In September and October, 2005, five alpacas were confirmed to have been fatally stricken with Eastern Equine Encephalitis. Three of these animals were from two different farms in south eastern New Hampshire, the other cases were from New York and New Jersey. NEAOBA and researchers from the Cummings School of Veterinary Medicine at Tufts University conducted a limited study in New Hampshire and New York during October to determine the prevalence of natural antibodies against EEE in alpacas living in areas with a high incidence of EEE associated death. In November a proposal to conduct a trial of an equine EEE and WEE vaccine in alpacas was approved for funding by the Alpaca Research Foundation (ARF). This trial began in December, 2005 and concluded in early summer 2006 (see update below).
Eastern equine encephalitis is a rare but serious disease caused by the Eastern equine encephalitis (EEE) virus, a member of the family Togaviridae, genus Alphavirus. Eastern equine encephalitis is found mainly along the eastern seaboard and Gulf coast of the United States. EEE is a mosquito borne infection of wild birds that can be transmitted to other birds, humans, horses and other animals by mosquitoes.
Infection with Eastern equine encephalitis virus can cause a range of illnesses. Most people infected with the virus have no symptoms; others get only a mild flu-like illness with fever, headache, and a sore throat. In rare cases, infection of the central nervous system occurs, causing sudden fever, muscle pains and a headache of increasing severity, often followed quickly by seizures and coma. In these rare instances, about one third of patients die from the disease. Of those who survive, many suffer permanent brain damage. Clearly, it is hard to know about mild cases in animals, but the severe neurological effects, including death, are known to occur in some infected horses (and now camelids). Reported clinical signs of EEE infection in llamas and alpacas are consistent with brain (CNS) disease and may include dullness, fever, incoordination, mentally inappropriate behavior, seizures, inability to rise, persistent tilting of the head, rapid eye movement and a swan-like backward flexion (opisthotonus) of the head and neck. Unfortunately, there are no disease specific findings for EEE infection, as these signs may mimic other CNS and spinal problems such as West Nile Virus infection, Equine Herpes Virus-1, rabies, trauma, bacterial meningitis, Thiamine (Vitamin B1) deficiency, tick paralysis and meningeal worm infection. Therefore, additional diagnostics such as a spinal tap and blood analysis are commonly required to make a diagnosis. To date, most reported cases have involved young camelids.
The Eastern equine encephalitis virus has a complex life cycle involving birds and a specific type of mosquito, Culiseta melanura, which lives in acid water swamps. These mosquitoes feed only on birds; they do not normally feed on humans or other mammals. Infected wild birds do not usually become ill with the disease. The natural cycle for EEE virus occurs nearly every year in some swamp habitats. In certain years the virus may become amplified in this bird-mosquito cycle, however, and other species of mosquitoes which feed on both birds and mammals act as bridge vectors and are capable of transmitting the virus to mammals, including horses and humans. Late summer of 2005 was such a year, with an outbreak of EEE in the north east causing several human fatalities in Massachusetts and New Hampshire. In fall of 2005 the state of New Hampshire was warning that 2006 could be as bad, or even worse. Fortunately, the outbreak in 2006 was less extensive than the one in 2005.
Prior to September, 2004, it was felt that camelids were not infected by the EEE virus, but necropsy of an alpaca at the Cummings School of Veterinary Medicine at Tufts University at that time confirmed EEE as the cause of death. In the course of preparing a publication on this case, researchers at Tufts discovered several other cases where EEE had been confirmed as the cause of death in camelids. The fact that alpacas were (and are) at risk from EEE was not widely disseminated; early in the 2005 outbreak described below the International Camelid Initiative (at Ohio State University) was strongly asserting that camelids could not be infected.
