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Vaccination of cattle against Mycobacterium bovis
Tuberculosis (2001) 81(1/2), 125I132 ^ 2001 Harcourt Publishers Ltd doi: 10.1054/tube.2000.0254, available online at http://www.idealibrary.com.on
Vaccination of cattle against Mycobacterium bovis B. M. Buddle AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
Summary Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistence of Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a ‘test and slaughter’ control programme. Early field trials with Bacille Calmette Guerin (BCG) M. bovis vaccine in cattle produced disappointing results, with induction of tuberculin skin-test reactivity following vaccination and low levels of protection. However, recent studies using a low dose of BCG vaccine in cattle have produced more encouraging results and field trials should now be carried out in developing countries to determine whether this low dose BCG vaccination strategy will reduce the spread of infection. The options for new candidate tuberculosis vaccines have increased markedly in the last decade with the advent of new attenuated strains of M. bovis, and sub-unit protein and recombinant DNA vaccines. Some of these new types of vaccines have recently been tested in cattle. New attenuated M. bovis vaccines induced greater protection than BCG vaccine in cattle which had been sensitized to environmental mycobacteria prior to vaccination. In contrast, it has proved difficult to stimulate appropriate immune responses in cattle necessary for protection with sub-unit protein and recombinant DNA vaccines and better immunological adjuvants are required for these types of vaccines. Progress in the development of new tuberculosis vaccines has been very rapid in the past decade and the prospects for vaccination to control and eradicate bovine tuberculosis are encouraging. ^ 2001 Harcourt Publishers Ltd
INTRODUCTION Bovine tuberculosis caused by Mycobacterium bovis is a major economic problem in several countries and constitutes a public health risk in a number of developing countries. The implementation of national bovine tuberculosis programmes, based on regular tuberculin testing and removal of infected animals, had led to the successful eradication or a major reduction in the incidence of bovine tuberculosis in cattle herds. However, these control measures have been only partially effective in countries such as the UK, Ireland and New Zealand, which have a wildlife reservoir of infected animals.1 Furthermore, this approach to control bovine tuberculosis is economically and socially unacceptable in many developing countries, particularly in Africa. It is in these
Correspondence to: B.M. Buddle, AgResearch, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand; Tel.:#64 4 9221418; Fax:#64 4 9221380; E-mail: [email protected] Financial assistance from New Zealand Ministry of Agriculture and Forestry.
developing countries where M. bovis infection is most likely to contribute to the prevalence of human tuberculosis.2 In industrialized countries where there is persistence of M. bovis infection in wildlife, and in many developing countries, the use of a vaccine against bovine tuberculosis warrants serious consideration. Recent advances in immunology and the molecular biology of mycobacteria have greatly increased the options for candidate vaccines against bovine tuberculosis. The two major groupings are live attenuated vaccines such as Bacille Calmette Guerin (BCG) and other attenuated strains of M. bovis, and subunit vaccines based on either mycobacterial protein or DNA. Live attenuated vaccines are advantageous as they promote strong cellular immune responses required for protective immunity against tuberculosis and may only need a single-shot delivery to induce life-long immunity. Subunit vaccines typically require a number of booster immunizations to obtain good levels of immunity. However, there are advantages in using sub-unit vaccines in cattle including fewer regulatory requirements and the possibility of animals not reacting in a tuberculin skin test following vaccination. The aim of this review is to look at the history of vaccination
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against bovine tuberculosis in view of the current knowledge of immunology and to investigate the prospects for development of new improved vaccines for control of this disease in cattle.
REQUIREMENTS OF A TUBERCULOSIS VACCINE FOR CATTLE The goal of vaccinating cattle against tuberculosis is to prevent the establishment of infection. Ideally, if vaccinated animals were subsequently exposed to M. bovis they would resist infection and would not react in a tuberculin skin test. Many vaccines such as BCG can induce a tuberculin skin test response and hence it will be important to develop differential diagnostic tests to distinguish vaccinated animals from those infected with M. bovis. Alternatively, a vaccine could be developed which does not induce a tuberculin skin test response. The vaccine would need to be safe and its use would have to be acceptable to countries importing beef and dairy products. In developing countries, the goal of vaccinating cattle may be less stringent, with the principal requirement being to reduce the spread of bovine tuberculosis.
EARLY STUDIES ON VACCINATION AGAINST BOVINE TUBERCULOSIS Calmette and Guerin commenced the development of the BCG strain in 1906 by attenuation of a virulent strain originally isolated from a cow with tuberculous mastitis. They found that immunization with a single dose of between 50 and 100 mg BCG (approximately 109}1010 colony forming units, CFU) protected cattle from arti9cial and natural infections, but protection was not long-lived.3 Attempts to con9rm these 9ndings in 9eld trials in the 1920s gave variable results.4, 5 Intradermal or oral routes of BCG administration were shown to give no better protection than the subcutaneous route. Particularly disappointing were the results of 9eld trials6 and the observation that re-vaccination had no effect. After several decades of vaccination trials in Canada,7 USA,4 Australia,5 Great Britain8 and Africa9 it was concluded that, although BCG could protect cattle from experimental infections it was ineffective as a vaccine in the 9eld against bovine tuberculosis. In the 1940s, vaccination trials in calves were also undertaken with a vole bacillus M. microti. From a series of trials, Young and Paterson10 reported that calves vaccinated intravenously with 5 mg of viable M. microti developed a relatively high degree of immunity to M. bovis. Immunity was evident one year after vaccination, but declined thereafter. No further 9eld trials were carried out with this vaccine, probably because some strains of Tuberculosis (2001) 81(1/2), 125I132
M. microti were more virulent than BCG and could cause disease in calves, and there was no indication that immunity produced was markedly different to that produced by BCG. The apparent failure of BCG to protect cattle in 9eld trials may be attributed to a number of factors. High doses of BCG (equivalent to 108}1010 CFU) were generally used for subcutaneous or intradermal vaccination of cattle and it is now known that these doses are less effective in stimulating protective immunity against tuberculosis.11 Vaccination was often carried out in areas with a very high prevalence of bovine tuberculosis9 and calves may have been exposed to M. bovis prior to vaccination through the consumption of milk from cows with tuberculous mastitis. In human vaccination trials, BCG has been less effective when used in the tropics where there is greater exposure to environmental mycobacteria. There is evidence in humans and cattle that prior exposure to environmental mycobacteria may lower the ef9cacy of BCG vaccine12,13 and this type of exposure needs to be considered in the evaluation of cattle trials.
DEVELOPMENT OF A CHALLENGE MODEL TO ASSESS VACCINE EFFICACY It is cost effective to undertake preliminary screening of bovine tuberculosis vaccines in small animal models such as mice and guinea-pigs with only the most promising vaccine candidates progressing for evaluation in cattle. Evaluation of vaccine ef9cacy in cattle should be carried out initially by experimentally challenging the vaccinated animals with virulent M. bovis. Field trials should be used only at the 9nal testing stage as they are expensive to run, with large group sizes needed due to the low prevalence or patchy distribution of disease and its slow progression. A requirement in a challenge model is that the lesions produced should mimic those seen in the natural disease. In cattle, lesions are predominantly found in the lungs or lymph nodes in the thorax.14 To mimic the type of lesion seen in the natural disease, the challenge dose needs to be kept as low as possible. Host factors including the condition of the animals and sensitization to environmental mycobacteria may in:uence how animals respond to vaccination. A low-dose respiratory challenge model has been established in cattle for testing vaccine ef9cacy using an intratracheal challenge of 103 CFU of virulent M. bovis.15 When the animals are killed and examined 4}5 months after challenge, tuberculous lesions are almost entirely restricted to the thoracic cavity with lesions in the lungs and thoracic lymph nodes. The lung lesions consist of small nodules 3}5 mm in diameter and the lymph node lesions vary in size from 2}30 mm in diameter. These types of lesions are typical of those seen in the natural disease. ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 127
Cattle challenged with higher doses (105}106 CFU) of M. bovis develop disease with multiple lymph node involvement and large lung lesions, which is atypical of that generally seen in the natural disease.16, 17
TYPES OF TUBERCULOSIS VACCINES Over recent years a number of different types of tuberculosis vaccines have shown promise in small animal models and have already or are about to be tested in cattle (Table 1). The bene9t of having a range of different types of vaccines is that they could be useful for different purposes.
