COUNTERMEASURES TO PREVENT AND CONTROL TUBERCULOSIS IN CATTLE AND WILDLIFE RESERVOIRS
Location: Infectious Bacterial Diseases Research Unit
Title: Bovine Tuberculosis Vaccine Efficacy Studies: Neonatal Calves and White-tailed Deer
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 30, 2010
Publication Date: October 30, 2010
Citation: Waters, W.R., Palmer, M.V., Thacker, T.C. 2010. Bovine Tuberculosis Vaccine Efficacy Studies: Neonatal Calves and White-tailed Deer [abstract]. American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology. p. 89.
Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti)(#1). Mycobacterium bovis is the species most often isolated from tuberculous cattle. Unlike M. tuberculosis, M. bovis has a wide host range, including several wildlife maintenance hosts (#2). While the mainstay of bovine TB control has been abattoir inspection plus targeted test / cull campaigns, vaccines are now being considered as an additional tool for control, both in cattle and wildlife (#3-4). The first known tuberculosis (TB) vaccine purposefully administered to cattle was M. bovis bacillus Calmette Guerin (BCG), circa 1911 (#5). The efficacy of BCG varies under both experimental and field conditions (#6); thus, other vaccine approaches have been evaluated (#7). An attenuated live vaccine with more consistent efficacy and/or improved safety as compared to BCG should prove invaluable as a bovine TB vaccine, either alone or in combination with subunit vaccines (#8-10).
Within the United States (i.e., northeast Michigan), free-ranging white-tailed deer are a wildlife reservoir of M. bovis (#11-14), presumably transmitting the disease to at least 44 cattle herds on 39 different farms. Control efforts such as limiting wildlife baiting / feeding practices and increased deer hunting have resulted in a reduced apparent prevalence of TB in deer (#15). However, there is increasing public resentment of ongoing control measures and targeted vaccination is an appealing option, particularly as a means to limit transmission from deer to cattle.
Our long-term goal is to develop a live attenuated DIVA (differentiate infected from vaccinated animals) vaccine with improved efficacy and safety as compared to BCG for use in cattle and associated wildlife hosts. Currently, ESAT-6 and CFP-10 are leading candidates for use as antigens in modern bovine TB diagnostic assays. ESAT-6 and CFP-10 are encoded in the RD1 region of the genomes of tuberculous mycobacteria and deletion of RD1 is the primary attenuating defect of BCG (#16). Thus, a Delta RD1 vaccine is a logical selection for evaluation. Our approach is two-fold: (A) characterize the safety and efficacy of BCG in white-tailed deer to expedite field application as this vaccine has a long track record of use in many species and (B) evaluate new generation TB vaccines (i.e., Delta RD1 mutants) in calves, as a model for use in humans and potentially for use in calves or other wildlife hosts of M. bovis.
Aerosol Challenge Model of Infection in neonatal calves
With our protocol, vaccines are administered to calves at ~2 wks of age and virulent M. bovis delivered via aerosol at ~3.5 months of age 17-19. The challenge inoculum is delivered to restrained calves by nebulization of ~10**3 cfu M. bovis strain 95-1315 in PBS into a mask (Trudell Medical International, London, ON, Canada) covering the nostrils and mouth. Upon inspiration, inoculum is inhaled through a one-way valve into the mask and directly into the lungs via the nostrils. The process continues until the inoculum, a 1 ml PBS wash of the inoculum tube, and an additional 2 ml PBS are delivered, a process taking ~10 min. Strict BL-3 safety protocols are followed to protect personnel from exposure to M. bovis. Variables used to evaluate vaccine efficacy include: gross pathology, radiograph morphometry, histopathology, mycobacterial culture (quantitative and qualitative), and various immune parameters (#18-19). An initial study was performed to compare the efficacy of BCG (originally attenuated circa 1913 by continued passage of a Nocard strain of M. bovis on potato slices / ox bile / glycerin media) to M. bovis Ravenel Delta RD1 (#18). Key findings from this calf vaccine trial were:
(A) vaccination of neonatal calves with Delta RD1 provided equivalent efficacy as did BCG against aerosol challenge with virulent M. bovis,
(B) mean central memory responses elicited by either Delta RD1 or BCG prior to challenge correlated with reduced pathology and bacterial colonization,
(C) vaccination with either Delta RD1 or BCG did not interfere with an IFN-gamma-based TB assay using ESAT-6 and CFP-10 antigens, and
(D) reduced in vitro recall responses after challenge were associated with effective Delta RD1 and BCG vaccines.
Thus, the Delta RD1 vaccine strain may prove useful as an alternative to M. bovis BCG for bovine TB vaccine programs, particularly if additional mutations are included for improved safety and immunogenicity. For future work, the inclusion of mutations to improve host immunity such as pro-apoptotic (#20) or IL-12 suppressing mutations 21 will be evaluated as a means to enhance the usefulness of this vaccine; thus, engendering a vaccine to limit colonization more dramatically than those observed for BCG and Delta RD1.
BCG Vaccination of White-tailed Deer: Efficacy, Persistence, Transmission and Safety
For efficacy studies, white-tailed deer are vaccinated at ~1 yr of age with BCG and virulent M. bovis (~300 – 500 cfu) delivered via direct instillation into the tonsilar crypts approximately 12 – 17 wks after vaccination. Intratonsilar inoculation of M. bovis results in lesion distribution similar to that detected with natural infection of white-tailed deer 22. The dose of M. bovis required to consistently produce lesions without overwhelming disease is much less for deer as compared to cattle. Vaccination with BCG by either subcutaneous (10**6 – 10**7 cfu) or oral (10**9 cfu) routes results in reduced M. bovis–associated pathology as compared to non-vaccinates. For subcutaneous vaccinated deer, lesion severity is reduced for both Danish and Pasteur strains of BCG as compared to non-vaccinates; however, superior protection against advanced lesion formation is seen in BCG Danish vaccinates (#23).
Prolonged BCG persistence, dissemination, and transmission of BCG are not described for cattle or other deer species. With both BCG Danish and Pasteur vaccination, the attenuated M. bovis persisted up to 250 days after subcutaneous vaccination, inducing microscopic lesions and disseminated to multiple sites (#23). BCG Pasteur was also detected within non-vaccinated pen mates, demonstrating apparent shedding and transmission (#23-24). In a subsequent study, BCG Danish persisted within white-tailed deer up to 3 months after oral vaccination and up to 9 months after subcutaneous vaccination (#25). A safety concern is that BCG may be transmitted from vaccinated deer to hunters upon consumption. BCG, however, has not been detected in meat, despite prolonged persistence and dissemination within multiple lymphoid organs (#25). These findings demonstrate the capability of M. bovis BCG to persist within tissues, disseminate to distant lymphoid tissues, and transmit from deer to deer.