Serological discrimination of dogs infected with gastric Helicobacter spp. and uninfected dogs

Abstract

Characterization of the humoral immune responses of people to Helicobacter pylori infection has facilitated the investigation of the host response to bacterial virulence factors and the development of sensitive and specific diagnostic tests. Dogs are commonly infected with gastric Helicobacter spp., but the presence of multiple Helicobacter spp. and possible coinfection in individual dogs have complicated serological evaluation. Evaluation of the antigenic homology of Helicobacter spp. revealed that the major protein bands of Helicobacter felis and Helicobacter bizzozeronii, two Helicobacter spp. that infect dogs, were very similar to UreA (29 to 31 kDa), UreB (63 to 66 kDa), and HSP (58 to 60 kDa) of H. pylori, and sera from infected and uninfected dogs bound in a similar way to each antigen. Immunoblotting and an enzyme-linked immunosorbent assay (ELISA) with H. felis ATCC 49179 antigen were performed with 101 serum samples (from 78 infected dogs and 23 uninfected dogs). Samples from uninfected dogs (median = 8) had fewer bands on immunoblotting than samples from infected dogs (median = 16) (P < 0.05). Combinations of the presence of any two of the low-molecular-mass bands (19, 25, 30, 32, and 37 kDa) or the high-molecular-mass bands (86 and 94 kDa) were found almost solely in samples from infected dogs (P < 0.0001). Kinetic ELISA results were significantly higher for samples from infected dogs (median = 0. 0802 optical density unit [OD]/min) than for samples from uninfected dogs (median = 0.01428 OD/min). The combination of ELISA and immunoblotting results gave a specificity of 95.6% and a sensitivity of 79.8%. No correlation between ELISA results, colonization density, degree of inflammation, and presence of lymphoid follicles was observed. The results indicate substantial antigenic homology between H. felis, H. pylori, and H. bizzozeronii. The combination of ELISA and immunoblotting was a highly specific and moderately sensitive indicator of infection. The degree of seropositivity assessed by ELISA was not related to bacterial colonization density, the degree of gastric inflammation, or the presence of lymphoid follicles.

FIG. 1

Detection of Helicobacter spp. DNA in endoscopic gastric biopsy specimens by PCR with primers directed against the 16S rRNA sequence. Lanes 1 to 5, DNAs from specimens from five dogs with HLOs on MS-stained stomach sections, respectively; lanes 6 to 10, DNAs from specimens from five dogs with no HLOs on MS-stained stomach sections, respectively; lane 11, DNA from H. bizzozeronii; lane 12, DNA from H. felis; M, 100-bp DNA ladder.

FIG. 2

Protein profiles (lanes 1) and immunoblot patterns (lanes 2 to 6) obtained with crude extracts of H. pylori 8826 (a lanes), H. felis ATCC 49179 (b lanes), and H. bizzozeronii ATCC 700030 (c lanes) with serum samples from one uninfected (lanes 2) and four infected (lanes 3 to 6) dogs. Lane M, molecular mass marker.

FIG. 3

Protein profiles of H. felis ATCC 49179 HM-CAP (lane A) and crude detergent extract (lane B). Immunoblotting patterns were obtained with the crude H. felis extract with the monoclonal antibody against H. pylori HSP (58 kDa) (lane C) and sera from uninfected (lanes 1 to 6) and infected dogs (lanes 7 to 12). Lane M, molecular mass marker.

FIG. 4

Immunoblotting patterns of monoclonal antibodies against H. pylori HSP (lane 1) and UreB (lane 2) and sera from uninfected (lanes 3 to 5) and infected (lanes 6 to 8) dogs, as obtained with the crude H. pylori 8826 extract.

FIG. 5

Immunoreactivity of sera from uninfected (□) and infected (▿, ○, ▵, ◊) dogs against crude extracts of H. bizzozeronii, H. felis, and H. pylori. Immunoreactivity is expressed as the total density (in square millimeters), which was measured with the NIH Image program on a digitalized image of the immunoblot. The solid bar indicates the mean total density of sera from the four infected dogs for each antigen. Differences between the means were not significant.

FIG. 6

Frequency plots of total number of bands per dog by immunoblotting (A) and ELISA (B) for 101 dogs. Solid bars indicate results for samples from infected dogs, and striped bars indicate results for samples from uninfected dogs. The arrows indicate the cutoff values that were chosen.

FIG. 7

Prevalence of at least one band in a molecular mass (MW) class (A), median number of bands in a molecular mass class (B), and prevalence of at least one of three of the most dense bands in a molecular mass class (C) in samples from infected dogs (solid bars) and samples from uninfected dogs (striped bars). Prevalence is defined as the percentage of dogs with at least one band. ∗, a statistically significant difference between infected and uninfected dogs (P < 0.05).

FIG. 8

Positive predictive values (PPVs) and negative predictive values (NPVs) of ELISA (dotted line), immunoblotting (striped line), and combined ELISA and immunoblotting (solid line) are plotted against the pretest anticipated prevalence of infection with gastric Helicobacter spp. The shaded area indicates the range of prevalence of infection in the dog population (see text for details). The arrow indicates the point prevalence of infection in the 101 dogs evaluated. Positive predictive value curves start at x = 0, while negative predictive value curves start at x = 100.