BCG The existing BCG vaccine may not be suf9ciently effective, but has many properties that are desirable for a good vaccine. It is cheap to produce, can be administered via a number of routes, is safe, relatively stable and is derived from M. bovis. A drawback of using BCG is that vaccinated cattle may react positively in a tuberculin skin test and on this basis, would be assumed to have bovine tuberculosis and would be slaughtered. Studies with BCG in cattle can be helpful in gaining an understanding of the mechanisms associated with protection against bovine tuberculosis which should lead to the rational design of new improved tuberculosis vaccines. In addition, studies with BCG assist in determining the optimal conditions for use of improved attenuated M. bovis vaccines and serve as a benchmark for comparison with other tuberculosis vaccines. Recent information suggests that lower doses of BCG may preferentially stimulate cellular immunity to mycobacterial antigens and may have minimal effect, in the
Table 1 Comparison of different types of vaccines for protection against experimental challenge with M. bovis or M. tuberculosis Protection against infectiona
Type of vaccine BCG Modification of BCG Attenuated M. bovis strain Killed M. vaccae Protein vaccine DNA vaccine
Small animal model Cattle
Tuberculin skin test reactivityb
#18 #20
#15,19 NT
Yes No
#21 $22 #23,24 #26
#13 !19 $25 NT
Yes No No No
a : Protection defined as a significant reduction in bacterial counts or in size and distribution of lesions. b: Reactivity to tuberculin induced by vaccine. #: Protection (reference in superscript). ! : No protection.
: Variable protection compared to BCG. NT: Not tested.
$
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long term, on skin testing.27 To determine whether low doses of BCG could protect cattle against bovine tuberculosis, three vaccination/challenge trials were undertaken using doses ranging from 104}106 CFU of BCG. In the 9rst trial, groups of calves were vaccinated subcutaneously with 104 CFU (low dose) or 106 CFU (medium dose) of BCG Pasteur.15 Eight weeks after vaccination, 15 animals from each group, together with 16 non-vaccinated calves were challenged intratracheally with virulent M. bovis. All animals were killed and necropsied 22 weeks after challenge. The proportion of animals with tuberculous lesions in the low dose BCG, medium dose BCG and non-vaccinated groups were 2/15, 4/15 and 10/16, respectively. BCG induced a signi9cant level of protection against development of tuberculous lesions compared to the non-vaccinated controls. In a second trial, groups of calves were vaccinated with low to medium doses of BCG by the subcutaneous or intratracheal route and challenged as in the 9rst trial.19 Vaccination with BCG again resulted in fewer animals developing lesions and a reduction in the number of lesions in the diseased animals compared to the nonvaccinated group. An advantage of vaccinating by the respiratory route was that non-challenged animals from the group produced a minimal reaction in the tuberculin skin test in contrast to the corresponding animals from the subcutaneous vaccinated group. In the third trial, a year later, the calves were of the same age and were sourced from the same farm as in the previous two trials, but subcutaneous vaccination with a low dose of BCG (105 CFU) induced no protection.13 Prior to being vaccinated, the calves had high IFN-c responses to avian PPD, indicative of exposure to environmental mycobacteria. Preexposure to environmental mycobacteria has been put forward as an explanation of the failure for BCG to induce protection in many human tuberculosis trials and may be a reason for the failure of BCG to protect the cattle in this trial. A feature of the immunological pro9le of BCG-vaccinated cattle is a release of high levels of IFN-c and IL-2 from bovine PPD-stimulated whole blood cultures peaking at 2}4 weeks after vaccination.15, 19 In the trial where BCG did not induce protection, there was a delay in the IFN-c response to bovine PPD with no increase in the response by week 2 following vaccination and a only small increase in the response by week 4 (Fig. 1). One can speculate, that in animals which have been exposed to environmental mycobacteria, BCG multiplication in the animals following vaccination was suppressed whereas there was no suppression in non-sensitized animals. One problem with BCG vaccine is that vaccination may induce tuberculin skin-test reactivity even when low doses of BCG are used. A number of mycobacterial antigens have recently been evaluated in a whole blood IFN-c Tuberculosis (2001) 81(1/2), 125I132
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supplemented with the appropriate amino acid. Their ability to grow in vivo is also reduced. Vaccination of mice with these auxotrophic mutants has induced protection against M. tuberculosis infection.20 Vaccination of cattle with a leucine auxotrophic mutant of BCG did not induce a skin-test response to bovine PPD (H.M. Vordermeier, personal communication), however, protection against bovine tuberculosis has yet to be evaluated.
Attenuation of virulent strains of M. bovis
Fig. 1 Interferon-c released from bovine PPD-stimulated peripheral blood lymphocytes of cattle vaccinated with BCG. Cattle were vaccinated with 106 (䉱), 105 (䉬) or 104 (䊉) CFU of BCG or were non-vaccinated (*). A and B represent two vaccination trials carried in different years. BCG vaccination induced a significant level of protection against challenge with virulent M. bovis in trial A, while no protection was observed in trial B. Data are expressed as mean optical density units (;1000).
test for differentiating BCG-vaccinated cattle from those infected with bovine tuberculosis.28 The protein, ESAT-6 which is produced by virulent M. bovis strains, but not by BCG has been shown to be a very useful reagent for the speci9c diagnosis of bovine tuberculosis,29 and was a suitable antigen for differentiating between BCG-vaccinated and M. bovis-infected cattle.
Modified BCG BCG has been modi9ed by inactivation or alteration of some of its own genes in an attempt to reduce tuberculin skin-test responses and to make a safer vaccine for immunocompromised individuals. Auxotrophic mutants of BCG have been produced which have mutations in the genes involved in the metabolism of leucine and methionine.30 These mutants can no longer grow in minimal medium and can only grow when this medium is Tuberculosis (2001) 81(1/2), 125I132
BCG was produced in an empirical manner and recent genetic analysis has indicated that BCG contains a number of gene deletions compared with virulent strains of M. bovis or M. tuberculosis. It should be possible to improve BCG by deleting from virulent strains of M. bovis or M. tuberculosis, speci9c genes, which are involved in virulence or encode enzymes for essential metabolic pathways. These mutants may more closely resemble virulent strains in terms of antigenic pro9le and expression of antigens than does BCG and hence, may have enhanced vaccine ef9cacy. Molecular biological techniques including transposon mutagenesis, illegitimate recombination and allelic exchange have now been developed to inactivate genes in M. bovis and screening techniques have been established to identify attenuated mutants.31 An attenuated vaccine for 9eld use would be required to have a deletion in two different genes, either of which caused it to be attenuated, in order to eliminate any possibility of back mutation to a virulent strain. It would be advantageous if an immunological screening test could be developed that would distinguish between vaccinated animals and those infected with M. bovis. If the new vaccine strain also had a gene or genes deleted, which induced DTH or another immunological reaction that could be tested, then an immunological test could be developed that would distinguish between vaccinated and infected animals. In preliminary studies, the esat-6 gene of a wild-type M. bovis strain has been deleted. Guineapigs inoculated with this mutant did not react in a skin test to ESAT-6 protein, but reacted strongly to bovine PPD. In contrast, animals inoculated with the wild-type M. bovis strain reacted strongly to both ESAT-6 and PPD.32 Auxotrophy or the inability to grow in minimal medium indicates that a strain has lost some metabolic function and this approach has been successfully used for several bacterial pathogens to develop attenuated strains with vaccine properties.33 As a 9rst step in determining the usefulness of this approach, several attenuated strains of M. bovis were developed by chemical mutagenesis of a liquid culture with nitrosoguanidine. Following the screening of strains for auxotrophy, the auxotrophs ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 129
were tested for virulence in guinea-pigs.34 Two of these auxotrophic M. bovis strains, which were shown to be attenuated in guinea-pigs, were tested for protection of cattle against bovine tuberculosis.13 Prior to vaccination, the calves in this trial had high IFN-c responses to avian PPD suggesting exposure to environmental mycobacteria. Vaccination with either of the two auxotrophic M. bovis strains resulted in signi9cantly fewer animals developing tuberculous lesions than in the animals from the BCGvaccinated and control groups. The reason BCG did not protect in this experiment may have been a consequence of previous exposure of the calves to environmental mycobacteria. The colony counts of live bacteria in the vaccines prepared from the auxotrophic strains were 1}2;106 CFU/dose, approximately 1 log10 higher than that in the BCG vaccine. It is unlikely that this contributed to the improvement in vaccine ef9cacy as doses of 104}106 CFU of BCG can induce similar levels of protection.15 Overall, it is encouraging that the newly-derived attenuated M. bovis strains appeared to perform better than BCG in this situation. Despite exhaustive efforts, the genetic alterations in these two auxotrophic strains could not be identi9ed. New attenuated M. bovis strains have been produced with de9ned gene deletions using molecular biological techniques. Vaccination with some of these strains has induced protection in guinea-pigs21 and possums (B.M. Buddle and G.W. de Lisle, unpublished observations) against virulent M. bovis which is at least equivalent to that induced by BCG. These vaccines have yet to be tested in cattle.
