Materials and Methods

Study protocol—The study was conducted as a nonrandomized clinical trial involving 3 groups of alpacas. All procedures were approved by the Institutional Animal Care and Use Committee at the Cummings School of Veterinary Medicine at Tufts University. Owners of animals included in the study provided their consent.

The first study group consisted of 39 healthy male alpacas in a single herd. Animals ranged from 1.3 to 14.5 years old (median, 2.2 years). All animals were vaccinated with a formaldehyde-inactivated, adjuvanted, bivalent (eastern and western equine encephalitis viral antigens) equine encephalitis vaccine^a administered IM in the semimembranosus and semitendinosus muscles with a 20- or 22-gauge, 1-inch needle. Three doses (1 mL/dose) of vaccine were administered at 4-week intervals (ie, on days 0, 28, and 56) on the basis of the manufacturer's recommendation for vaccination of horses.

Venous blood samples (3 to 5 mL) were collected from all alpacas immediately prior to vaccination (ie, day 0) and every other week thereafter for 12 weeks (ie, days 14, 28, 42, 56, 70, and 84). Serum was separated within 3 hours of blood sample collection and frozen at −70°C until assayed for EEE virus neutralizing antibody titer. All blood samples were collected during the winter and early spring, when natural exposure to EEE virus in New England was considered negligible.

The second study group consisted of 86 alpacas (43 males and 43 females) in a separate herd and was included to determine whether age, sex, or breed had an effect on humoral responses following vaccination. Animals ranged from 0.4 to 13.9 years old (median, 5 years); there were 61 Huacaya and 25 Suri alpacas. All animals in the second study group received 3 doses, 4 weeks apart, of the same killed-virus vaccine used for animals in the first study group. A single blood sample was collected 70 days after the initial vaccine dose was administered (ie, 2 weeks after the third dose was administered), and serum was assayed to determine virus neutralizing antibody responses. Blood samples were collected during the winter, approximately 10 months after samples had been collected from animals in the first study group.

The third study group consisted of 30 male alpacas ranging from 0.2 to 14.9 years old (median, 3.3 years) that were not vaccinated. Blood samples were collected for determination of virus neutralizing antibody titers against EEE at the same time blood samples were collected from vaccinated animals in the second study group. Unvaccinated animals in the third study group were located on a separate farm, in proximity to but without direct contact with vaccinated animals in the second study group.

Determination of virus neutralizing antibody titer—Serum samples were submitted to the National Veterinary Services Laboratory in Ames, Iowa, for determination of neutralizing antibody titers against EEE virus. Titers were determined with a plaque reduction neutralization assay, as described.^1,4 In brief, undiluted samples and samples diluted 1:10, 1:100, 1:200, and 1:400 in cell culture medium were incubated at 37°C for 75 minutes with 100 plaque-forming units of EEE virus. The serum-virus mixture was then added to a confluent monolayer of Vero 76 cells in a 25-cm² flask. After 1 hour, cells were overlaid with agar containing medium and neutral red. Flasks were incubated for 72 hours, and virus plaques were enumerated. The recorded titer represented the highest serum dilution capable of neutralizing at least 90% of the virus.⁵ Animals were considered seronegative if undiluted serum did not neutralize at least 90% of the virus.

Seroprevalence of natural EEE virus exposure—Blood samples were collected during the fall of 2005 from 73 clinically normal, unvaccinated alpacas in New Hampshire to estimate the seroprevalence of natural EEE virus exposure. A single blood sample was obtained from 27 animals on 2 alpaca farms that suffered losses attributed to EEE that year and from 46 animals living at 4 other farms within 10 miles of affected farms. There were 43 females, 23 sexually intact males, and 7 castrated males. Median age was 3 years (range, 0.1 to 14 years). Samples were processed as described for the other study groups and submitted for determination of neutralizing antibody titers.

