The original habitats of Rhesus macaques range from Central to Southeast Asia. However, populations of Rhesus macaques have been living in captivity for generations, since they are the most important non-human primates used for medical and biological research[32–36]. For this study, we analyzed gastric biopsies and blood samples from the animal facility at the Uniformed Services University of the Health Sciences, to establish reliable cut-off values for detecting H. pylori antibodies (Cohort 1). To investigate the sero-prevalence of responses to H. pylori and C. jejuni antigens in social Rhesus macaques, we studied blood samples routinely taken from a population of animals living together in one outdoor cage in the Caribbean Primate Research Center since 1984 (Cohort 2). We sought to determine how prevalent H. pylori and C. jejuni-specific antibodies are in a group of Rhesus macaques to evaluate whether the colonization rates of monkeys are similar to human populations.
Although H. pylori and C. jejuni are phylogenetically close, they differ substantially in the nature of their antigens, even including conserved proteins like their heat-shock protein 60. Among other factors, flagellae proteins, adhesins and outer membrane proteins differ considerably between C. jejuni and H. pylori. In this study we used water-extracted proteins as antigens. Potential cross-reactions between H. pylori and Campylobacter antigens have been addressed in previous studies[41, 42] and are considered to be of minor significance. Moreover, the immune response to CagA is specific for H. pylori and no cross-reactions have been described.
Using the biopsy and blood samples from Cohort 1, we were able to establish ELISAs that were 100% specific and 93% sensitive for H. pylori IgG detection, and 100% sensitive for CagA IgG. In contrast, the IgA ELISA was much less specific and sensitive and as is the case with humans, not sufficiently accurate for classification of H. pylori status in Rhesus macaques.
The determined ELISA parameters were used to evaluate Cohort 2 for H. pylori prevalence. In developed countries, H. pylori is disappearing[1, 2] but in developing countries, H. pylori prevalence remains high[3, 4]. The prevalence of H. pylori in monkeys clearly increases with age. In the oldest monkeys, positivity increased to 94%. Even using a more stringent IgG and IgA double positive criterion to classify monkeys as H. pylori-colonized, the trends remain the same and the same age-dependency was observed for the CagA IgG ELISA. Parallel age trends are commonly observed in studies conducted in human populations[27, 45]. However, in contrast to humans, younger Rhesus macaques have the same likelihood to have a CagA-positive strain as do the macaques in the oldest group. Comparing double- (44%) and triple- (29%) positive rates, about 67% of the H. pylori-positive monkeys were colonized with a CagA + strain. This is comparable to the situation among humans in developing countries[27, 45]. In summary, the socially living Rhesus macaques in captivity resemble the pre-modern situation for H. pylori colonization. One possible route of acquiring H. pylori in younger monkeys is by oral-oral contact with older monkeys. Free-living monkeys and monkeys in captivity have close contact with each other, making it impossible to trace infection routes back to parents, siblings, aunts or playmates, without the ability to genotype the strains. How the Rhesus macaques originally acquired H. pylori also remains unclear. Genotyping would help to elucidate whether the strains are of human origin, and whether such strains were introduced into the Rhesus macaque population before or after they were captured in 1938 and brought to Cayo Santiago Island (i.e. carrying an Asian strain). Strain isolation from gastric biopsies and multi-locus sequence typing (MLST) analysis could help answer these questions.
To determine the cut-off values for the C. jejuni ELISA, we used blood samples from humans of known C. jejuni status. The tested 94 monkeys did not show any signs of diarrhea at the time of sampling and had no evidence of acute infection. In contrast to H. pylori, there was no correlation between age and C. jejuni sero-positivity. In general, the prevalence of C. jejuni IgG was equally high in all the groups, with a significant drop of prevalence of IgG antibodies in juvenile Rhesus macaques. However, a high prevalence of C. jejuni IgA was observed in that group. In areas in which C. jejuni infection is hyper-endemic and infection is recurrent, young children develop high levels of specific serum IgG antibodies. With continued exposure, IgG levels wane and IgA levels rise[29, 30], and the duration of colonization diminishes reflecting development of gut immunity. The drop in IgG between infant and juvenile macaques that we observed is consistent with this phenomenon. As in humans, C. jejuni causes transient infection in Rhesus macaques and specific antibodies are detected in convalescence.