Killed species of mycobacteria Killed mycobacterial vaccines have perceived advantages in being safer to use than live attenuated M. bovis vaccines. However, traditional killed mycobacterial vaccines mixed in an oil adjuvant are not protective.11 Killed Mycobacterium vaccae has been advocated for the immunoprophylaxis of tuberculosis.35 The protective effects of M. vaccae are considered to arise from stimulation of cellular immune responses to common mycobacterial antigens and from switching off the tissue-necrotising aspects of the Koch phenomena. To assess vaccine ef9cacy of killed M. vaccae, a group of calves were vaccinated intradermally with 109 killed M. vaccae and subsequently challenged with M. bovis.19 The calves showed no protection against tuberculosis in comparison to calves vaccinated with BCG. A recent study in possums showed that a combination of killed M. vaccae and live BCG administered intranasally and intraconjunctivally induced protection against M. bovis which was greater than with BCG alone administered in a similar manner (M.A. Skinner, unpublished observations). This raises the possibility that killed M. vaccae may enhance the vaccine ef9cacy of BCG. ^ 2001 Harcourt Publishers Ltd
Mycobacterial protein vaccines An alternative approach focuses on the use of protective protein antigens secreted from live mycobacteria. Such sub-unit vaccines have the potential of not compromising diagnostic tests and their ef9cacy may not be affected by prior sensitization of animals to environmental mycobacteria. Culture 9ltrates prepared from M. tuberculosis have been shown to contain proteins that are highly stimulatory to T cells of human tuberculosis patients,36 mice23, 37 and cattle38 experimentally infected with tuberculosis. Several studies using small animal models have demonstrated the protective potential of antigens contained in culture 9ltrates. Immunization of mice and guinea-pigs with culture 9ltrate proteins (CFP) from M. tuberculosis gave high levels of protection against aerogenic challenge with M. tuberculosis.23, 39 Similarly, a CFP vaccine derived from M. bovis has been shown to induce signi9cant protection in mice against aerogenic challenge with virulent M. bovis.24 Vaccines prepared from culture 9ltrates of M. tuberculosis or M. bovis have recently been tested in cattle. Use of an adjuvant such as dimethyldeoctadecylammonium chloride (DDA) with M. tuberculosis CFP induced cellular immune responses to mycobacterial antigens in mice, but not in cattle. When a DEAE dextran adjuvant was used in cattle with the CFP, strong antigen-speci9c antibody and IL-2 responses were produced, while only a very weak IFN-c response was detected (D.N. Wedlock and B.M. Buddle, unpublished observations). The addition of the cytokine IL-2 to M. tuberculosis CFP vaccine in a mouse model has increased the level of protection against tuberculosis.39 In cattle, the incorporation of bovine IL-2 in a M. bovis CFP vaccine with lipid A adjuvant markedly enhanced antigen-speci9c antibody responses and induced weak antigen-speci9c IFN-c responses.25 This vaccine reduced the mean tuberculous lung lesion score in challenged cattle and induced no tuberculin skin-test reactivity, but these animals had a higher prevalence of extra-thoracic spread of disease than non-vaccinated and BCG-vaccinated animals. Possibly, the presence of antibodies in the CFP-vaccinated cattle may have served to opsonise the M. bovis, promoting the phagocytosis of the bacteria by monocytes. The infected cells could readily traf9c through the lymphatics, leading to the development of tuberculous granulomata in lymph nodes outside the thoracic cavity. In small animal models, a vaccination strategy comprising a combination of M. tuberculosis CFP vaccine plus IL-12 in the 9rst vaccination and CFP plus IL-2 in the second vaccination induced a signi9cant level of protection in the lungs following an aerosol challenge with M. tuberculosis.39 A similar protocol was recently tried in cattle using recombinant human IL-12 and bovine IL-2, Tuberculosis (2001) 81(1/2), 125I132
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but no protection was observed following challenge with M. bovis (D.N. Wedlock and B. Vesosky, unpublished observations). More detailed studies are now required to investigate the use of a wider range of doses of these cytokines to stimulate protective immunity. These studies highlight the dif9culty of stimulating strong antigen-speci9c IFN-c responses and protection against tuberculosis in cattle using mild adjuvants. Furthermore these same adjuvants have been used successfully to induce protection against tuberculosis in small animal models and illustrate how the effectiveness of adjuvants may vary markedly between different animal species. To date, none of the sub-unit vaccines have induced antigen-speci9c IFN-c responses in cattle comparable to those induced by attenuated M. bovis vaccines. Improvements might be made by formulation with better adjuvants and incorporating cytokines such as IL-12 or IL-18 at appropriate doses to promote antigen-speci9c IFN-c responses and protection against tuberculosis.
DNA vaccines Mycobacterial DNA vaccines have shown considerable promise in inducing protection against tuberculosis in small animal models.26, 39 Studies in mice have shown that tuberculosis DNA vaccines can stimulate IFN-c and cytotoxic T cell responses, elicit strong memory responses and protect when encoding a single protein or epitope. Protection can be enhanced when the DNA vaccine encodes for several proteins or epitopes40 and when adjuvant molecules such as DNA CpG motifs are incorporated in the vaccine.41 A recent study using a mouse model has shown that tuberculosis DNA vaccines can help eliminate existing infections.42 A disadvantage is that multiple inoculations appear necessary. In a recent cattle study, two of these vaccines induced CD4 T cell responses, but only weak IFN-c responses to bovine PPD and the vaccine antigens, as well as IgG1-biased humoral responses. Encouragingly, no tuberculin skin test reactivity was observed in vaccinated animals.43 Further studies are in progress to assess the protective ef9cacy of these vaccines.
DEVELOPMENT OF MORE EFFECTIVE HUMAN TUBERCULOSIS VACCINES Since M. tuberculosis and M. bovis are closely related species and the pathogenesis of tuberculosis in humans and cattle is similar, cattle could provide useful information on the development of more effective human tuberculosis vaccines. Both cattle and humans are most commonly infected via the respiratory route and the disease course is chronic with progression over a number of Tuberculosis (2001) 81(1/2), 125I132
years. The pathology in both species is characterized by a marked granulomatous response containing numerous giant cells and mineralisation with 9brous encapsulation of the lesion.14 There are also similarities in the immune response against tuberculosis including strong tuberculin skin test and IFN-c responses from PPD-stimulated blood cultures.17 Antibodies to M. bovis antigens are usually produced only in the late phase of the disease. Th-1 and Th-2 type immune responses are not mutually exclusive, as in mice. Information from cattle studies, which could have particular relevance to vaccination against human tuberculosis, includes adjuvant formulation, correlates of protective immunity and protection against tuberculosis under a variety of environmental conditions.
CONCLUSIONS The increase in our knowledge of mycobacterial genetics and our understanding of immune responses during the last decade suggest that the development of an effective vaccine for bovine tuberculosis is a realistic goal. Studies using relatively low doses of BCG in cattle indicate that this vaccine has potential to reduce economic losses in developing countries, although protection may only be partial. Trials should be carried out in these countries to determine whether a low dose BCG vaccination strategy could reduce the spread of bovine tuberculosis. Studies with newly attenuated strains of M. bovis have indicated that these vaccines could be better than BCG for protection against bovine tuberculosis when cattle have been previously exposed to environmental mycobacteria. Molecular biological techniques are now available to speci9cally delete genes from a M. bovis strain including deletion of two different genes, either of which caused it to be attenuated, to ensure that there is no chance of a vaccine strain reverting to virulence. Furthermore, it is possible to devise differential diagnostic tests to distinguish animals which have been vaccinated with attenuated M. bovis vaccine strains from those infected with M. bovis. New generation tuberculosis vaccines such as sub-unit protein vaccines and recombinant DNA vaccines are now being evaluated. One of the advantages of these types of vaccines is that they may not induce a tuberculin skin test response. They are, however, poorly immunogenic in cattle when administered alone and there is a lack of appropriate adjuvants to stimulate the IFN-c responses in cattle required for protective immunity. There is a need for improved immunological adjuvants that are potent, safe and compatible with new-generation vaccines. A better understanding of immune networks involved in the host response to tuberculosis should lead to further developments in novel vaccination strategies for bovine tuberculosis. The recent progress in the development of ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 131
tuberculosis vaccines has provided increasing optimism that vaccines will play an important role in the control and eventual eradication of bovine tuberculosis.
