Campylobacter jejuni (C. jejuni) is the leading cause of acute gastroenteritis in humans worldwide, posing potential risk to susceptible human and animals due to their zoonotic transmission. Watery or bloody diarrhea, abdominal pain, headache, fever, chills, and dysentery are the main symptoms of campylobacteriosis (Black et al., 1988). Gastroenteritis and Guillain–Barré syndrome (GBS) are the two representative diseases associated with human infection of C. jejuni (Jackson et al., 2014). Over the last 10 years, the incidence of campylobacteriosis has increased in both developed and developing countries (Ruiz-Palacios, 2007), which has generated significant threats to the public’s health. However, the precise role of C. jejuni in the development of clinical condition is largely unknown. Unlike other intestinal pathogens, C. jejuni does not harbor pathogen-defining toxins that explicitly contribute to the disease in humans. Moreover, the majority of campylobacteriosis cases are self-limiting, which makes the understanding of Campylobacter pathogenesis challenging (Crofts et al., 2018).

Polysaccharide capsule is an important virulence factor of clinical importance. Highly variable structures of C. jejuni capsular are the basis of classical Penner serotyping scheme, dividing isolates into 47 serotypes, which has been considered as the gold standard for C. jejuni serotyping (Penner and Hennessy, 1980). The rise of molecular diagnostic method has promoted the development capsular genotyping, re-enforcing the strong correlation between capsular polysaccharide (CPS) and Penner serotypes. This method is not sensitive to the variations in capsule gene expression or influenced by genes or gene products outside the capsule locus (Poly et al., 2011). Consequently, it has been introduced as a fast, readily available and reliable method to assess the serotypes in C. jejuni (Poly et al., 2015). Particular serotypes in the antecedent infection of C. jejuni could contribute to the development of GBS (Poly et al., 2011). HS19 (Kuroki et al., 1993), HS23/36 c (Islam et al., 2009), and HS41 (Lastovica et al., 1997; Zhang et al., 2010a) were the major serotypes commonly represented by the isolates from GBS patients in Japan, Bangladesh, South Africa, and China, respectively. HS4 (Saida et al., 1997), HS2, and HS1 were commonly prevalent in human isolates from sporadic GBS cases all over the world (Guerry et al., 2012). In contrast, HS4 complex, HS2, HS3, HS5/31, and HS8/17 were consistently prevalent in enteritis cases across all regions (Rojas et al., 2019).

Sialylated lipooligosaccharide (LOS) is another virulence factor associated with C. jejuni associated GBS (Godschalk et al., 2004). Molecular mimicry between sialylated C. jejuni LOS and the ganglioside presented on human peripheral nerve drives a cross-reactive immune response, which could result into immune-mediated nerve damage (Yuki et al., 2004). The variability of gene content in C. jejuni LOS biosynthesis locus has led to the assignment of several LOS locus classes (A to S) (Parker et al., 2008). Genes harbored in LOS A, B, and C classes are involved in the synthesis of sialylated LOS. In particular, LOS A class is highly dominant in GBS-associated C. jejuni populations, whereas LOS B, C, and E classes show over-representation in enteritis-associated C. jejuni populations (Ellström et al., 2016; Hameed et al., 2020).

Currently, capsular genotype and LOS class are mainly used to characterize human clinical isolates, but not animal isolates, even though campylobacteriosis is a foodborne disease in which foods of animal origin, especially poultry and cattle, play an important role (Guirado et al., 2020). In this study, a collection of animal strains sampled from a long time span and a wide range of putative hosts was identified by capsular genotyping and LOS classification, compared with the control isolates from enteritis patients. Correlation between LOS class and disease associated capsular genotype in food animal isolates were analyzed. Genetic relationship between the animal isolates with disease associated capsular genotypes and human clinical isolates worldwide were further investigated.