Statistical analysis—Plaque reduction neutralization test titers (1:10, 1:100, 1:200, and 1:400) from individual animals were averaged by means of a logarithmic (base 10) transformation. Geometric mean titers and their 95% confidence intervals were calculated for each time interval, and the Friedman test followed by the Wilcoxon rank test was used to compare median GMTs among time points. Animals were then classified as juveniles (0 to 2 years old), young adults (> 2 to 6 years old), and mature adults (> 6 years old) on the basis of their utility as breeding animals, and median GMTs were compared among age groups by means of the Kruskal-Wallis test followed by the Mann-Whitney U test. In addition, the Pearson χ² test was used to determine whether age group was significantly associated with seroreactivity (seropositive vs seronegative). All analyses were performed with standard software.^b Values of P < 0.05 were considered significant.

Results

All 39 alpacas in the first study group were seronegative for antibodies against EEE virus prior to administration of the first vaccine dose (ie, day 0; Table 1). No adverse local or systemic effects were observed following vaccination of alpacas.

On both day 14 and day 28, only 1 alpaca was seropositive, and median GMTs on days 42, 56, 70, and 84 were significantly higher than median GMTs on days 0, 14, and 28. In addition, median GMT on day 42 (ie, 2 weeks after administration of the second vaccine dose) was significantly higher than median GMT on day 28, and median GMT on day 70 (ie, 2 weeks after administration of the third vaccine dose) was significantly higher than median GMT on day 56. On day 70, median GMT for juvenile animals was significantly (P = 0.023) higher than median GMT for mature adult animals. On days 56 and 84, percentage of juvenile animals that were seropositive was significantly higher than percentage of mature adult animals that were seropositive. On day 70, percentages of juvenile and young adult animals that were seropositive were significantly higher than percentage of mature adult animals that were seropositive.

For animals in the second study group, median GMT on day 70 was not significantly different between Huacaya alpacas and Suri alpacas or between male and female alpacas (Table 2). However, the GMT of juvenile animals was significantly higher than the GMT of both young and mature adult animals. Additionally, the percentage of juvenile animals that were seropositive was significantly higher than the percentage of mature adult animals that were seropositive. All 30 alpacas in the unvaccinated group (ie, group 3) were seronegative for antibodies against EEE virus.

Seroprevalence of natural EEE virus exposure—Seven of the 73 (10%) clinically normal, unvaccinated alpacas living on the same farm as or within 10 miles of alpacas that had died of natural EEE infection in 2005 were seropositive for antibodies against EEE virus, with GMTs ranging from 1 to 2.6.

Discussion

Results of the present study suggested that inoculation of alpacas with 3 doses of a bivalent, killed-virus equine encephalitis vaccine induced a humoral antibody response, with GMT peaking 2 weeks after administration of the third dose. Titers did not vary significantly on the basis of breed or sex. However, median GMT and percentage of seropositive animals were significantly higher for juvenile than for mature adult animals.

In the present study, we elected to use a bivalent (eastern and western equine encephalitis viral antigens), killed-virus equine encephalitis vaccine approved for use in horses because no commercially available encephalitis vaccines were labeled for use in New Word camelids. In addition, monovalent, killed-virus EEE vaccines approved for use in horses were not being sold in the United States at the time of the study.

Antibody titers in response to vaccination in juvenile alpacas in the present study were similar to those recently reported in a study⁶ involving 10 adult horses given 2 doses of a trivalent, killed-virus equine encephalitis vaccine 4 weeks apart. In that study, however, median GMT peaked 4 weeks after the second vaccine dose was administered, whereas in the present study, comparable titers were not observed until after the third vaccine dose. Similarly, in a study⁵ of humoral responses in alpacas after West Nile virus vaccination, 3 doses of vaccine were needed to induce neutralizing antibody titers comparable to those observed following administration of 2 doses in horses. It has been speculated that New World camelids respond differently to vaccination than do other domestic animals.⁵ However, a recent study⁷ demonstrated that neither serum antibody titers nor serum inhibitory capacity was significantly different between rabbits and llamas following immunization with recombinant bacterial E-lactamase. Clearly, immunologic responses of New World camelids to vaccination require further investigation. Interestingly, it has been reported that immunoglobulin responses in camelids are unique in that both conventional heterotetrameric IgG (ie, paired heavy and light polypeptide chains) common to all vertebrates and homodimeric heavy-chain antibodies that lack light chains are produced.⁸ Although the real role of these homodimeric heavy-chain antibodies remains unknown, it has been suggested that unconventional antibodies may represent an evolutionary advantage and could be more efficient than conventional antibodies at inhibiting microbial enzymes thus providing a more protective immune response against pathogens.⁹

