Vaccine delivery to wildlife: Assessment of efficacy



Vaccine delivery to wildlife: Assessment of efficacy


By: Daniella Dvilanski, veterinary student final year Glazgo UK, E.M.S at Dr Neri veterinary surgeon


Vaccination of wildlife populations is important in the control of many infectious diseases that have both economic and public health implications. It can also be valuable in the management of the overabundance of certain wildlife species through immunocontraception. Vaccination of wildlife can be costly, difficult and even dangerous in its implementation, but it has been effective in many areas of the world. Immunisation schemes can aid the conservation of wildlife, especially if it is an endangered species; important examples include the control of plague (Yersinia pestis infection) in the black-footed ferret and control of anthrax (Bacillus anthracis) in Roan antelope and black rhinoceroses. 

Wildlife species are also an important reservoir for many zoonotic diseases, so vaccination of these species against zoonotic infections is of major public health concern. The highest risk of transmission of diseases from wildlife to humans is through domestic species, such as cattle in the case of badgers infected with tuberculosis or cats and dogs in the case of wild canids such as foxes infected with rabies. This essay will discuss the formulations, modes of delivery, and assessment of efficacy of vaccines against infectious diseases and for immunocontraception in wildlife. 

Broadcast vaccination of wildlife is currently an important research area. Scientists are trying to determine the benefits, efficacy and problems of vaccination compared to other methods used, in order to offer the best approach for disease control. The goal of immunization is to increase the individual’s ability to fight disease, and to slow disease transmission (1). In order for a vaccine to offer a valid alternative to other means of disease control, it should provide long-term protection, be safe, stable and cost-effective, and feasible for administration in the wild.

This section of the report aims to cover various experiments and approaches to the measurement of wildlife vaccination efficacy. 

Oral bait evaluation  

Wildlife vaccine administration using oral baits is the commonest feasible way for wildlife mass vaccination (1). A study held on piglets, aimed to find and assess new oral baits, used the European wild boar specie; a reservoir host for Mycobacterium bovis, swine fever virus and porcine herpesvirus (2). Physical stability and bacterial viability of baits, as well as generating effective antibody response, were tested. The vaccine was a recombinant E.coli cells expressing a pMBXAF3 plasmid. Following induction, fusion protein was formed, as part of immunogenic peptides on the bacteria outer membrane (3). E.coli cells were dipped in polyethylene capsules for bait incorporation. To test bait stability in diverse field conditions, pressure was applied on four bait groups, each incubated in different temperatures or in water; and height differences were calculated. In 42°C the baits showed stability for a minimum of 3 days, whereas water had a damaging effect. High temperatures (53°C) did not affect E.Coli viability, proving the paraffin to be a protective vaccine matrix. When placed in selective feeders, the baits were completely consumed within 24 hours; with the majority of the vaccine capsules chewed and swallowed. Antibodies in fecal samples were determined using ELISA, and presence of plasmid in bacterial colonies was checked using western blot and PCR. Recombinant E.coli cells, recovered from fecal samples of vaccinated piglets four weeks post immunization, confirmed the vaccine reached the alimentary tract. Likewise, significant IgG titers against the fusion protein were observed in the vaccinated piglets. The study established palatability and thermo-stability of the tested baits, allowing the vaccine to be successfully delivered to the oropharangeal lymphoid tissues. This study, like many others, supports the claim that the oral delivery of vaccination is the most effective and applicable delivery method.


Bovine Tuberculosis

Bovine Tuberculosis (bTB) is a bacterial zoonotic infectious disease in cattle, caused by Mycobacterium bovis, rendering economic loss and having a detrimental effect on animal's health and welfare (4). Great effort has been made in order to find effective disease eradication methods, focusing on wildlife host reservoirs. Studies centered mainly on wildlife oral vaccination with the human BCG vaccine. Nevertheless, BCG losses its efficacy if delivered in a non-viable state caused by acidic stomach environment (5,6). Hence, formulating a stable vaccine remaining viable throughout the alimentary tract - is vital.  

