Serologic host response to Helicobacter pylori and Campylobacter jejuni in socially housed Rhesus macaques (Macaca mulatta)

Cohort 1 was used to establish reliable cut-off values for H. pylori sero-positivity to analyze Cohort 2. According to endoscopy performed on the 30 animals in Cohort 1, 13 macaques were negative for H. pylori and 17 were positive. Specific plasma IgG to H. pylori was substantially higher in animals who had been shown by endoscopy to be colonized compared to negative macaques (Table1). Having a positive IgG antibody (ODR >0.340) determination was 100% sensitive but it was only 70% specific for colonization when endoscopy was used as the gold standard (Table2). Because of presumed falsely negative endoscopic results observed in the initial samples, we defined H. pylori-positivity by either a positive endoscopy or IgG ODR >0.340 on the initial plasma. When we tested these combined criteria for 40 follow-up plasma obtained from the same animals, we found that this combination was highly accurate (93% sensitive, 100% specific). Four macaques were H. pylori biopsy-negative on the initial examination but had high CagA, IgG, and IgA values as well as high gastric inflammation scores (Table1). The CagA assay was highly specific (100%) and 61% of the H. pylori-positive macaques were CagA-positive, which is similar to the prevalence in humans[25‐27]. In contrast, the IgA ELISA was only 82% specific and 61% sensitive (Table2). In total, we conclude that determination of the H. pylori IgG status is highly accurate in Rhesus macaques, reflecting the actual H. pylori colonization status. As such, we could use it to assess H. pylori status in monkeys without endoscopy.

First, we examined the ODR-values obtained for the Cohort 2 monkeys. Since the cut-offs obtained from the Cohort 1 macaques and from Cohort 2 were very similar (Table3), we considered them to be reliable. We then examined the H. pylori IgG status in 89 macaques in Cohort 2, using the determined cut-off. Summaries of the data are shown in Table4 and Figure1. A total of 58 (62%) of the 89 Rhesus macaques were H. pylori sero-positive and the sero-prevalence of H. pylori increased with age (Cochran-Armitage Trend Test, p < 0.0001).

The serum of the 89 animals was also tested for the presence of serum IgA antibodies to H. pylori antigens using the IgG-determined status as a standard. An IgA cut-off at 0.39 was considered predictive to determine the H. pylori status since it yielded the same percent (64%) of positivity. Of the 57 macaques determined to be IgA-positive (Figure1), in the youngest group, only 2 (7%) monkeys were both IgA and IgG positive, but 9 (38%) group 2 monkeys, 14 (67%) group 3 monkeys and 14 (82%) monkeys of the oldest group showed double-positive status (Figure1). Thus, IgG antibodies become more prevalent with age in the H. pylori-positive monkeys.

When the Rhesus macaques of Cohort 2 were analyzed with the CagA cut-off established at 0.200, 52% of the macaques were CagA+, similar to the 61% of CagA + monkeys of Cohort 1. As with the H. pylori IgG, the prevalence of CagA antibodies increased with the age of the monkeys (Table4 and Figure1) (Cochran-Armitage Trend Test, p < 0.0001). With the exception of the youngest group of Rhesus macaques, the H. pylori status closely correlated with the CagA status. Next, we compared the percent of macaques positive for both H. pylori and CagA with those that only were positive for H. pylori (Table4). The ratio of double-positive monkeys increased with age (Figure1). In total, 36 (40%) of the 89 animals were positive in both assays and 12 (13%) were only positive for H. pylori.

If our analysis is correct and sero-prevalence is truly higher in older monkeys, ODR-values and monkey age should be correlated. Linear regression analysis to examine H. pylori IgG and IgA and CagA IgG values in relation to monkey age were performed (Figure2A-C). For both H. pylori IgG and IgA, there were significant trends of higher values with age. The same relationship was obtained for CagA IgG. This remains true after adjusting for gender in the linear regression analysis. Moreover, H. pylori IgG levels in female monkeys appear to increase faster as they age compared with males values. Females also had elevated H. pylori IgA and CagA IgG levels compared to males throughout their lifetime (not shown). There also were significant gender differences in H. pylori IgG sero-prevalence between females and males (Odd ratio = 2.56, 95% CI: 1.04-6.32, p = 0.04) when the total number of monkeys was analyzed. In summary, older Rhesus macaques have higher H. pylori IgG and IgA and CagA IgG ODR-values, consistent with a higher prevalence of H. pylori and of CagA-positive strains in older monkeys.