In late September, 2005 a cluster of cases raised the visibility of EEE in alpacas significantly. Four animals from three different farms in south eastern NH presented to a single veterinary practice within a 2 week period. The first case was a 10 day old cria that had progressed to recumbency and seizures before reaching the vet. This animal died soon after being admitted to the clinic, no postmortem analyses were conducted. However, when a 16 day old cria from a nearby farm presented at the same clinic with similar symptoms, alarm bells went off and intensive supportive measures were initiated. Despite these efforts, case 2 succumbed within 48 hours. Days later cases 3 (another cria from a third farm) and 4 (healthy yearling from same farm as case 2) also presented at the same clinic. These animals died within 4 and 3 days, respectively. PCR analysis (polymerase chain reaction, a DNA test) of brain tissue from cases 2-4 confirmed EEE for all cases, The first death is also suspected to be due to EEE but was never confirmed.
During this period the NEAOBA board of directors initiated several efforts to learn more about EEE in camelids. First step was to ascertain whether the outbreak was localized or more widespread. Mass emails to all NEAOBA members requested “private” reporting of any similar cases. This revealed one additional fatal EEE case in an alpaca from central New York, as well as a cria in south western NH that displayed neurological problems including seizure, but survived. Analysis of serum from this animal subsequently confirmed high titer of EEE antibodies. It was also during this time that NEAOBA learned of the earlier cases at Tufts and established initial contact with Dr. Daniela Bedenice (also Drs. Karen Baum and Patricia Craven, members of the ARF BOD).
News of the outbreak spread beyond the north east through forwarded email messages and several of the industry chat sites. In October a breeder in New Jersey reported that a breeding male had died from EEE, in this case the attending vet felt that stress from elective surgery had weakened the animal’s immune system and made it easier for the virus to overcome his defenses.
NEAOBA decided to launch two studies, in collaboration with Dr. Bedenice and Dr. Amy Bright (a clinical vet at the practice that treated the four cases in south east NH described above). To establish seroprevalence (fraction of the population with natural antibodies) of EEE antibodies in naturally exposed alpacas we collected serum samples on the 22nd of October from 14 surviving animals on the NY farm that lost an animal to EEE, 27 survivors on the two NH farms that suffered losses, and 46 animals living on 4 different NH farms that were within 10 miles of the impacted farms. Serum neutralization (by PRNT) tests were conducted at the US Department of Agriculture National Veterinary Services Laboratory (NVSL). The seroprevalence of EEE antibodies (PRNT titer > 1:10) was as high as 29% on one of the farms with an overall prevalence of 10% in this study.
The second study is the vaccine trial, “Humoral response to EEE vaccination in healthy alpacas” supported by ARF. Dr. Daniela Bedenice is the Principal Investigator, collaborating researchers include: Jack Dibb, NEAOBA treasurer, Drs. Tim Fallon and Amy Bright, Cornerstone Vet Hospital, and Dr. Doug Pedersen, NVSL. NEAOBA breeders provided 39 subject animals, spread between two farms in Maine, two farms in NH and one in Vermont. We selected a bivalent (eastern and western equine encephalitis) inactivated vaccine because no monovalent EEE vaccines are currently commercially available. Dr. Craig Barnett at Intervet arranged for donation of the Intervet vaccine sold as Encevac™.
Specific objectives declared in the proposal are reproduced here:
1. Evaluation of EEE serum-antibodies via a semi-quantitative plaque reduction neutralization test (PRNT) following 3 intramuscular vaccinations with Encevac™ at 4-week intervals in 45 healthy alpacas.
2. Assessment of local and systemic reactions following EEE vaccination.
We hypothesized that alpacas would develop virus-neutralizing antibody titers similar to those reported in horses following repeated EEE vaccination.
In plainer English, objective 1 is to determine whether the vaccine leads to antibody titers that are similar to those proven to provide protection against EEE infection in other animals and objective 2 states that we want to be sure the vaccine has minor or no side effects. It is important to recognize that this trial did not intend to prove that the Encevac™ vaccine is actually protective against EEE virus infection in alpacas, since proving this would require deliberately exposing vaccinated animals to the live virus. Such a study would have to be performed on a small scale in specialized and approved facilities due to the risks involved when working with the EEE virus.
As noted in the objectives, each subject animal received an initial vaccination, followed by boosters 4 and 8 weeks later. Serum samples were collected immediately before the first vaccination, and then at 2 week intervals for a total of 12 weeks.