In a UK 2008 research, different formulations efficacies were tested in guinea pigs and mice models for the primary Eurasian badger (Meles Meles) wildlife host; testing two different doses and a single dose respectively. It was possible to use a rodent model since its respiratory rout, being the chief exposure route, is analogous to that of badgers (7). Formulation of live BCG in lipid matrix was compared to BCG in alginate beads, and to microcapsules designed from lipid and alginate combined. The hypothesis offered that the lipid formulation will be exposed to the acidic gastric environments, whereas the alginate will not.

 After administrating the vaccine to assigned groups, they were challenged via the aerosol route with M. Bovis and euthanized five weeks later. Those receiving the lipid and the microcapsules formulations showed strong PPD (Purified Protein Derivative); inducible IFN-g; elevated proliferative lymphocytes; and presence of BCG in the GIT lymphatic’s, In contrast to those receiving the alginate beads formulation. The study concluded that even in low doses, best levels of immunoprotection were seen in vaccines formulated with lipids. These were successful in achieving protection similar to that induced by vaccines administrated by injection.

Later that year, a following study of the BCG efficacy in captive possums was held in New Zealand (8). New Zealand's possums (Trichosurus vulpecula) are a main TB wildlife reservoir, highly susceptible to M. bovis.  Previous finding verified wild possums responsiveness to mucosal BCG vaccination, administrated intra- nasally or intra-conjunctively (9). In captivity, lipid based BCG vaccination provided protection, both when given as intra-tracheal instillation of liquid suspension, and when given via inhaled microdroplets (10,11,12,13). This study explored the resilience and the capability of the specific vaccine formulation to protect from M.bovis challenge in the wild, as well as in captivity.

 In the field study, mature possums were captured, marked, and assigned for control and vaccine groups. Following oral BCG vaccination, possums were released and recaptures three months later. Blood lymphocytes levels and lymphocytes proliferation response were higher in the vaccinated group at that point in time. Captures animals were challenged with intra-tracheal installation of M. bovis, after which they were released, bearing a mortality sensing radio-collar. The relative death risk of vaccinated animals was 2.4 times lower than that of the control group. Moreover, the survival probability was higher for the vaccinated group, as shown in the diagram taken from the study (8).

The vaccine has achieved a 60% fall in the relative mortality risk, both in possums with poor body condition as in those with good body conditions in the wintertime, showing stability and efficacy under non-optimal fiend conditions. However, on average, challenged possums survived 2.5 months less than possums naturally infected with TB (4.7 months); stressing the problem of finding a true reflecting assay method that would accurately mimic wild TB disease infection.  

Concurrently, a captivity study was held, using thirteen trapped possums. The vaccine group was given palatable oral vaccine formulation for two nights in a row, intended to increase intake chances. Possums were challenged eight weeks later with M.bovis- containing aerosolized microdroplets. The vaccinated group expressed reduced pulmonary pathology and bacterial counts, produced from infected organs, relatively to the control group. Furthermore, fewer animals in the vaccination group exhibited extra-pulmonary macroscopic TB lesions than the control group.

Results demonstrated unexpectedly high protection response levels, much higher than previous captivity studies (12,13,14), setting the upper bar for the protection the vaccine can achieve under optimal conditions. 