ACKNOWLEDGEMENTS The author thanks D. N. Wedlock, M. A. Skinner, G. W. de Lisle, D. M. Collins, M. A. Chambers, H. M. Vordermeier and B. Vesosky for helpful advice and for kindly providing unpublished data. The New Zealand Ministry of Agriculture and Forestry provided 9nancial assistance.
REFERENCES 1. O’Reilly L M, Daborn C J. The epidemiology of Mycobacterium bovis infection in animals and man: a review. Tubercle Lung Dis 1995; 76 (Suppl. 1): 1}46. 2. Cosivi O, Grange J M, Daborn C J et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998; 4: 59}70. 3. Calmette A, GueH rin C. Tuberculosis in man and animals. In: (A. Boquet, L. Ne` gre, eds.). L’infection bacillaire et la tuberculose, 4th edn, Masson et Cie, Paris 1936; pp 1024. 4. Haring C M, Traum J, Hayes F M, Henry BS. Vaccination of calves against tuberculosis with BCG. Hilgardia 1930; 4: 307}394. 5. Woodruff H A, Gregory T S. The prevention of tuberculosis in cattle. Further investigations to determine the value of the BCG vaccine for the prevention of tuberculosis. Journal of the Council for Scienti9c and Industrial Research 1929; 2: 1}14. 6. Watson E A. Bovine tuberculosis and BCG vaccination. Proceedings of the 12th International Veterinary Congress 1934; 2: 2}14. 7. Watson E A. La tuberculose, avec reH feH rence speH ciale a` la vaccination contre la tuberculose au moyen du BCG Bulletin Of9ce International Des ED pizootie 1935; 11: 718}720. 8. Doyle T M, Stuart P. Vaccination of cattle with BCG. Br Vet J 1958; 114: 310. 9. Berggren S A. Field experiment with BCG vaccine in Malawi. Br Vet J 1981; 137: 88}96. 10. Young J A, Paterson J S. Studies on the vaccination of cattle as a measure against infection with tuberculosis with the living vole acid-fast bacillus. J Hyg 1949; 4: 39}78. 11. Grif9n J F T, Mackintosh C G, Slobbe L, Thomson A J, Buchan G S. Vaccine protocols to optimise the protective ef9cacy of BCG. Tubercle Lung Dis 1999; 79: 135}143. 12. Fine P E, Vynnycky E. The effect of heterologous immunity upon the apparent ef9cacy of (eg. BCG) vaccines. Vaccine 1998: 16: 1923}1928. 13. Buddle B M, Aldwell F E, Wedlock D N. Vaccination of cattle and possums against bovine tuberculosis. In: F. Grif9n and G de Lisle, eds. Tuberculosis in Wildlife and Domestic Animals. Otago Conference Series no. 3, University of Otago Press, Dunedin, New Zealand 1995; 113}115. 14. Jubb K V F, Kennedy P C, Palmer N. Pathology of domestic animals Vol. 2. Academic Press, Orlando, Florida, 582 1985. 15. Buddle B M, de Lisle G W, Pfeffer A, Aldwell F E. Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine 1995; 13: 1123}1130. 16. Buddle B M, Aldwell F E, Pfeffer A, de Lisle G W, Corner L A. Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions. NZ Vet J 1994; 42: 167}172.
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17. Wedlock D N, Aldwell F E, Collins D M, de Lisle G W, Wilson T, Buddle B M. Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayedtype hypersensitivity with ability to grow in macrophages. Infect Immun 1999; 67: 2172}2177. 18. Collins F M. Protection against mycobacterial disease by means of live vaccines tested in experimental animals. In GP Kubica, LG Wayne (eds) The Mycobacteria: a Source book. Marcell Dekker, Inc., New York 1984; pp. 787}839. 19. Buddle B M, Keen D, Thomson A et al. Protection of cattle from bovine tuberculosis by vaccination with BCG by the respiratory or subcutaneous route, but not by vaccination with killed Mycobacterium vaccae. Res Vet Sci 1995; 59: 10}16. 20. Guleria I, Teitelbaum R, McAdam R A, Kalpana G, Jacobs W R Jr, Bloom B R. Auxotrophic vaccines for tuberculosis. Nature Medicine 1996; 2: 334}337. 21. de Lisle G W, Wilson T, Collins D M, Buddle B M. Vaccination of guinea pigs with nutritionally impaired avirulent mutants of Mycobacterium bovis protects against tuberculosis. Infect Immun 1999; 67: 2624}2626. 22. Abou-Zeid C, Gares M-P, Inwald J et al. Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae. Infect Immun 1997; 65: 1856}1862. 23. Andersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 1994; 62: 2536}2544. 24. Bosio C M, Orme I M. Effective, nonsensitizing vaccination with culture 9ltrate proteins against Mycobacterium bovis infections in mice. Infect Immun 1998; 66: 5048}5051. 25. Wedlock D N, Vesosky B, Skinner M A, de Lisle G W, Orme I M, Buddle B M. Vaccination of cattle with Mycobacterium bovis culture 9ltrate proteins and interleukin-2 against bovine tuberculosis. Infect Immun 2000; 68: 5809}5815. 26. Lowrie D B, Silva C L, Colston M J, Rango S, Tascon R E. Protection against tuberculosis by a plasmid DNA vaccine. Vaccine 1997; 15: 834}838. 27. Bretscher P A. A strategy to improve the ef9cacy of vaccination against tuberculosis and leprosy. Immunol Today 1992; 13: 342}345. 28. Buddle B M, Parlane N A, Keen D L et al. Differentiation between Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle by using recombinant mycobacterial antigens. Clin Diag Lab Immunol 1999; 6: 1}5. 29. Pollock J M, Girvin R M, Lightbody K A et al. Assessment of de9ned antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle. Vet Rec. 2000; 146: 659}665. 30. McAdam R A, Weisbrod T R, 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. 31. Collins D M. New tuberculosis vaccines based on attenuated strains of Mycobacterium tuberculosis complex. Immun Cell Biol 2000; 78: 342}348. 32. Wards B J, de Lisle G W, Collins D M. An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will be useful for development of a live tuberculosis vaccine. Tubercule Lung Dis 2000; 80: 185}189. 33. Hondalus M K, Bardarov S, Russell R, Chan J, Jacobs W R Jr, Bloom B R. Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000; 68: 2888}2898. 34. Wards B, Collins D, de Lisle G. Isolation and characterisation of auxotrophs of Mycobacterium bovis. In: F Grif9n, G W de Lisle eds.
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35.
36.
37.
38.
Tuberculosis in wildlife and domestic animals. Otago Conference Series No 3, Dunedin, New Zealand 1995; 7}10. Stanford J L, Rook G A, Bahr G M et al. Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis. Vaccine 1990; 8: 525}530. Demissie A, Pavn P, Olobo J et al. T-cell recognition of Mycobacterium tuberculosis culture 9ltrate fractions in tuberculosis patients and their household contacts. Infect Immun 1999; 67: 5867}5971. Roberts A D, Sonnenberg M G, Ordway D L et al. Characteristics of protective immunity engendered by vaccination of mice with puri9ed culture 9ltrate protein antigens of Mycobacterium tuberculosis. J Immunol 1995; 85: 502}508. Pollock J M, Andersen P. Predominant recognition of the ESAT-6 protein in the 9rst phase of infection with Mycobacterium bovis in cattle. Infect Immun 1997; 65: 2587}2592.
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39. Baldwin S L, D’Souza C, Roberts A D et al. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun 1998; 66: 2951}2959. 40. Kamath A T, Feng C G, MacDonald M, Briscoe H, Britton W J. Differential protective ef9cacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun 1999; 67: 1702}1707. 41. Kreig A M. CpG DNA: a novel immunomodulator. Trends Microbiol 1999; 7: 64}65. 42. Lowrie D B, Tascon R E, Bonato V L et al. Therapy of tuberculosis in mice by DNA vaccination. Nature 1999; 400: 269}271. 43. Vordermeier H M, Cockle P J, Whelan A O et al. Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the speci9city of the comparative intradermal skin test. Vaccine 2000; 19: 1246 }1255.