Using a very stringent criterion in which only IgA and IgG double-positive macaques were considered as H. pylori-positive (Figure1), there were 39 (44%) monkeys that were double-positive. Of these, 26 (67%) were triple-positive (H. pylori and CagA IgG, and H. pylori IgA).

We also analyzed C. jejuni positivity by using a cut-off value obtained by both testing 26 human samples of known C. jejuni status[28], and by using statistical evaluation of Cohort 2 values. A total of 59 monkeys (52%) showed serum IgG against C. jejuni (Figure3). There was no correlation between age and infection status (p = 0.23). Using stringent criteria, counting only IgA and IgG double-positive monkeys as C. jejuni positive (Figure3), a total of 31 monkeys (35%) were thus positive. The IgA and IgG status were not correlated, and C. jejuni sero-positivity also was not age-related (Cochran-Armitage Trend Test, p = 0.79). There was no positive or negative association at any age between H. pylori and C. jejuni IgG status.

As described for H. pylori, using regression analysis, we determined whether IgG and IgA ODR-values change with age. In contrast to H. pylori, C. jejuni IgG or IgA values are not higher in older macaques (Figure2D-E). When C. jejuni IgG mean values for each group were analyzed separately, there was a significant difference (p = 0.004) between groups 1 and 2, but there was no drop for IgA (Figure2F). A similar trend has been described in humans[29, 30]. The IgA median stayed constant for the first three groups and rose in the oldest Rhesus macaques. In contrast to H. pylori colonization, C. jejuni positivity did not substantially increase with age.

The studied social group of monkeys (Cohort 2) consisted of 94 Rhesus macaques housed in the Caribbean Primate Research Center in Sabana Seca, Puerto Rico. These animals are descendants of monkeys brought to the Research Center in 1984 from the free-ranging colony on Cayo Santiago Island. They are co-housed and are in constant contact with one another. Serum samples were collected between December 2008 and April 2010. The studies were performed in a cross-sectional design for determination of H. pylori and C. jejuni status, according to age. The Rhesus macaques (Cohort 2, N = 94, 60 females, 34 males) were assigned to groups according to age: group 1 (Infant) includes monkeys <1 year [N = 32 (17 females/15 males)]; group 2 (Juvenile) monkeys were 1.0-2.9 years [N = 24(18/6)]; group 3 (Young Adult) monkeys were 3.0-9.9 years [N = 21(12/9)] and group 4 (Adult) consisted of all monkeys ≥10 years of age [N = 17(13/4)]. Of the 32 monkeys in group 1, five (3 female, 2 male) <0.5 years were excluded from serological analysis, due to potential maternal antibodies.

The 30 Rhesus macaques of Cohort 1 underwent gastroduodenal endoscopic examination under general anesthesia, essentially as described[13]. From each animal, six pinch biopsies of the gastric corpus and six from gastric antral mucosa were obtained. Two biopsies from each region were fixed in neutral 10% buffered formalin and embedded in paraffin. Biopsy sections were stained with haematoxylin and eosin or Genta’s method and viewed under 100x to 1,000x magnification. The presence of inflamamatory cells in the biopsies was scored on coded slides, as described[47]; as were the presence of stained H. pylori organisms, as described[13]. Two other biopsies from antrum and corpus were immediately placed in 0.1 ml sterile 0.9% NaCl on ice and processed for H. pylori isolation, as described[47]. H. pylori isolates were identified as pinhead-colonies, urease-, oxidase- and catalase-positive, and were Gram-negative curved or “gull-wing” rods.