The question regarding the efficacy of the oral BCG vaccination was raised once more in Australia, where wild brush-tail possums (Trichosurus vulpecula) are the TB wildlife host. A New Zealanders’ group focused on the vaccine's performance in the face of natural challenge (15). The chosen experiment area proved to be TB positive since 1980. Trapped possums were tested for TB status by superficial lymph nodes palpation. In the two years study, 51 animals were vaccinated and 71 were used as control animals. Immune response to the vaccine was tested two months post vaccination, when no significant statistical response was expressed in lymphocytes proliferation assay. Boosters were given to the vaccinated group every six months (13). All possums were killed and tested for TB using spleen, lungs, liver and lymph nodes, in addition to bacteriological cultures retrieved from suspected TB lesions.  In face of the same infection force, TB was culture-confirmed in twelve control animals versus one vaccinated animal. All infected control possums showed classic lungs and or lymph nodes lesions, while the singular infected vaccinated animal showed small liver lesions, and was negative for Mycobacterum in lymph node culture.  In Spain, pathology and genetic profiles following BCG vaccination were studied in TB wild reservoir, Eurasian wild boar (16). Oral baits were manually administrated to the vaccinated group. They were later challenged with M.bovis via the oropharangeal route, each animal with different CFU concentrations. Euthanasia took place 114 days post challenge.  Lymph nodes were tested for presence of TB lesions, in addition to ZN staining. PCR was formed on immunoregulatory genes associated with TB protection: IFN-g, RANTES, C3, IL-4 and MUT. M. bovis was found in 75% of the non-vaccinated challenged animals and in 50% of the vaccinated group, with fewer organisms. In contrast to 100% unvaccinated challenged piglets showing TB lesions, only 50% vaccinated animals developed lesions with lower score. Severe TB lesions in vaccinated piglets were only found in those most strongly challenged. Vaccinated animals showed up- regulation of C3 and MUT, 46 dpi (days post immunization) and 186 dpi respectively; suggesting the involvement of these genes in immunoprotection against M. bovis infection in the wild boar. These different studies manage to demonstrate that with advanced bait delivery methods and excellent efficacy rating, oral, lipid based BCG vaccination is the most practical and valid way to eradicate TB, starting from wildlife.  



Efforts to find the best way for wildlife vaccine delivery are also markedly seen in the elaborate studies evolving the Rabies disease, a zoonotic viral neuroinvasive disease. In order to find the best vaccination for wild mongoose, a chief wildlife Rabies reservoir, efficacy of two vaccines was tested (17); the common recombinant V-RG vaccine (based on Rabies glycoprotein) and the new SPBNGA-S vaccine.

When efficacy of the V-RG vaccine was tested using a rapid fluorescent focus inhibition test for antibody detection, (RFFIT), no virus neutralizing antibodies (VNA) were detected in either vaccinated or control groups. Subsequently to challenge, all experiment animals were affected from the virus, including the vaccinated group, developing the disease.

Next, the SPBNGA-S recombinant virus vaccine was tested for its efficacy, and the entire vaccinated mongoose were VNA positive. In contrast to the non-vaccinated group, showing Rabies clinical signs, none were seen in the vaccinated group, and no viral antigens were detected in a direct fluorescent antibody test. This study successfully showed the superiority of the newly developed SPBNGA-S recombinant vaccine over the

V-RG one.

In light of the SPBNGA vaccine success, further developments took place, generating the SPBNGAS-GAS vaccine, containing an additional copy of Rabies virus glycoprotein gene (18). Efficacy was tested in wild raccoons (Procyon lotor), the most reported rabies reservoir in the North America. SPBNGAS-GAS was found to be better than both original V-RG and the SPBNGA vaccines, both in efficacy and safety, having quicker VNA production with less morbidity and mortality (18,19).

An especially large-scale research was held in Estonia, where raccoon dogs (Nyctereutes procyonoides) and the red fox (Vulpues vulus) consist of the major rabies wildlife reservoirs (20). As part of the EU rabies control program, the study was devised in order to measure the efficacy of the oral SAG2 vaccine: modified live attenuated vaccine, by looking at rabies epidemiology status before and after vaccination in 2005-06 covering 25,000km 2 of forest.

 In 2004 the vaccination program feasibility was tested in a small island with two oral vaccination rounds, spring and fall. In 2005 a bigger trial covering 25,540 km2 was performed in the north of Estonia, applying 505,600 baits, dropped from airplanes.


 During the spring and fall of 2006 the whole of Estonia was vaccinated for rabies, using 850,000 baits over 42,992 km2. For the efficacy testing, Fluorescent antibody test (FAT), culture virus isolation and PCR were used, and animals were tested for the presence of Tetracycline in the mandible: biomarker for oral bait uptake. Tetracycline was detected by using inverse microscopy under ultraviolet light, looking at teeth (canine and surrounding alveolar bone) and jawbones.

Finally, Rabies antibodies titres were detected using indirect ELISA.