Campylobacteriosis is a worldwide public health problem with numerous socio-economic impacts, ranging from mild symptoms to fatal illness (Hansson et al., 2018), and it is clear that not all C. jejuni strains are equally important as human pathogens (Nichols et al., 2012). To our knowledge, this is the first comprehensive genomic epidemiological study to reveal the genetic diversity and pathogenic correlation of C. jejuni isolates from various animals in China, within a long sampling time span. The characterization of the genetic pathogenicity of C. jejuni is essential for better infection control practice and clinical treatment in humans. Here, we observed that part of animal isolates shared disease associated capsular genotypes with human clinical isolates. Notably, these zoonotic isolates also belonged to the dominant CCs which were over-represented in the clinical isolates from GBS and enteritis patients, specifically CC-22 and CC-21. Notably, CC-21 is among the most common causes of acute human infection (Sheppard et al., 2009), while CC-22 isolates account for up to a third of infections among patients who developed GBS following campylobacteriosis (Islam et al., 2009). Notably, ST22 clonal complex has also been described as “high-risk” lineage, which was also over-represented in isolates that lead to the development of Campylobacter enterocolitis associated post-infection-Irritable Bowel Syndrome in United States (Peters et al., 2021). In this study, the same “high-risk” lineage was also observed among the animal isolates from China and the GBS isolates worldwide (Netherlands, United States, Mexico, Japan, and China), indicating the pathogenic potential of zoonotic isolates need to be highlighted.

Our current data revealed that disease associated serotype reached the highest proportional representation in C. jejuni isolates from cattle, which enhanced our understanding of the pathogenic potential from zoonotic isolates. Recently, human isolates have been reported being closely related to cattle isolates, although the epidemiological meta data necessary to determine causality were unavailable (Hsu et al., 2020). Based on the idea of “one health” (Wolfe et al., 2007), human infection of zoonotic disease needs to be controlled from animals. The source of human infection is thought to be the massive reservoir of C. jejuni in animal population (Parker et al., 2005), since C. jejuni isolates colonize in the intestines of various hosts. While exposure to retail chicken meat has been reported as the major risk factor of campylobacteriosis (Huang et al., 2017), many isolates from poultry carcasses and products did not show genetic relatedness to human pathogenic strains (Zhang et al., 2010b). Notably, drinking of unpasteurized milk and contact with domestic animals could be the other ways of C. jejuni propagation (Wilson et al., 2008). Our results showed that the frequency of disease associated serotypes combined was statistically higher in cattle isolates compared to isolates of other animals. HS19 was especially over-represented in cattle isolates, which has been reported as the major serotype prevalent in C. jejuni isolates from GBS patients (Heikema et al., 2015).

Capsular genotype and LOS class have been concerned the two direct indicators of campylobacteriosis. Association between LOS class and disease associated capsular genotype was of considerable interest for a better understanding of C. jejuni pathogenesis mechanism. Striking genetic correlation has been found between human clinical isolates with GBS associated serotypes and LOS classes (Penner et al., 1983). In our research, this correlation was not only confirmed by human clinical isolates but also observed among animal isolates. Specially, HS19 was prevalent in the enteritis isolates with LOS classes AE, which was also over-represented in cattle isolates with LOS AB classes. In contrast, HS23/26 was dominant in enteritis isolates with LOS E class, which was also present in cattle and poultry isolates with LOS B class. Moreover, GBS maker serotype HS41 was only found in the monkey isolates belonging to the LOS A class, indicating a potential hazard of animal origin.

Although no evident genetic linkage between LOS classification and capsular genotyping in enteritis cases has been previously reported (Karlyshev et al., 2005), our results showed that LOS E was the second dominated LOS class in enteritis isolates, followed by LOS B. Enteritis associated serotype HS3 was solely associated with enteritis isolates of LOS E class, indicating a correlation attributed to the genes shared by LOS and capsules. The bi-functionality of some enzymes involved in polysaccharide biosynthesis may additionally explain why particular LOS and capsule genotypes are linked, associated genes included cj1152 and gmhA2 (Karlyshev et al., 2005). More in-depth studies on gene functionalities of the biosynthesis genes of LOS and capsule are needed to explain why certain LOS classes and capsules are genetically linked together (Heikema et al., 2015). Moreover, our results showed that, unlike the sialylation of isolates with LOS class A could trigger GBS, LOS class E is not characterized as sialylation, indicating sialylation is not required for human diarrheal disease, which was is consistent with the previously reported one (Hameed et al., 2020), both the sialylated and non-sialylated LOS can be used for vaccine design.