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Vaccination of cattle against Mycobacterium bovis B. M. Buddle AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand
Summary Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistence of Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a ‘test and slaughter’ control programme. Early field trials with Bacille Calmette Guerin (BCG) M. bovis vaccine in cattle produced disappointing results, with induction of tuberculin skin-test reactivity following vaccination and low levels of protection. However, recent studies using a low dose of BCG vaccine in cattle have produced more encouraging results and field trials should now be carried out in developing countries to determine whether this low dose BCG vaccination strategy will reduce the spread of infection. The options for new candidate tuberculosis vaccines have increased markedly in the last decade with the advent of new attenuated strains of M. bovis, and sub-unit protein and recombinant DNA vaccines. Some of these new types of vaccines have recently been tested in cattle. New attenuated M. bovis vaccines induced greater protection than BCG vaccine in cattle which had been sensitized to environmental mycobacteria prior to vaccination. In contrast, it has proved difficult to stimulate appropriate immune responses in cattle necessary for protection with sub-unit protein and recombinant DNA vaccines and better immunological adjuvants are required for these types of vaccines. Progress in the development of new tuberculosis vaccines has been very rapid in the past decade and the prospects for vaccination to control and eradicate bovine tuberculosis are encouraging. ^ 2001 Harcourt Publishers Ltd
INTRODUCTION Bovine tuberculosis caused by Mycobacterium bovis is a major economic problem in several countries and constitutes a public health risk in a number of developing countries. The implementation of national bovine tuberculosis programmes, based on regular tuberculin testing and removal of infected animals, had led to the successful eradication or a major reduction in the incidence of bovine tuberculosis in cattle herds. However, these control measures have been only partially effective in countries such as the UK, Ireland and New Zealand, which have a wildlife reservoir of infected animals.1 Furthermore, this approach to control bovine tuberculosis is economically and socially unacceptable in many developing countries, particularly in Africa. It is in these
Correspondence to: B.M. Buddle, AgResearch, Wallaceville Animal Research Centre, P.O. Box 40063, Upper Hutt, New Zealand; Tel.:#64 4 9221418; Fax:#64 4 9221380; E-mail: [email protected] Financial assistance from New Zealand Ministry of Agriculture and Forestry.
developing countries where M. bovis infection is most likely to contribute to the prevalence of human tuberculosis.2 In industrialized countries where there is persistence of M. bovis infection in wildlife, and in many developing countries, the use of a vaccine against bovine tuberculosis warrants serious consideration. Recent advances in immunology and the molecular biology of mycobacteria have greatly increased the options for candidate vaccines against bovine tuberculosis. The two major groupings are live attenuated vaccines such as Bacille Calmette Guerin (BCG) and other attenuated strains of M. bovis, and subunit vaccines based on either mycobacterial protein or DNA. Live attenuated vaccines are advantageous as they promote strong cellular immune responses required for protective immunity against tuberculosis and may only need a single-shot delivery to induce life-long immunity. Subunit vaccines typically require a number of booster immunizations to obtain good levels of immunity. However, there are advantages in using sub-unit vaccines in cattle including fewer regulatory requirements and the possibility of animals not reacting in a tuberculin skin test following vaccination. The aim of this review is to look at the history of vaccination
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against bovine tuberculosis in view of the current knowledge of immunology and to investigate the prospects for development of new improved vaccines for control of this disease in cattle.
REQUIREMENTS OF A TUBERCULOSIS VACCINE FOR CATTLE The goal of vaccinating cattle against tuberculosis is to prevent the establishment of infection. Ideally, if vaccinated animals were subsequently exposed to M. bovis they would resist infection and would not react in a tuberculin skin test. Many vaccines such as BCG can induce a tuberculin skin test response and hence it will be important to develop differential diagnostic tests to distinguish vaccinated animals from those infected with M. bovis. Alternatively, a vaccine could be developed which does not induce a tuberculin skin test response. The vaccine would need to be safe and its use would have to be acceptable to countries importing beef and dairy products. In developing countries, the goal of vaccinating cattle may be less stringent, with the principal requirement being to reduce the spread of bovine tuberculosis.
EARLY STUDIES ON VACCINATION AGAINST BOVINE TUBERCULOSIS Calmette and Guerin commenced the development of the BCG strain in 1906 by attenuation of a virulent strain originally isolated from a cow with tuberculous mastitis. They found that immunization with a single dose of between 50 and 100 mg BCG (approximately 109}1010 colony forming units, CFU) protected cattle from arti9cial and natural infections, but protection was not long-lived.3 Attempts to con9rm these 9ndings in 9eld trials in the 1920s gave variable results.4, 5 Intradermal or oral routes of BCG administration were shown to give no better protection than the subcutaneous route. Particularly disappointing were the results of 9eld trials6 and the observation that re-vaccination had no effect. After several decades of vaccination trials in Canada,7 USA,4 Australia,5 Great Britain8 and Africa9 it was concluded that, although BCG could protect cattle from experimental infections it was ineffective as a vaccine in the 9eld against bovine tuberculosis. In the 1940s, vaccination trials in calves were also undertaken with a vole bacillus M. microti. From a series of trials, Young and Paterson10 reported that calves vaccinated intravenously with 5 mg of viable M. microti developed a relatively high degree of immunity to M. bovis. Immunity was evident one year after vaccination, but declined thereafter. No further 9eld trials were carried out with this vaccine, probably because some strains of Tuberculosis (2001) 81(1/2), 125I132
M. microti were more virulent than BCG and could cause disease in calves, and there was no indication that immunity produced was markedly different to that produced by BCG. The apparent failure of BCG to protect cattle in 9eld trials may be attributed to a number of factors. High doses of BCG (equivalent to 108}1010 CFU) were generally used for subcutaneous or intradermal vaccination of cattle and it is now known that these doses are less effective in stimulating protective immunity against tuberculosis.11 Vaccination was often carried out in areas with a very high prevalence of bovine tuberculosis9 and calves may have been exposed to M. bovis prior to vaccination through the consumption of milk from cows with tuberculous mastitis. In human vaccination trials, BCG has been less effective when used in the tropics where there is greater exposure to environmental mycobacteria. There is evidence in humans and cattle that prior exposure to environmental mycobacteria may lower the ef9cacy of BCG vaccine12,13 and this type of exposure needs to be considered in the evaluation of cattle trials.
DEVELOPMENT OF A CHALLENGE MODEL TO ASSESS VACCINE EFFICACY It is cost effective to undertake preliminary screening of bovine tuberculosis vaccines in small animal models such as mice and guinea-pigs with only the most promising vaccine candidates progressing for evaluation in cattle. Evaluation of vaccine ef9cacy in cattle should be carried out initially by experimentally challenging the vaccinated animals with virulent M. bovis. Field trials should be used only at the 9nal testing stage as they are expensive to run, with large group sizes needed due to the low prevalence or patchy distribution of disease and its slow progression. A requirement in a challenge model is that the lesions produced should mimic those seen in the natural disease. In cattle, lesions are predominantly found in the lungs or lymph nodes in the thorax.14 To mimic the type of lesion seen in the natural disease, the challenge dose needs to be kept as low as possible. Host factors including the condition of the animals and sensitization to environmental mycobacteria may in:uence how animals respond to vaccination. A low-dose respiratory challenge model has been established in cattle for testing vaccine ef9cacy using an intratracheal challenge of 103 CFU of virulent M. bovis.15 When the animals are killed and examined 4}5 months after challenge, tuberculous lesions are almost entirely restricted to the thoracic cavity with lesions in the lungs and thoracic lymph nodes. The lung lesions consist of small nodules 3}5 mm in diameter and the lymph node lesions vary in size from 2}30 mm in diameter. These types of lesions are typical of those seen in the natural disease. ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 127
Cattle challenged with higher doses (105}106 CFU) of M. bovis develop disease with multiple lymph node involvement and large lung lesions, which is atypical of that generally seen in the natural disease.16, 17
TYPES OF TUBERCULOSIS VACCINES Over recent years a number of different types of tuberculosis vaccines have shown promise in small animal models and have already or are about to be tested in cattle (Table 1). The bene9t of having a range of different types of vaccines is that they could be useful for different purposes.
