Association Between Serological Responses to Two Zoonotic Ruminant Pathogens and Esophageal Squamous Cell Carcinoma

Abstract

Questionnaire data have linked contact with ruminants to the risk of esophageal squamous cell carcinoma (ESCC) in high-risk Asian populations. To better understand this observed association, we investigated exposure to two major zoonotic ruminant pathogens relative to ESCC risk. Using enzyme-linked immunosorbent assay, immunofluorescence assay, and Brucella microagglutination test assays, we measured immunoglobulin G anti-Coxiella burnetii and anti-Brucella spp. antibodies in patients with ESCC (n = 177) and population-based controls (n = 177) matched by age, gender, and residence area from the Golestan case–control study in Iran. We found a similarly high seroprevalence of C. burnetii in ESCC cases and controls (75% and 80%, respectively), and a similarly low seroprevalence of Brucella spp. (0% and 0.6%, respectively). While documenting a high exposure to one of two zoonotic ruminant infections, this exposure failed to explain the observed association of ruminant contact and ESCC risk in this high-risk population.

Introduction

Esophageal cancer has two histological subtypes, adenocarcinoma and esophageal squamous cell carcinoma (ESCC), and represents the sixth most common cause of cancer death worldwide (Wang et al. 2018). ESCC is an important public health problem in areas of Asia, including China and Iran (Rafiemanesh et al. 2016, Wang et al. 2018).

Coxiella burnetii and Brucella spp. are zoonotic bacterial pathogens shed from infected domestic ruminants such as goats, sheep, and cattle. These infections can be found worldwide and are primarily an occupational hazard. In humans, C. burnetii is the causative agent of Q fever, and has been associated with non-Hodgkin B-cell lymphoma (Melenotte et al. 2016). Similarly, brucellosis has been linked to brain tumors (Zhang et al. 2011).

Investigators from Iran (Nasrollahzadeh et al. 2015) and India (Dar et al. 2014) have reported a strong association between ruminant animal exposure and ESCC risk based on questionnaire data. This finding suggests a potential role for zoonotic infections in the etiology of ESCC. Our serology study aimed to explore this association further by looking for differential humoral responses to C. burnetii and Brucella spp. We considered these antibody data as stand-alone sensitive and specific measure of exposure to these two zoonotic diseases.

Materials and Methods

Sample procurement

Our study was nested within the Golestan case–control study of ESCC in Iran (Nasrollahzadeh et al. 2015). In brief, 300 newly diagnosed ESCC cases and 571 population-based controls matched by age (±2 years), gender, and residence area were recruited between 2003 and 2007. For this analysis, we selected 177 case–control pairs with at least 500 μL of serum and one control available. The original study was approved by the institutional review boards (IRBs) at Tehran University of Medical Sciences and the U.S. National Cancer Institute (NCI). Written informed consent was obtained from all participants. This study was exempt from IRB review under 45 CFR 46.102(f) by the Human Research Protection Office at the NCI and the Centers for Disease Control and Prevention (CDC).

Antibody titer determination

Serum samples were tested for the presence of anti-C. burnetii immunoglobulin G antibodies using an enzyme-linked immunosorbent assay (ELISA; Virion/Serion, Wurzburg, Germany) followed by an immunofluorescence assay (IFA), as described previously (Miller et al. 2017). Samples were considered positive if they were positive or borderline on the ELISA and had titers ≥1:16 against phase I or phase II antigens by IFA. Assay reproducibility was excellent based on 20 masked replicates, all samples were concordant by ELISA (12 positive and 8 negative). Serum samples were tested for anti-Brucella species antibodies by the Brucella microagglutination test, as previously described, with minor changes (Brown et al. 1981). Total antibody titers ≥1:160 were considered positive and titers between 1:20 and 1:80 were inconclusive. All 20 masked replicates were concordant within one dilution. Laboratory staff at both reference CDC laboratories were blinded to the case–control status.

Statistical analysis

Seropositivity is presented as proportions across categorical variables. Geometric mean titer (GMT) for serology variables was calculated. Conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals for the association of seropositivity to each zoonotic agent with ESCC. All regression models were adjusted for age, gender, and residence area. Kappa statistics were used to assess the level of agreement between serology results and self-reported exposure to ruminants. For the agreement with Brucella, inconclusive and positive results were combined into one category. Two-sided p values <0.05 were considered statistically significant. All analyses were performed using Stata version 14 (Stata Corp, College Station, TX).