For both groups of vaccinated animals in the present study, the percentage of seropositive alpacas and median GMT on day 70 were significantly higher among juvenile animals than mature adult animals. These differences were seen even though animals as young as 0.4 years old were included in the juvenile group. These results were somewhat unexpected because human studies¹⁰ suggest that the immune system is not fully developed in young children and infants, but may further mature with age. However, the magnitude of humoral responses is generally dependent on priming rather than age.¹¹

It is possible that the higher median GMT in juvenile animals in the present study was related, in part, to the higher amount of antigen administered in relation to body size. All animals received 1 mL of the vaccine at each immunization, regardless of animal size. It has previously been demonstrated in people that immunoglobulin responses to influenza vaccine could be improved by the use of high-dose vaccines.¹²

Maternally derived antibodies would not be expected to interfere with vaccination in alpacas > 4 months old because the mean half-life of maternal IgG in alpaca crias has been documented to be 15.7 days.¹³ To date, it has not been established whether administration of an equine encephalitis vaccine to alpaca dams during the last third of pregnancy would lead to development of colostral antibodies that could interfere with vaccine responses in young crias. Vaccination of pregnant sows with an equine encephalitis vaccine leads to development of maternal antibodies that are transmitted via colostrum to piglets and are protective against clinical EEE-related disease after experimental challenge with EEE virus.¹⁴

In contrast to results for juvenile animals, no significant differences in median GMT were found between young adult and mature adult alpacas in the present study. Several human studies^15,16 have also been unable to verify significant age-related differences among serologic responses and protection rates in elderly versus young adults following influenza vaccination.

Importantly, we did not in the present study assess whether vaccination with an equine encephalitis vaccine would protect alpacas from EEE. In horses, vaccination titers that will protect against EEE are not known. However, in a previous study,¹⁷ seronegative horses remained refractory to challenge with EEE virus 3, 8, and 12 months after vaccination. Similarly, a commercially available polyvalent equine encephalitis vaccine capable of inducing detectable neutralizing antibody titers in emus protected the birds against subsequent exposure to an otherwise lethal dose of EEE virus.² Additionally, an equine encephalitis vaccination program appeared to be efficacious in a whooping crane population.¹⁸ Nevertheless, it remains unknown whether neutralizing antibody responses in camelids are associated with vaccine efficacy.

In the present study, seroprevalence of naturally occurring EEE antibodies among clinically normal alpacas residing on or within 10 miles of a farm where alpacas had died of EEE 2 months previously was 10% (7/73). This was similar to results of previous studies that have estimated the seroprevalence of antibodies to EEE virus in white-tailed deer,^19,20 mules,²⁰ free-ranging ruminants,²¹ and feral and domestic swine.²² Eastern equine encephalitis generally occurs near habitats suitable for breeding enzootic and bridge vectors and where amplifying avian hosts are abundant,²³ and in these areas, seroprevalence has been used to estimate the relative risk of exposure to EEE virus. Although the use of serologic findings to establish information on distribution of viral diseases has substantial limitations, seroprevalence surveys may still be useful in detecting geographic areas where the virus is active.

The present study has several limitations. Juvenile alpacas from the first farm (mean age, 1 year; range, 4.8 months to 2 years) were significantly (P < 0.001) younger than those from the second farm (mean age, 1.54 years; range, 15.5 months to 2 years). These age differences may partly account for the higher GMT observed for juvenile animals in the second study group, compared with juvenile animals in the first study group. Additionally, blood samples were collected from the animals in the second study group approximately 10 months after samples were collected from animals in the first study group (winter 2006 vs winter 2005). Control animals in the present study remained negative for antibodies against EEE virus, and no naturally occurring cases of EEE were reported in alpacas in New Hampshire in the intervening time. However, we could not be completely certain whether EEE virus was endemic during this period, as the persistence of neutralizing antibodies against EEE virus following natural exposure in alpacas has not been determined.