BCG The existing BCG vaccine may not be suf9ciently effective, but has many properties that are desirable for a good vaccine. It is cheap to produce, can be administered via a number of routes, is safe, relatively stable and is derived from M. bovis. A drawback of using BCG is that vaccinated cattle may react positively in a tuberculin skin test and on this basis, would be assumed to have bovine tuberculosis and would be slaughtered. Studies with BCG in cattle can be helpful in gaining an understanding of the mechanisms associated with protection against bovine tuberculosis which should lead to the rational design of new improved tuberculosis vaccines. In addition, studies with BCG assist in determining the optimal conditions for use of improved attenuated M. bovis vaccines and serve as a benchmark for comparison with other tuberculosis vaccines. Recent information suggests that lower doses of BCG may preferentially stimulate cellular immunity to mycobacterial antigens and may have minimal effect, in the
Table 1 Comparison of different types of vaccines for protection against experimental challenge with M. bovis or M. tuberculosis Protection against infectiona
Type of vaccine BCG Modification of BCG Attenuated M. bovis strain Killed M. vaccae Protein vaccine DNA vaccine
Small animal model Cattle
Tuberculin skin test reactivityb
#18 #20
#15,19 NT
Yes No
#21 $22 #23,24 #26
#13 !19 $25 NT
Yes No No No
a : Protection defined as a significant reduction in bacterial counts or in size and distribution of lesions. b: Reactivity to tuberculin induced by vaccine. #: Protection (reference in superscript). ! : No protection.
: Variable protection compared to BCG. NT: Not tested.
$
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long term, on skin testing.27 To determine whether low doses of BCG could protect cattle against bovine tuberculosis, three vaccination/challenge trials were undertaken using doses ranging from 104}106 CFU of BCG. In the 9rst trial, groups of calves were vaccinated subcutaneously with 104 CFU (low dose) or 106 CFU (medium dose) of BCG Pasteur.15 Eight weeks after vaccination, 15 animals from each group, together with 16 non-vaccinated calves were challenged intratracheally with virulent M. bovis. All animals were killed and necropsied 22 weeks after challenge. The proportion of animals with tuberculous lesions in the low dose BCG, medium dose BCG and non-vaccinated groups were 2/15, 4/15 and 10/16, respectively. BCG induced a signi9cant level of protection against development of tuberculous lesions compared to the non-vaccinated controls. In a second trial, groups of calves were vaccinated with low to medium doses of BCG by the subcutaneous or intratracheal route and challenged as in the 9rst trial.19 Vaccination with BCG again resulted in fewer animals developing lesions and a reduction in the number of lesions in the diseased animals compared to the nonvaccinated group. An advantage of vaccinating by the respiratory route was that non-challenged animals from the group produced a minimal reaction in the tuberculin skin test in contrast to the corresponding animals from the subcutaneous vaccinated group. In the third trial, a year later, the calves were of the same age and were sourced from the same farm as in the previous two trials, but subcutaneous vaccination with a low dose of BCG (105 CFU) induced no protection.13 Prior to being vaccinated, the calves had high IFN-c responses to avian PPD, indicative of exposure to environmental mycobacteria. Preexposure to environmental mycobacteria has been put forward as an explanation of the failure for BCG to induce protection in many human tuberculosis trials and may be a reason for the failure of BCG to protect the cattle in this trial. A feature of the immunological pro9le of BCG-vaccinated cattle is a release of high levels of IFN-c and IL-2 from bovine PPD-stimulated whole blood cultures peaking at 2}4 weeks after vaccination.15, 19 In the trial where BCG did not induce protection, there was a delay in the IFN-c response to bovine PPD with no increase in the response by week 2 following vaccination and a only small increase in the response by week 4 (Fig. 1). One can speculate, that in animals which have been exposed to environmental mycobacteria, BCG multiplication in the animals following vaccination was suppressed whereas there was no suppression in non-sensitized animals. One problem with BCG vaccine is that vaccination may induce tuberculin skin-test reactivity even when low doses of BCG are used. A number of mycobacterial antigens have recently been evaluated in a whole blood IFN-c Tuberculosis (2001) 81(1/2), 125I132
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supplemented with the appropriate amino acid. Their ability to grow in vivo is also reduced. Vaccination of mice with these auxotrophic mutants has induced protection against M. tuberculosis infection.20 Vaccination of cattle with a leucine auxotrophic mutant of BCG did not induce a skin-test response to bovine PPD (H.M. Vordermeier, personal communication), however, protection against bovine tuberculosis has yet to be evaluated.
Attenuation of virulent strains of M. bovis
Fig. 1 Interferon-c released from bovine PPD-stimulated peripheral blood lymphocytes of cattle vaccinated with BCG. Cattle were vaccinated with 106 (䉱), 105 (䉬) or 104 (䊉) CFU of BCG or were non-vaccinated (*). A and B represent two vaccination trials carried in different years. BCG vaccination induced a significant level of protection against challenge with virulent M. bovis in trial A, while no protection was observed in trial B. Data are expressed as mean optical density units (;1000).
test for differentiating BCG-vaccinated cattle from those infected with bovine tuberculosis.28 The protein, ESAT-6 which is produced by virulent M. bovis strains, but not by BCG has been shown to be a very useful reagent for the speci9c diagnosis of bovine tuberculosis,29 and was a suitable antigen for differentiating between BCG-vaccinated and M. bovis-infected cattle.
Modified BCG BCG has been modi9ed by inactivation or alteration of some of its own genes in an attempt to reduce tuberculin skin-test responses and to make a safer vaccine for immunocompromised individuals. Auxotrophic mutants of BCG have been produced which have mutations in the genes involved in the metabolism of leucine and methionine.30 These mutants can no longer grow in minimal medium and can only grow when this medium is Tuberculosis (2001) 81(1/2), 125I132
BCG was produced in an empirical manner and recent genetic analysis has indicated that BCG contains a number of gene deletions compared with virulent strains of M. bovis or M. tuberculosis. It should be possible to improve BCG by deleting from virulent strains of M. bovis or M. tuberculosis, speci9c genes, which are involved in virulence or encode enzymes for essential metabolic pathways. These mutants may more closely resemble virulent strains in terms of antigenic pro9le and expression of antigens than does BCG and hence, may have enhanced vaccine ef9cacy. Molecular biological techniques including transposon mutagenesis, illegitimate recombination and allelic exchange have now been developed to inactivate genes in M. bovis and screening techniques have been established to identify attenuated mutants.31 An attenuated vaccine for 9eld use would be required to have a deletion in two different genes, either of which caused it to be attenuated, in order to eliminate any possibility of back mutation to a virulent strain. It would be advantageous if an immunological screening test could be developed that would distinguish between vaccinated animals and those infected with M. bovis. If the new vaccine strain also had a gene or genes deleted, which induced DTH or another immunological reaction that could be tested, then an immunological test could be developed that would distinguish between vaccinated and infected animals. In preliminary studies, the esat-6 gene of a wild-type M. bovis strain has been deleted. Guineapigs inoculated with this mutant did not react in a skin test to ESAT-6 protein, but reacted strongly to bovine PPD. In contrast, animals inoculated with the wild-type M. bovis strain reacted strongly to both ESAT-6 and PPD.32 Auxotrophy or the inability to grow in minimal medium indicates that a strain has lost some metabolic function and this approach has been successfully used for several bacterial pathogens to develop attenuated strains with vaccine properties.33 As a 9rst step in determining the usefulness of this approach, several attenuated strains of M. bovis were developed by chemical mutagenesis of a liquid culture with nitrosoguanidine. Following the screening of strains for auxotrophy, the auxotrophs ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 129
were tested for virulence in guinea-pigs.34 Two of these auxotrophic M. bovis strains, which were shown to be attenuated in guinea-pigs, were tested for protection of cattle against bovine tuberculosis.13 Prior to vaccination, the calves in this trial had high IFN-c responses to avian PPD suggesting exposure to environmental mycobacteria. Vaccination with either of the two auxotrophic M. bovis strains resulted in signi9cantly fewer animals developing tuberculous lesions than in the animals from the BCGvaccinated and control groups. The reason BCG did not protect in this experiment may have been a consequence of previous exposure of the calves to environmental mycobacteria. The colony counts of live bacteria in the vaccines prepared from the auxotrophic strains were 1}2;106 CFU/dose, approximately 1 log10 higher than that in the BCG vaccine. It is unlikely that this contributed to the improvement in vaccine ef9cacy as doses of 104}106 CFU of BCG can induce similar levels of protection.15 Overall, it is encouraging that the newly-derived attenuated M. bovis strains appeared to perform better than BCG in this situation. Despite exhaustive efforts, the genetic alterations in these two auxotrophic strains could not be identi9ed. New attenuated M. bovis strains have been produced with de9ned gene deletions using molecular biological techniques. Vaccination with some of these strains has induced protection in guinea-pigs21 and possums (B.M. Buddle and G.W. de Lisle, unpublished observations) against virulent M. bovis which is at least equivalent to that induced by BCG. These vaccines have yet to be tested in cattle.
