The objectives we sought to determine in this study were the length of the incubation period, if a viremic state exists, and if PCR of saliva is an effective ante mortem diagnostic tool after rabies virus infection in night owl monkeys (Aotus nancymaae). The average incubation period of rabies was observed as 11.5 days for the two monkeys that developed clinically apparent disease. However, the study was terminated at 134 days (4.5 months) post-inoculation, at which time the remaining six study subjects did not have clinically apparent disease. Based on this information, it is possible the incubation period for infection in the A. nancymaae is greater than our termination point.
The presence of viral RNA in blood and saliva post-inoculation might suggest viremia and viral shedding occurs, respectively, in A. nancymaae and serve as an effective ante mortem diagnostic test; however, no viremia or viral shedding was detected in any of the study subjects. The PCR assay used in this study is highly sensitive and specific for the detection of rabies viral RNA
. Viral shedding in salivary sections presumably should occur irrespective of viremia, as rabies virus spread to glandular tissue is unlikely to occur via blood or epithelial cells. Previous studies have established that even with direct rabies virus inoculation, salivary gland infection is dependent on centrifugal neuronal spread and preceding CNS involvement
. We would not expect to measure viral shedding from salivary glands without simultaneous CNS involvement
[27, 28]. Our study results for the two clinical cases show that there was no detection of viral shedding pre-onset or post-onset of clinical symptoms; however, observation was only continued for three days post-onset of symptoms in the clinically ill monkeys. In contrast, viral shedding was detected in a 55-year old patient in the U.K. using RT-PCR, but this was 4 days post-onset of symptoms
. Also, RT-PCR successfully detected rabies virus in 5 saliva samples from 28 cases after the onset of symptoms
. Therefore, clinical cases may have progressed too rapidly to allow sufficient time for salivary gland infection prior to euthanasia. Other possible explanations for the lack of viral shedding include a failure of CNS involvement and centrifugal neuronal spread in non-clinical cases, inability of glandular cells to support infection, and immune response in these monkeys. Despite the development of serum neutralizing antibody, an inoculum dose-related factor may also be a possibility. Studies in foxes, dogs, and mice indicate high inoculum doses cause rapidly progressing infections leading to death prior to salivary gland infection
, but this differs from our study as there were six cases without clinical rabies or CNS involvement.
For both the lack of viremia and viral shedding, viral neutralization, interference, and restricted infection to the site of inoculation by interferons or other non-specific immune mechanisms may have been factors
. Although these findings suggest there is no viremia or viral shedding of rabies virus in clinically ill Aotus nancymae, it is difficult to make a conclusion with such a small sample size since only two monkeys exhibited clinical symptoms. Interestingly, this study can be used to observe the time course of immunologic action between inoculation sites with different distances from the CNS. The clinically ill rabies cases were inoculated in the neck and exhibited a larger and more rapid IgG antibody response compared to the non-clinical rabies cases. Clinically apparent disease could be caused by the brain’s virus-specific T-cell response that fights against viral entry into the blood brain barrier of the monkey
. Since the clinically ill cases exhibited a greater response more quickly, this could simply be indicative of the virus gaining access to the CNS more rapidly. In a study challenging mice with either an avirulent or virulent strain of rabies, the antibody response was higher after seven days in the avirulent strain group, which did not develop clinical symptoms
. The prompt induction of neutralizing antibodies, inflammation and the inhibition of viral spread are necessary for recovery from rabies virus
. Once in the CNS, the virus continually replicates and leads to the onset of classical rabies symptoms
. Therefore, at the point of detection of high antibody titers, it would be too late to clear the virus. However, if this is the case, antibody responses in the neck inoculated monkeys that successfully overcame the rabies challenge should have also been high earlier; this suggests the influence of some other factor. The overall pattern of antibody response for the non-clinical group did not vary significantly; however, the footpad inoculated group tended to have a more robust initial IgM response and a prolonged maximum IgG response compared to the neck inoculated group. The rate of antibody response seems more important than the magnitude. A possible explanation could be that in the footpad inoculated group, the slowly developing antibody response allowed sufficient time to develop antibodies with greater avidity and counter virus in relation to its replication rate preventing it from entering the brain, essentially preventing clinical disease. This explanation is further supported by the fact that none of the brain tissue sections in the non-clinically ill cases exhibited immunoreactivity for rabies virus or histological evidence of inflammation or Negri bodies. With rabies virus infection in humans, Negri bodies are most commonly found in the hippocampus, pyramidal cells of the cerebral cortex, and Purkinje cells of the cerebellum
. Unfortunately, brain sections from the clinically ill rabies cases were not available for histologic and immunoreactivity analysis for comparison.
Serological testing did provide predictive value into the onset of disease. However, serological testing is of limited value because seroconversion occurs late in the course of the disease
. Studies in different animal models such as mice have shown that antibodies can be detected in sera
, but this response requires the collaboration of inflammatory responses from B-cells and T-cells such as IFN-gamma
. Skunks experimentally-infected intramuscularly with wild-type rabies developed a detectable neutralizing antibody response 7–12 days post- inoculation but not without CNS involvement
. One study has shown that virus-neutralizing antibodies for rabies can be developed around 122 days after vaccination in capuchin monkeys using suckling mouse brain rabies vaccine
. Virus-neutralizing antibody detected in the serum can only really indicate exposure to the rabies virus; the outcome of the invasion relies on a complex interplay of viral and host factors
. Antibody immune response following viral inoculation relies on the replication of virus in animal tissue, in contrast to response from high levels of viral particles in commonly used inactivated vaccines
. However, serum neutralizing antibodies in our study were likely in response to viral antigen in the original inoculum since immunoflorescence was not detected in any pathology tissue, including the sites of infection. Extraneuronal infection in muscle is short lived, being eliminated by immune response
[27, 28]. This may explain the negative immunofluorescence findings at tissue inoculation sites since pathology testing was not done until 134 days post inoculation.