In the pilot 2004 trial, no rabies was found in the area, based on a sample of 11 animals- raccoons and fox, in which bait intake was 82%. In 2005, out of 97 rabies cases reported in the country, only 16 cases stemmed from the vaccinated area. 73% of tested fox were positive for Tetracycline. In the 2006 cross country trail, only six cases out of the total low number of 17 rabies cases, originated in the vaccinated area. 85% animals were Tetracycline positive and 64% had significant rabies antibodies levels.

Examining the 2006 data, the scientists encountered a disparity between the Tetracycline and the antibody serology results, shown in the table taken from the article (20).

According to the study, a possible reason for the differences between the positive Tetracycline results and the negative antibody results, could be that although the bait case was ingested, the actual vaccine was not, a common occurrence found in tested cubs. Furthermore, the fox’s tendency to hide food and consume it later, when the vaccine is inactive, could explain the differences. Finally, an explanation might be found in the biomarker properties. The Tetracycline antibiotic is a reliable physical marker, used widely in wildlife research. Tetracycline deposits are seen as a pale yellow ring in tooth cementum. While reliable, Tetracycline may not be seen, despite bait intake, in older animals and in young, due to slowed bone and teeth growth in the former, or bone reformation in the latter (21). Lyme, Brucella and Plague Finding the appropriate immunization protocol was the focus of many more disease studies.  A few examples of such studies are summarized in the following chart (22,23,24): 


Pathogen Disease  Animal tested Vaccine tested ExperimentDesign Administration method Methods of assessment Conclusion
Brucella abortus Bovine brucellosis Sprague-Dawley rats (females and offspring) RB51 Captivity Intraperitoeal injection - Cultures isolates- PCR- Antibody response (RBPAT)- ELISA- Standard tube agglutination Vaccine showed high protection from both individual infection and vertical transmission.
Borrelia burgdorferi Lyme disease White-footed mice (Permyscus leucopus) OspA Wild Injection - Enzyme immunosorbent assays (EIAs)- PCR- Infected tick prevalence (tick collection)   Vaccination reduced pathogen prevalence by disrupting the infectious life cycle, lowering prevalence of the infectious vector.
Yersinia Pestis Plague Prairie dog RCN-F1 Captivity Oral baits - PCR- Anti-RCN and F1 antibody test (ELISA)  The vaccine - based RCN vector (Orthopoxvirus) was shown to be safe and effective, allowing for higher survivor rates.   




Manipulation of wildlife animals by vaccination, is a form of preventative means for disease management. Immunization creates a situation whereby population becomes resistant to disease, and disease transmission rate is reduced.However, suitable vaccination program is challenging to develop, taking into account the finding of a suitable vaccine, a proper means of delivery, cost and technical matters. Currently, the most effective method for wildlife mass vaccination is via oral baits. However, concerns such as safety to non targeted species still remain unsolved. Essentially, vaccination is suited for a fairly small numbers of pathogens, with low reproductive rate (Ro); in a low- turnover population; and where disease effects mature animals (1).

Once a suitable vaccine formulation and delivery method has been chosen and applied, efficacy trials must be conducted in order to verify the effectiveness of the specific immunization program. Such studies were held worldwide covering different vaccination programs, aimed to protect from disease like Rabbis; Tuberculosis; Anthrax; Plague and Lyme, focusing on serology and pathology. Most of the studies covered in this report, have shown positive results of disease control following vaccination. However, factors such as sample size; sex of the animals; sampling and capturing techniques; environmental condition; climate; season and study duration, should all be considered when analyzing the results. The ultimate test for a vaccine efficacy is the population resistance throughout a long period of time, which cannot be reached during the course of standard scientific experiment. The Estonia Rabies study was an exception, covering a perios of three years, thus succeeding in being a reliable source for regarding the efficacy of the vaccine in use.It is interesting to note that little reference of epidemiology and disease breakout modeling has been incorporated in the studies. It could suggested that the use of mathematical modeling would contribute to the understanding and prediction of the disease behavior, thus overcoming some of the time limitation. The above mention models should consider factors such as population movement; climate changes; disease behavior; and population size. By using forecasting information, it might be possible to reach a more accurte vaccination programs, considering that

vaccination is a continuous process wich should be re-assessed, in terms of formulation and delivery.