Killed species of mycobacteria Killed mycobacterial vaccines have perceived advantages in being safer to use than live attenuated M. bovis vaccines. However, traditional killed mycobacterial vaccines mixed in an oil adjuvant are not protective.11 Killed Mycobacterium vaccae has been advocated for the immunoprophylaxis of tuberculosis.35 The protective effects of M. vaccae are considered to arise from stimulation of cellular immune responses to common mycobacterial antigens and from switching off the tissue-necrotising aspects of the Koch phenomena. To assess vaccine ef9cacy of killed M. vaccae, a group of calves were vaccinated intradermally with 109 killed M. vaccae and subsequently challenged with M. bovis.19 The calves showed no protection against tuberculosis in comparison to calves vaccinated with BCG. A recent study in possums showed that a combination of killed M. vaccae and live BCG administered intranasally and intraconjunctivally induced protection against M. bovis which was greater than with BCG alone administered in a similar manner (M.A. Skinner, unpublished observations). This raises the possibility that killed M. vaccae may enhance the vaccine ef9cacy of BCG. ^ 2001 Harcourt Publishers Ltd
Mycobacterial protein vaccines An alternative approach focuses on the use of protective protein antigens secreted from live mycobacteria. Such sub-unit vaccines have the potential of not compromising diagnostic tests and their ef9cacy may not be affected by prior sensitization of animals to environmental mycobacteria. Culture 9ltrates prepared from M. tuberculosis have been shown to contain proteins that are highly stimulatory to T cells of human tuberculosis patients,36 mice23, 37 and cattle38 experimentally infected with tuberculosis. Several studies using small animal models have demonstrated the protective potential of antigens contained in culture 9ltrates. Immunization of mice and guinea-pigs with culture 9ltrate proteins (CFP) from M. tuberculosis gave high levels of protection against aerogenic challenge with M. tuberculosis.23, 39 Similarly, a CFP vaccine derived from M. bovis has been shown to induce signi9cant protection in mice against aerogenic challenge with virulent M. bovis.24 Vaccines prepared from culture 9ltrates of M. tuberculosis or M. bovis have recently been tested in cattle. Use of an adjuvant such as dimethyldeoctadecylammonium chloride (DDA) with M. tuberculosis CFP induced cellular immune responses to mycobacterial antigens in mice, but not in cattle. When a DEAE dextran adjuvant was used in cattle with the CFP, strong antigen-speci9c antibody and IL-2 responses were produced, while only a very weak IFN-c response was detected (D.N. Wedlock and B.M. Buddle, unpublished observations). The addition of the cytokine IL-2 to M. tuberculosis CFP vaccine in a mouse model has increased the level of protection against tuberculosis.39 In cattle, the incorporation of bovine IL-2 in a M. bovis CFP vaccine with lipid A adjuvant markedly enhanced antigen-speci9c antibody responses and induced weak antigen-speci9c IFN-c responses.25 This vaccine reduced the mean tuberculous lung lesion score in challenged cattle and induced no tuberculin skin-test reactivity, but these animals had a higher prevalence of extra-thoracic spread of disease than non-vaccinated and BCG-vaccinated animals. Possibly, the presence of antibodies in the CFP-vaccinated cattle may have served to opsonise the M. bovis, promoting the phagocytosis of the bacteria by monocytes. The infected cells could readily traf9c through the lymphatics, leading to the development of tuberculous granulomata in lymph nodes outside the thoracic cavity. In small animal models, a vaccination strategy comprising a combination of M. tuberculosis CFP vaccine plus IL-12 in the 9rst vaccination and CFP plus IL-2 in the second vaccination induced a signi9cant level of protection in the lungs following an aerosol challenge with M. tuberculosis.39 A similar protocol was recently tried in cattle using recombinant human IL-12 and bovine IL-2, Tuberculosis (2001) 81(1/2), 125I132
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but no protection was observed following challenge with M. bovis (D.N. Wedlock and B. Vesosky, unpublished observations). More detailed studies are now required to investigate the use of a wider range of doses of these cytokines to stimulate protective immunity. These studies highlight the dif9culty of stimulating strong antigen-speci9c IFN-c responses and protection against tuberculosis in cattle using mild adjuvants. Furthermore these same adjuvants have been used successfully to induce protection against tuberculosis in small animal models and illustrate how the effectiveness of adjuvants may vary markedly between different animal species. To date, none of the sub-unit vaccines have induced antigen-speci9c IFN-c responses in cattle comparable to those induced by attenuated M. bovis vaccines. Improvements might be made by formulation with better adjuvants and incorporating cytokines such as IL-12 or IL-18 at appropriate doses to promote antigen-speci9c IFN-c responses and protection against tuberculosis.
DNA vaccines Mycobacterial DNA vaccines have shown considerable promise in inducing protection against tuberculosis in small animal models.26, 39 Studies in mice have shown that tuberculosis DNA vaccines can stimulate IFN-c and cytotoxic T cell responses, elicit strong memory responses and protect when encoding a single protein or epitope. Protection can be enhanced when the DNA vaccine encodes for several proteins or epitopes40 and when adjuvant molecules such as DNA CpG motifs are incorporated in the vaccine.41 A recent study using a mouse model has shown that tuberculosis DNA vaccines can help eliminate existing infections.42 A disadvantage is that multiple inoculations appear necessary. In a recent cattle study, two of these vaccines induced CD4 T cell responses, but only weak IFN-c responses to bovine PPD and the vaccine antigens, as well as IgG1-biased humoral responses. Encouragingly, no tuberculin skin test reactivity was observed in vaccinated animals.43 Further studies are in progress to assess the protective ef9cacy of these vaccines.
DEVELOPMENT OF MORE EFFECTIVE HUMAN TUBERCULOSIS VACCINES Since M. tuberculosis and M. bovis are closely related species and the pathogenesis of tuberculosis in humans and cattle is similar, cattle could provide useful information on the development of more effective human tuberculosis vaccines. Both cattle and humans are most commonly infected via the respiratory route and the disease course is chronic with progression over a number of Tuberculosis (2001) 81(1/2), 125I132
years. The pathology in both species is characterized by a marked granulomatous response containing numerous giant cells and mineralisation with 9brous encapsulation of the lesion.14 There are also similarities in the immune response against tuberculosis including strong tuberculin skin test and IFN-c responses from PPD-stimulated blood cultures.17 Antibodies to M. bovis antigens are usually produced only in the late phase of the disease. Th-1 and Th-2 type immune responses are not mutually exclusive, as in mice. Information from cattle studies, which could have particular relevance to vaccination against human tuberculosis, includes adjuvant formulation, correlates of protective immunity and protection against tuberculosis under a variety of environmental conditions.
CONCLUSIONS The increase in our knowledge of mycobacterial genetics and our understanding of immune responses during the last decade suggest that the development of an effective vaccine for bovine tuberculosis is a realistic goal. Studies using relatively low doses of BCG in cattle indicate that this vaccine has potential to reduce economic losses in developing countries, although protection may only be partial. Trials should be carried out in these countries to determine whether a low dose BCG vaccination strategy could reduce the spread of bovine tuberculosis. Studies with newly attenuated strains of M. bovis have indicated that these vaccines could be better than BCG for protection against bovine tuberculosis when cattle have been previously exposed to environmental mycobacteria. Molecular biological techniques are now available to speci9cally delete genes from a M. bovis strain including deletion of two different genes, either of which caused it to be attenuated, to ensure that there is no chance of a vaccine strain reverting to virulence. Furthermore, it is possible to devise differential diagnostic tests to distinguish animals which have been vaccinated with attenuated M. bovis vaccine strains from those infected with M. bovis. New generation tuberculosis vaccines such as sub-unit protein vaccines and recombinant DNA vaccines are now being evaluated. One of the advantages of these types of vaccines is that they may not induce a tuberculin skin test response. They are, however, poorly immunogenic in cattle when administered alone and there is a lack of appropriate adjuvants to stimulate the IFN-c responses in cattle required for protective immunity. There is a need for improved immunological adjuvants that are potent, safe and compatible with new-generation vaccines. A better understanding of immune networks involved in the host response to tuberculosis should lead to further developments in novel vaccination strategies for bovine tuberculosis. The recent progress in the development of ^ 2001 Harcourt Publishers Ltd
Vaccination of cattle against Mycobacterium bovis 131
tuberculosis vaccines has provided increasing optimism that vaccines will play an important role in the control and eventual eradication of bovine tuberculosis.