From each Rhesus macaque at the time of each endoscopy, five milliliters of blood was collected in tubes containing 10.5 mg of EDTA and centrifuged; the supernatant plasma was frozen at −70°C. The monkey samples were diluted 1:800 for IgG and 1:100 for IgA. Anti-H. pylori immunoglobulin G (IgG) and A (IgA) levels in the plasma were determined, using a modification of a method with high sensitivity and specificity for human H. pylori positivity, as described[13, 41]. In brief, the H. pylori antigens, composed of a mixture of protein and lipopolysaccharides from five H. pylori strains (ATCC 53722, 53721, 53725, 53726, and 53727), were obtained by water extraction and sonication, as described[41]. The mixture was diluted in 0.05 M carbonate buffer (pH 9.6) to coat each well of a flat-bottom ELISA-plate with 1.0 mg antigen. Plates were coated overnight at 4°C and then blocked for 3 h with 1x PBS containing 0.05% Tween-20, 0.1 mg/ml thimerosal, and 0.1% gelatin. Serum samples were added and plates were incubated for 1 h at 37°C. All washing steps were performed with 1x PBS containing 0.05% Tween-20 and 0.1 mg/ml thimerosal. Goat anti-monkey IgG (gamma chain) or IgA (alpha chain) conjugated to horseradish peroxidase (Rockland Immunochemicals Inc., Gilbertsville, PA) was used as second antibody for detection of responses. A cut-off value was established to distinguish between positive and negative results in the IgG enzyme-linked immunosorbent assay (ELISA) by determining ODR-values (optical density ratio). ODRs were determined for each sample by dividing the OD-value of the sample by the OD-value of two positive controls for H. pylori and C. jejuni IgA and IgG and one positive control for H. pylori CagA, which were included on each ELISA plate as reference specimens. Using 9 Rhesus macaques found to be H. pylori negative by tissue examination (negative culture or histology), the mean ODR-value plus 3 intervals of standard deviation (SD) from those animals was used to define the threshold for negativity (0.34). For IgA, the mean value of 9 uninfected monkeys plus 2 intervals of SD was used to define the threshold for negativity (0.44). An ELISA to detect anti-CagA IgG in plasma from the monkeys was performed using a purified recombinant CagA antigen, as described[48]. The assay was modified with the use of a goat anti-monkey IgG (gamma chain) conjugate. The monkey plasma was diluted 1:100. A cut-off value was established to distinguish between positive and negative CagA results based on the 9 monkeys found by tissue exam to be H. pylori-negative. The mean value plus 2 intervals of SD for the 9 animals was used to define the threshold for positivity (0.20).

For cohort 2, ELISAs were performed as described above. Each sample was tested twice and in the case of disparate results, a third measurement was performed. If samples had discordant ODR-values in the two runs (values close to the negative and positive controls, respectively), we assumed that at least one value was an artifact, and the assay repeated. An assay also was repeated when the ODR-values were close to the threshold values. Resulting ODRs were averaged and evaluated using the determined cut-off values from the reference values from Cohort 1. Additionally, Cohort 2 values were statistically analyzed to evaluate whether the same cut-off values could be obtained without using the reference group. To obtain the cut-off for positive samples, the mean of ODRs between 0.4-1.0 was calculated and 2 times the SD was subtracted, providing a cut-off for positivity (a summary of all values is provided in Table3). To determine the negative cut-off, the mean of ODRs between 0.000-0.299 was calculated and two times the SD was added. Values between 0.300-0.399 were only excluded for the statistical analysis of the cut-off values. After establishing the cut-off values for positivity and negativity, ODRs between 0.300-0.399 were assigned according to these values.

Since antigens from sonicated whole C. jejuni cells did not yield reliable results for serology (not shown), the McCoy antigen[49] (from PEN1, 2, and 3 strains), as described[49, 50], was used for IgG and IgA determination. In brief, C. jejuni cells were harvested in sterile water, washed twice with water and 0.1 g of wet cells were suspended in 2.5 ml 0.2 M glycine-hydrochloride buffer (pH 2.2). Suspensions were stirred at 25°C for 15 min and centrifuged at 11,000 x g for 15 min. The supernatant was collected and sodium hydroxide was added to neutralize the suspension. Next, the suspension was dialyzed against water for 24 h at 4°C and the protein concentration was determined. ELISA was performed as described for H. pylori. Monkey sera were diluted 1:200 and goat anti-monkey IgG (gamma chain) or IgA (alpha chain) conjugated to horseradish peroxidase (Rockland Immunochemicals Inc., Gilbertsville, PA) (1:2000) was used as the secondary antibody. OD₄₀₅ values were normalized using a known positive human control serum (1:200) to compare ODR. Each sample was tested 2–3 times, as described above.

To initially determine a range to examine seropositivity, the mean of sera with ODRs between 0.000-0.299 was calculated and 2 intervals of SD were added, obtaining an initial negative cut-off of 0.338. Next, the mean of ODRs between 0.4-1.0 was calculated and 2 SD intervals substracted resulting in a positive cut-off of 0.318. We also used 10 serum samples from C. jejuni-positive persons and 16 serum samples from C. jejuni-negative persons. The calculated negative cut-off was 0.362 and the positive was 0.418. We calculated the mean value for these three tentative cut-offs, and established a cut-off of 0.340 as an alternative means to define the thresholds for positivity and negativity.

IgA cut-offs were calculated as described for IgG. Since the negative cut-off was 0.368 and the positive cut-off was 0.326, we used a cut-off of 0.350 to analyze Cohort 2.