Capsule multiplex PCR approach has been introduced as a fast, readily available and reliable method, allowing high-throughput genotyping of a large dataset of isolates within several hours (Poly et al., 2015). In contrast, whole genome sequencing (WGS) has emerged as an effective method to examine the genomic characteristic of Campylobacter isolates with high-resolution, novel types of “non-typable” isolates could be possibly identified. Multiplex PCR approach and WGS analysis offer complementary strengths for isolates identification. WGS could provide isolates with huge genomic information, at present, it takes at least 1 month for isolates sequencing, including the preparation of bacterial DNA, establishment of DNA library, sequencing, reads assembly, and genome annotation. In this study, since we mainly focused on the prevalence of disease associated genotypes among a large dataset of C. jejuni isolates, we chose PCR approach for serotype characterization. Besides of, the serotyped isolates, the left isolates could belong to the uncommon serotypes exist in Penner serotyping scheme but not be involved in this study, as well as the undiscovered serotypes. In fact, non-typable isolates have been consistently reported since Penner first introduced serotyping scheme. The phase variable nature of CPS expression in C. jejuni, especially the exchange of capsular genes by horizontal gene transfer could generate new capsular genotypes (Bacon et al., 2001; Karlyshev et al., 2005; Clarke et al., 2021). In the future, isolates with undiscovered genotypes will be sequenced, CPS gene clusters of these isolates will be investigated using comparative genomics techniques. Paralog or unique sequences in CPS gene cluster could be extracted and be employed as the amplification target for capsular genotypes identification.

Epidemiological data could provide accurate assessment of the burden of campylobacteriosis. In this study, for a better understanding of the pathogenic potential of food animal isolates, C. jejuni isolates collected from representative putative animal hosts within a long sampling time span were genotyped. One drawback of this study is that the quantity of human isolates was much smaller than animal isolates. Largely due to most of the campylobacteriosis cases are self-limiting as well as the rigorous cultivation condition of C. jejuni, disease associated human strain has been still insufficient in Africa and Asia, not to mention about the prevalence information of capsular types (Islam et al., 2009). In view of the quantity of human isolates, the reported human disease associated capsule and lipooligosaccharide types were especially identified, domestic and foreign human isolates from enteritis and GBS patients were involved in this study as the control isolates. As C. jejuni associated food safety problems have been increasingly highlighted, more and more human isolates will be monitored and characterized through the collaboration between hospitals and research institutes. More human isolates will be identified in our future research to confirm or disprove the assumption generated from this study. Moreover, animal isolates from a wider geographically area (other regions in China and other countries) will also be characterized using genotyping and in vitro pathogenic experiment.

Campylobacteriosis is a mainly foodborne disease, food animal plays a primary role. Firstly, this study contributed to a better understanding of the pathogenicity potential of representative food animal isolates, which were collected from a long sampling time span and a wide range of putative hosts. Secondly, distributions of disease associated capsular genotypes and LOS classes in animal isolates were firstly studied. Correlations between LOS class, capsular genotypes and CCs were investigated. High-risk lineages were found dominated in the isolates with disease associated capsular types, including the isolates from GBS patients worldwide and food animals in China, suggesting the possibility of clonal spread of the disease associated capsular genotype isolates across different regions and hosts. Last but not least, our results not only confirmed the previously reported genetic relatedness between cattle isolates and human pathogenic strains, but also indicated that disease associated capsular genotypes and LOS classes all reached a higher frequency in cattle isolates than poultry isolates, providing genetic evidence for these food animal isolates harbor human clinical isolates alike pathogenic characteristics. Generally speaking, this study provided critical supporting data to understand the hazard of C. jejuni isolates from food animals, suggesting cattle isolate with disease associated capsular genotypes is especially need to be eliminated for food safety control, which will lay a foundation for the development of campylobacteriosis biocontrol in animal sector.

All data needed to evaluate the conclusions of this article are present in the article. MLST profiles of 65 isolates in this study have been uploaded in PubMLST2, with new ID: 110484-110548. Five new sequence types include ST-11157, ST-11159, ST-11160, ST-11161, and ST-11162.

This study was carried out in accordance with the principles of the Basel Declaration and recommendations of the institutional administrative committee and ethics committee of laboratory animals. License was issued by the Science and Technology Department of Jiangsu Province in China [SYXK (Su) 2017-0044]. The sampling procedure was supervised and inspected by the Animal Welfare and Ethics Committee of Yangzhou University, with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication number 80–23).

XZ: conceptualization, writing – original draft preparation, writing – review and editing, and visualization. XZ and HT: methodology and formal analysis. XZ and HL: investigation. JH and XJ: project administration. JH: funding acquisition. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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