Results

Eighty percent of population-based controls and 75% of ESCC cases were seropositive to C. burnetii, with an overall seroprevalence of 77%. Among population-based controls, seropositivity to C. burnetii was significantly higher in individuals residing in rural areas (83%) than in those residing in urban areas (68%), and in individuals who had ever been exposed (self-reported history) to equines (85%) or canines (84%) than those who had never had contact with those animals (62%) (Table 1).

Table 1.

Anti-Coxiella burnetii Seropositivity Among Population-Based Controls and Esophageal Squamous Cell Carcinoma Cases

	Population-based controls (n = 177)		ESCC (n = 177)
	N	Seropositive, %	N	Seropositive, %
Residence area
Rural	136	83	136	80
Urban	41	68	41	59
Education
Illiterate	147	80	160	78
Primary school/higher	30	77	17	53
Exposure to equines
Never	39	62	77	71
Ever	131	85	90	79
Missing	7	86	10	70
Exposure to ruminants
Never	11	73	9	44
Ever	159	80	158	77
Missing	7	86	10	70
Exposure to canines
Never	37	62	57	65
Ever	133	84	110	81
Missing	7	86	10	70
No. of animal types
1	9	67	19	53
2	19	53	35	64
3	41	73	41	80
4	101	88	72	79
Missing	7	86	10	70

ESCC, esophageal squamous cell carcinoma.

ESCC cases were less likely to be seropositive for C. burnetii (adjusted OR: 0.76, p = 0.30; Table 2). For both anti-C. burnetii phase I and phase II antibodies, GMTs were similar between seropositive cases and controls (Table 2).

Table 2.

Anti-Coxiella burnetii and Anti-Brucella Antibody Associations with Esophageal Squamous Cell Carcinoma Risk

	Controls (N = 177)	ESCC (N = 177)	Adjusted ORa(95% CI)
	n (%)	n (%)
Anti-Coxiella burnetii antibodies
Negative	36 (20.3)	44 (24.9)	1
Positiveb	141 (79.7)	133 (75.1)	0.76 (0.46–1.27)
IFA phase I GMT (range)	1:128 (1:16–1:2048)	1:128 (1:16–1:2048)
IFA phase II GMT (range)	1:128 (1:16–1:2048)	1:128 (1:16–1:2048)
Anti-Brucella antibodies
Negative (<20)	97 (54.8)	120 (67.8)	1
Inconclusive (20–80)	80 (45.2)	56 (31.6)	0.60 (0.40–0.91)
Positive (≥160)	0	1 (0.6)

^aAdjusted for matching variables (age, gender, and residence area).

^bSamples positive or borderline against phase II antigen by enzyme-linked immunosorbent assay and validated by IFA with phase I or phase II titer of ≥1:16 were considered positive.

CI, confidence interval; GMT, geometric mean titer; IFA, immunofluorescence assay; OR, odds ratio.

None of the controls were seropositive for Brucella, 45% were inconclusive, and 55% were seronegative (Table 2). Similarly, only 0.6% (1 of 177) of ESCC cases were seropositive for Brucella, 32% were inconclusive, and 68% were seronegative. The adjusted OR for inconclusive status was 0.60 (p = 0.03; Table 2).

There was low agreement between the serology results and self-report exposure to ruminants (Kappa coefficients: 0.08 and 0.008 for C. burnetii and Brucella, respectively).

Discussion

Our findings demonstrate a high seropositivity for C. burnetii among individuals in Golestan Province, consistent with increased exposure to livestock. However, seroprevalences and GMTs were similar between cases and controls, indicating that this zoonotic infection is not a risk factor for ESCC. Based on our findings, there is not sufficient evidence to suggest that exposure to Brucella spp. is associated with ESCC risk; however, due to the transient nature of anti-Brucella antibody titers, additional studies may be warranted.

To better understand the ruminant association with ESCC that has been observed in self-reported questionnaire data, additional zoonotic agents should be evaluated. In particular, some ruminant viruses may have etiological significance in ruminant tumors localized to the alimentary and upper aerodigestive tracts (Cousens et al. 2009). Future studies should also address noninfectious hypotheses and the possibility that the ruminant association could be a proxy for other unidentified risk factors.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This study was supported by the U.S. Centers for Disease Control and Prevention and the Intramural Research Program of the U.S. National Cancer Institute.