February 2007 update:
The EEE vaccine trial ended as planned in mid March, 2006 with final results from the lab received in June. A manuscript describing the detailed findings is in preparation. Dr. Bedenice has agreed that this summary should be provided now, for breeders and their veterinarians to consider.
One firm finding was that the vaccine appeared to be safe in the adult males tested, with no adverse reactions reported in any of the subject animals. Just over three quarters of the trial animals developed titers after the three shot series, with the highest number of seropositive animals, and highest mean titers, observed 2 weeks after the third shot. Younger (less than 4 years old) animals in the trial tended to respond “better” than older animals, but the sample size was limited, especially for the older groups.
Drs. Bedenice and Bright recommended that farms in high risk areas strongly consider vaccinating in 2006, and several in NH took this advice (including the two farms with all of the animals that developed titers in Fall 2005 from natural exposure). Several hundreds of additional serum samples were privately analyzed for EEE antibodies at NVSL through the summer and fall of 2006. They may be of interest because, unlike the trial, they included pregnant females and neonates. The number of animals was small, and the timing of sample collection could not ensure that additional natural exposure to EEE had not caused antibody responses, so the findings are unlikely to pass peer review. However, I offer them to the community.
We resampled the naturally exposed animals several times through late 2005 and up to June of 2006. In all cases where the alpacas had developed titers higher than 1:100 when first sampled in October, 2005 they maintained titers at this level or higher through June, 2006. (Most were then vaccinated to boost their immunity prior to peak mosquito season.)
One of the naturally exposed females delivered a healthy cria in late May, the cria had a positive titer (1:100) at 9 days of age (we presume the antibodies came from mom, since it was very early in the year for this animal to have been exposed to the live virus).
A total of 29 breeding females and their 2006 cria were vaccinated on the 2 NH farms that owned all of the animals with natural antibodies to EEE in 2005 (these farms were clearly at “high risk”). Ten of the females were relatively advanced in their pregnancies, but none reacted adversely to the 3 shot series. Nine of them delivered healthy full term cria during the summer and fall (the tenth lost a breech delivery at term).
Results of PRNT on serum samples collected 3.5 to 4.5 months after the first shot (for animals that were negative in fall 2005) were broadly similar to the formal trial. Overall, 65% of the vaccinated animals developed titers and maintained them at least until they were sampled about 2 months after final booster. Like the males in the trial, measurable responses (titers) were less common, and lower when found at all, in the oldest girls.
Neonates (first shot given 3-5 weeks post partum) as a group also responded poorly, compared both to the young boys in the trial and the younger breeding girls in their herds. The cria that had the presumed natural titer, transferred from mom, at 9 days of age tested negative at 6 months of age, despite receiving a 4-shot vaccination series. Pat Long, DVM and ARF treasurer, speculates that the maternal antibodies may be antagonistic to the vaccine, and recommends waiting till 3 months of age before vaccinating crias, especially if mom is known to be seropositive.
We are deeply appreciative of the support provided by NEAOBA members during the seroprevalence study, and thank the farms (Crown Point Alpacas, Milesview Alpacas, Ande’s Acres Farm, Cynjim Alpacas, Fraggle Rock Farm, Tamarack Meadow Farm, Sallie’s Fen Alpacas) that allowed serum to be collected from their animals. The vaccine trial would not have been possible without the financial support of ARF, but the willingness of NEAOBA breeders to provide subject animals for the study was also critical (special thanks to New Aim Farm, Half Horse Farm, Cas Cad Nac, Cynjim Alpacas, Tamarack Meadow, Crown Point Alpacas, A Craftman’s Touch Alpacas and Sallie’s Fen Alpacas). Drs. Tamy Doty and Amy Bright donated their time to vaccinate and collect samples from the test groups in Maine and New Hampshire, and Ian and Jennifer Lutz took care of the group from their farm. ARF exists solely to support research into issues impacting alpacas and their caregivers, and depends on donations to fund this important work.
An earlier version of this article was previously printed in Alpacas Magazine.