61. Khare S, Hondalus MK, Nunes J, Bloom BR, Garry Adams L. Mycobacterium bovis DeltaleuD auxotroph-induced protective immunity against tissue colonization, burden and distribution in cattle intranasally challenged with Mycobacterium bovis Ravenel S. Vaccine 2007;25:1743–1755.

62. Mustafa T, Wiker HG, Morkve O, Sviland L. Reduced apoptosis and increased inflammatory cytokines in granulomas caused by tuberculous compared to non-tuberculous mycobacteria: role of MPT64 antigen in apoptosis and immune response. Clin Exp Immunol 2007;150:105–113.

63. Mustafa T, Wiker HG, Morkve O, Sviland L. Differential expression of mycobacterial antigen MPT64, apoptosis and inflammatory markers in multinucleated giant cells and epithelioid cells in granulomas caused by Mycobacterium tuberculosis. Virchows Arch 2008;452:449–456.

64. Hinchey J, Lee S, Jeon BY, et al. Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis. J Clin Invest 2007;117:2279–2288.

65. Ghosh P, Wu CW, Talaat AM. Key role for the alternative sigma factor, SigH, in the intracellular life of Mycobacterium avium subsp. paratuberculosis during macrophage stress. Infect Immun 2013;81:2242–2257.

66. Bannantine JP, Hines ME 2nd, Bermudez LE, et al. A rational framework for evaluating the next generation of vaccines against Mycobacterium avium subspecies paratuberculosis. Front Cell Infect Microbiol 2014;4:126.

67. Lamont EA, Talaat AM, Coussens PM, et al. Screening of Mycobacterium avium subsp. paratuberculosis mutants for attenuation in a bovine monocyte-derived macrophage model. Front Cell Infect Microbiol 2014;4:87.

68. Rosseels V, Huygen K. Vaccination against paratuberculosis. Expert Rev Vaccines 2008;7:817–832.

69. Koets AP, Rutten VP, Hoek A, et al. Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis. Vet Immunol Immunopathol 1999;70:105–115.

70. Shin SJ, Chang CF, Chang CD, et al. In vitro cellular immune responses to recombinant antigens of Mycobacterium avium subsp. paratuberculosis. Infect Immun 2005;73:5074–5085.

71. Huntley JF, Stabel JR, Bannantine JP. Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein. BMC Microbiol 2005;5:3.

72. Rigden RC, Jandhyala DM, Dupont C, et al. Humoral and cellular immune responses in sheep immunized with a 22 kilodalton exported protein of Mycobacterium avium subspecies paratuberculosis. J Med Microbiol 2006;55(Pt 12):1735–1740.

73. Nagata R, Muneta Y, Yoshihara K, Yokomizo Y, Mori Y. Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis. Infect Immun 2005;73:3778–3782.

74. Olsen I, Reitan LJ, Holstad G, Wiker HG. Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp. paratuberculosis. Infect Immun 2000;68:801–808.

75. Koets A, Hoek A, Langelaar M, et al. Mycobacterial 70 kD heat-shock protein is an effective subunit vaccine against bovine paratuberculosis. Vaccine 2006;24:2550–2559.

76. Santema W, Hensen S, Rutten V, Koets A. Heat shock protein 70 subunit vaccination against bovine paratuberculosis does not interfere with current immunodiagnostic assays for bovine tuberculosis. Vaccine 2009;27:2312–2319.

77. Santema W, Overdijk M, Barends J, Krijgsveld J, Rutten V, Koets A. Searching for proteins of Mycobacterium avium subspecies paratuberculosis with diagnostic potential by comparative qualitative proteomic analysis of mycobacterial tuberculins. Vet Microbiol 2009;138:191–196.

78. Vrieling M, Santema W, Vordermeier M, Rutten V, Koets A. Hsp70 vaccination-induced primary immune responses in efferent lymph of the draining lymph node. Vaccine 2013;31:4720–4727.

79. Huygen K. Plasmid DNA vaccination. Microbes Infect 2005;7:932–938.

80. Huygen K. DNA vaccines against mycobacterial diseases. Future Microbiol 2006;1:63–73.

81. Bull TJ, Gilbert SC, Sridhar S, et al. A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis. PLoS One 2007;2:e1229.

82. Bull TJ, Vrettou C, Linedale R, et al. Immunity, safety and protection of an Adenovirus 5 prime: modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves. Vet Res 2014;45:112.

83. Guzman E, Cubillos-Zapata C, Cottingham MG, et al. Modified vaccinia virus Ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation. J Virol 2012;86:5452–5466.

84. Norbury CC, Malide D, Gibbs JS, Bennink JR, Yewdell JW. Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells in vivo. Nat Immunol 2002;3:265–271.

85. Reyes-Sandoval A, Rollier CS, Milicic A, et al. Mixed vector immunization with recombinant adenovirus and MVA can improve vaccine efficacy while decreasing antivector immunity. Mol Ther 2012;20:1633–1647.

86. Chiodini RJ. Abolish Mycobacterium paratuberculosis strain 18. J Clin Microbiol 1993;31:1956–1958.

87. Windsor P. Research into vaccination against ovine Johne's disease in Australia. Small Rumin Res 2006;62:139–142.

88. Eppleston J, Reddacliff L, Windsor P, Links I, Whittington R. Preliminary observations on the prevalence of sheep shedding Mycobacterium avium subsp paratuberculosis after 3 years of a vaccination program for ovine Johne's disease. Aust Vet J 2005;83:637–638.

89. Windsor PA, Eppleston J, Dhand NK, Whittington RJ. Effectiveness of Gudair vaccine for the control of ovine Johne's disease in flocks vaccinating for at least 5 years. Aust Vet J 2014;92:263–268.

90. Stringer LA, Wilson PR, Heuer C, Mackintosh CG. A randomised controlled trial of Silirum vaccine for control of paratuberculosis in farmed red deer. Vet Rec 2013;173:551.

91. Kohler H, Gyra H, Zimmer K, et al. Immune reactions in cattle after immunization with a Mycobacterium paratuberculosis vaccine and implications for the diagnosis of M. paratuberculosis and M. bovis infections. J Vet Med B Infect Dis Vet Public Health 2001;48:185–195.

92. Muskens J, van Zijderveld F, Eger A, Bakker D. Evaluation of the long-term immune response in cattle after vaccination against paratuberculosis in two Dutch dairy herds. Vet Microbiol 2002;86:269–278.

93. Santema W, Rutten V, Koets A. Bovine paratuberculosis: recent advances in vaccine development. Vet Q 2011;31:183–191.

94. Good M, Duignan A. Perspectives on the History of Bovine TB and the Role of Tuberculin in Bovine TB Eradication. Vet Med Int 2011;2011:410470.

95. Stabel JR, Waters WR, Bannantine JP, Lyashchenko K. Mediation of host immune responses after immunization of neonatal calves with a heat-killed Mycobacterium avium subsp. paratuberculosis vaccine. Clin Vaccine Immunol 2011;18:2079–2089.

96. Patterson CJ, LaVenture M, Hurley SS, Davis JP. Accidental self-inoculation with Mycobacterium paratuberculosis bacterin (Johne's bacterin) by veterinarians in Wisconsin. J Am Vet Med Assoc 1988;192:1197–1199.