Zoonotic Transmission of Campylobacter jejuni to Caretakers From Sick Pen Calves Carrying a Mixed Population of Strains With and Without Guillain Barré Syndrome-Associated Lipooligosaccharide Loci

Abstract

Campylobacter jejuni causes foodborne gastroenteritis and may trigger acute autoimmune sequelae including Guillain Barré Syndrome. Onset of neuromuscular paralysis is associated with exposure to C. jejuni lipooligosaccharide (LOS) classes A, B, C, D, and E that mimic and evoke antibodies against gangliosides on myelin and axons of peripheral nerves. Family members managing a Michigan dairy operation reported recurring C. jejuni gastroenteritis. Because dairy cattle are known to shed C. jejuni, we hypothesized that calves in the sick pen were the source of human infections. Fecal samples obtained from twenty-five calves, one dog, and one asymptomatic family member were cultured for Campylobacter. C. jejuni isolates were obtained from thirteen calves and the family member: C. coli from two calves, and C. hyointestinalis from two calves. Some calves had diarrhea; most were clinically normal. Typing of lipooligosaccharide biosynthetic loci showed that eight calf C. jejuni isolates fell into classes A, B, and C. Two calf isolates and the human isolate possessed LOS class E, associated mainly with enteric disease and rarely with Guillain Barré Syndrome. Multi-locus sequence typing, porA and flaA typing, and whole genome comparisons of the thirteen C. jejuni isolates indicated that the three LOS class E strains that included the human isolate were closely related, indicating zoonotic transmission. Whole-genome comparisons revealed that isolates differed in virulence gene content, particularly in loci encoding biosynthesis of surface structures. Family members experienced diarrheal illness repeatedly over 2 years, yet none experienced GBS despite exposure to calves carrying invasive C. jejuni with LOS known to elicit antiganglioside autoantibodies.

Keywords: Campylobacter jejuni; Guillain Barré Syndrome; autoimmunity; gastrointestinal inflammation; outbreak investigation; zoonoses.

FIGURE 1

Cluster analysis of LOS locus content in calf and human isolates. Clustering was performed In PAST 2.12 (Hammer et al., 2001) using the Sorensen coefficient and the unweighted pair groups with arithmetic averages method on presence/absence data for 55 LOS loci in LOS classes A through W as detailed by Parker et al. (2005, and Richards et al. (2013). Numbers at nodes indicate the percentage of 1000 bootstrap replicates that support that node. Full data appear in Supplementary Table 1.

FIGURE 2

Alignment of translated calf isolate cstII/cstIII homologs to known loci. Clustal Omega alignment of cstII/cstIII homologs to known cstII and cstIII sequences (AF215659, AF257460, Cj26094_1202, and AL111168 Cj1140, respectively). The position of residue Asn51 is indicated.

FIGURE 3

Comparison of percent protein identity of one representative strain of each ST to reference strain C. jejuni RM1221 as determined in RAST. Colors indicate protein percent identity of open reading frames within the strains of each sequence type in this study and C. jejuni 11168 (Parkhill et al., 2000) to reference strain C. jejuni RM1221 (Parker et al., 2006), which does not appear in the graph.

FIGURE 4

Cluster analysis of content of 1270 unambiguously identified open reading frames in calf and human isolates. UPGMA clustering was performed as in Figure 1 using the Sorensen coefficient (presence/absence of homologs) on data for 1270 open reading frames in reference strain LM01 that had unambiguously defined functions as determined in RAST using isolate LM01 as the reference strain. Numbers at nodes indicate the percentage of 1000 bootstrap replicates that supported that node. Detailed data used in the cluster analyses are given in Supplementary Table 4. (A) Twelve calf isolates and TW16941; (B) twelve calf isolates, TW16491, and six clinical isolates: 11168 and 81-176 (enteritis); 84-25 (meningitis); 260.94 and HB93-13 (Guillain Barré Syndrome); and CF93-13 (Miller Fisher Syndrome). Full data appear in Supplementary Table 2.

FIGURE 5

Virulence-associated ORFs in calf and human isolates. (A) UPGMA clustering was performed as in Figure 1 using the Sorensen coefficient (presence/absence of homologs) on data for 342 ORFs associated with virulence in C. jejuni. (B) Heat map showing protein% identity of open reading frames in calf isolates to the C. jejuni virulome; variation within and between sequence types. Full data appear in Supplementary Table 3.