ACKNOWLEDGEMENTS The author thanks D. N. Wedlock, M. A. Skinner, G. W. de Lisle, D. M. Collins, M. A. Chambers, H. M. Vordermeier and B. Vesosky for helpful advice and for kindly providing unpublished data. The New Zealand Ministry of Agriculture and Forestry provided 9nancial assistance.
REFERENCES 1. O’Reilly L M, Daborn C J. The epidemiology of Mycobacterium bovis infection in animals and man: a review. Tubercle Lung Dis 1995; 76 (Suppl. 1): 1}46. 2. Cosivi O, Grange J M, Daborn C J et al. Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 1998; 4: 59}70. 3. Calmette A, GueH rin C. Tuberculosis in man and animals. In: (A. Boquet, L. Ne` gre, eds.). L’infection bacillaire et la tuberculose, 4th edn, Masson et Cie, Paris 1936; pp 1024. 4. Haring C M, Traum J, Hayes F M, Henry BS. Vaccination of calves against tuberculosis with BCG. Hilgardia 1930; 4: 307}394. 5. Woodruff H A, Gregory T S. The prevention of tuberculosis in cattle. Further investigations to determine the value of the BCG vaccine for the prevention of tuberculosis. Journal of the Council for Scienti9c and Industrial Research 1929; 2: 1}14. 6. Watson E A. Bovine tuberculosis and BCG vaccination. Proceedings of the 12th International Veterinary Congress 1934; 2: 2}14. 7. Watson E A. La tuberculose, avec reH feH rence speH ciale a` la vaccination contre la tuberculose au moyen du BCG Bulletin Of9ce International Des ED pizootie 1935; 11: 718}720. 8. Doyle T M, Stuart P. Vaccination of cattle with BCG. Br Vet J 1958; 114: 310. 9. Berggren S A. Field experiment with BCG vaccine in Malawi. Br Vet J 1981; 137: 88}96. 10. Young J A, Paterson J S. Studies on the vaccination of cattle as a measure against infection with tuberculosis with the living vole acid-fast bacillus. J Hyg 1949; 4: 39}78. 11. Grif9n J F T, Mackintosh C G, Slobbe L, Thomson A J, Buchan G S. Vaccine protocols to optimise the protective ef9cacy of BCG. Tubercle Lung Dis 1999; 79: 135}143. 12. Fine P E, Vynnycky E. The effect of heterologous immunity upon the apparent ef9cacy of (eg. BCG) vaccines. Vaccine 1998: 16: 1923}1928. 13. Buddle B M, Aldwell F E, Wedlock D N. Vaccination of cattle and possums against bovine tuberculosis. In: F. Grif9n and G de Lisle, eds. Tuberculosis in Wildlife and Domestic Animals. Otago Conference Series no. 3, University of Otago Press, Dunedin, New Zealand 1995; 113}115. 14. Jubb K V F, Kennedy P C, Palmer N. Pathology of domestic animals Vol. 2. Academic Press, Orlando, Florida, 582 1985. 15. Buddle B M, de Lisle G W, Pfeffer A, Aldwell F E. Immunological responses and protection against Mycobacterium bovis in calves vaccinated with a low dose of BCG. Vaccine 1995; 13: 1123}1130. 16. Buddle B M, Aldwell F E, Pfeffer A, de Lisle G W, Corner L A. Experimental Mycobacterium bovis infection of cattle: effect of dose of M. bovis and pregnancy on immune responses and distribution of lesions. NZ Vet J 1994; 42: 167}172.
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17. Wedlock D N, Aldwell F E, Collins D M, de Lisle G W, Wilson T, Buddle B M. Immune responses induced in cattle by virulent and attenuated Mycobacterium bovis strains: correlation of delayedtype hypersensitivity with ability to grow in macrophages. Infect Immun 1999; 67: 2172}2177. 18. Collins F M. Protection against mycobacterial disease by means of live vaccines tested in experimental animals. In GP Kubica, LG Wayne (eds) The Mycobacteria: a Source book. Marcell Dekker, Inc., New York 1984; pp. 787}839. 19. Buddle B M, Keen D, Thomson A et al. Protection of cattle from bovine tuberculosis by vaccination with BCG by the respiratory or subcutaneous route, but not by vaccination with killed Mycobacterium vaccae. Res Vet Sci 1995; 59: 10}16. 20. Guleria I, Teitelbaum R, McAdam R A, Kalpana G, Jacobs W R Jr, Bloom B R. Auxotrophic vaccines for tuberculosis. Nature Medicine 1996; 2: 334}337. 21. de Lisle G W, Wilson T, Collins D M, Buddle B M. Vaccination of guinea pigs with nutritionally impaired avirulent mutants of Mycobacterium bovis protects against tuberculosis. Infect Immun 1999; 67: 2624}2626. 22. Abou-Zeid C, Gares M-P, Inwald J et al. Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae. Infect Immun 1997; 65: 1856}1862. 23. Andersen P. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 1994; 62: 2536}2544. 24. Bosio C M, Orme I M. Effective, nonsensitizing vaccination with culture 9ltrate proteins against Mycobacterium bovis infections in mice. Infect Immun 1998; 66: 5048}5051. 25. Wedlock D N, Vesosky B, Skinner M A, de Lisle G W, Orme I M, Buddle B M. Vaccination of cattle with Mycobacterium bovis culture 9ltrate proteins and interleukin-2 against bovine tuberculosis. Infect Immun 2000; 68: 5809}5815. 26. Lowrie D B, Silva C L, Colston M J, Rango S, Tascon R E. Protection against tuberculosis by a plasmid DNA vaccine. Vaccine 1997; 15: 834}838. 27. Bretscher P A. A strategy to improve the ef9cacy of vaccination against tuberculosis and leprosy. Immunol Today 1992; 13: 342}345. 28. Buddle B M, Parlane N A, Keen D L et al. Differentiation between Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle by using recombinant mycobacterial antigens. Clin Diag Lab Immunol 1999; 6: 1}5. 29. Pollock J M, Girvin R M, Lightbody K A et al. Assessment of de9ned antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle. Vet Rec. 2000; 146: 659}665. 30. McAdam R A, Weisbrod T R, 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. 31. Collins D M. New tuberculosis vaccines based on attenuated strains of Mycobacterium tuberculosis complex. Immun Cell Biol 2000; 78: 342}348. 32. Wards B J, de Lisle G W, Collins D M. An esat6 knockout mutant of Mycobacterium bovis produced by homologous recombination will be useful for development of a live tuberculosis vaccine. Tubercule Lung Dis 2000; 80: 185}189. 33. Hondalus M K, Bardarov S, Russell R, Chan J, Jacobs W R Jr, Bloom B R. Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000; 68: 2888}2898. 34. Wards B, Collins D, de Lisle G. Isolation and characterisation of auxotrophs of Mycobacterium bovis. In: F Grif9n, G W de Lisle eds.
Tuberculosis (2001) 81(1/2), 125I132
132 Buddle
35.
36.
37.
38.
Tuberculosis in wildlife and domestic animals. Otago Conference Series No 3, Dunedin, New Zealand 1995; 7}10. Stanford J L, Rook G A, Bahr G M et al. Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis. Vaccine 1990; 8: 525}530. Demissie A, Pavn P, Olobo J et al. T-cell recognition of Mycobacterium tuberculosis culture 9ltrate fractions in tuberculosis patients and their household contacts. Infect Immun 1999; 67: 5867}5971. Roberts A D, Sonnenberg M G, Ordway D L et al. Characteristics of protective immunity engendered by vaccination of mice with puri9ed culture 9ltrate protein antigens of Mycobacterium tuberculosis. J Immunol 1995; 85: 502}508. Pollock J M, Andersen P. Predominant recognition of the ESAT-6 protein in the 9rst phase of infection with Mycobacterium bovis in cattle. Infect Immun 1997; 65: 2587}2592.
Tuberculosis (2001) 81(1/2), 125I132
39. Baldwin S L, D’Souza C, Roberts A D et al. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun 1998; 66: 2951}2959. 40. Kamath A T, Feng C G, MacDonald M, Briscoe H, Britton W J. Differential protective ef9cacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect Immun 1999; 67: 1702}1707. 41. Kreig A M. CpG DNA: a novel immunomodulator. Trends Microbiol 1999; 7: 64}65. 42. Lowrie D B, Tascon R E, Bonato V L et al. Therapy of tuberculosis in mice by DNA vaccination. Nature 1999; 400: 269}271. 43. Vordermeier H M, Cockle P J, Whelan A O et al. Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the speci9city of the comparative intradermal skin test. Vaccine 2000; 19: 1246 }1255.
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