Discrimination of Major Capsular Types of Campylobacter jejuni by Multiplex PCR

Poly, Frédéric; Serichatalergs, Oralak; Schulman, M. E.; Ju, Jennifer; Cates, Cory N.; Kanipes, Margaret I.; Mason, Carl J.; Guerry, Patricia

doi:10.1128/jcm.02348-10

Cited by 52 publications

(82 citation statements)

References 46 publications

(43 reference statements)

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“…At the whole-genome level, the reference strains for serovars 5 and 12 are not identical, with the serovar 12 strain sharing 81.7% nucleotide identity with the complete SH0165 strain (serovar 5) (our unpublished data). Further differences between these serovars may be due to a modification of the capsule encoded by a gene outside the main capsule locus or differential expression of capsule genes (29,69,70).…”

Section: Resultsmentioning

confidence: 99%

“…There are some problems with the serotyping assay, including the difficulty of consistently producing specific antisera against several reference strains, variation in growth conditions, cross-reactions between serovars, and the very small number of laboratories that currently perform this test (8,18,20,26). Molecular serotyping systems have been developed for other bacteria based on the genes involved in biosynthesis of extracellular polysaccharide structures such as LPS O antigens or capsular polysaccharides (27)(28)(29)(30).…”

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confidence: 99%

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Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Howell

Weinert

Luan

et al. 2013

Journal of Bacteriology

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gHaemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3= end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Howell

Weinert

Luan

et al. 2013

Journal of Bacteriology

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“…These techniques have been developed for other bacteria based on the genes involved in biosynthesis of extracellular polysaccharide structures, such as lipopolysaccharides (LPS) or capsules (36)(37)(38)(39). These are also likely to be the dominant components of the serotyping antigens for H. parasuis based on the antigen preparation techniques for both the GID and IHA methods (22,23,30,31).…”

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confidence: 99%

Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

et al. 2015

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iHaemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 ؋ 10 5 ng/l bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis. Haemophilus parasuis is a Gram-negative bacterium commonly found in the upper respiratory tract of the pig, and it was identified in 1910 as the causative agent of a globally prevalent systemic disease of pigs known as Glässer's disease. The more severe presentations of this disease include arthritis, meningitis, polyserositis, septicemia, and pneumonia (1-5). Based on statistics from the United States, H. parasuis is the leading cause of mortality (alongside the porcine reproductive and respiratory syndrome [PRRS] virus) in nursery herds, and it is the third most important bacterial pathogen affecting finisher herds (6). H. parasuis also contributes to a multifactorial porcine respiratory disease complex, the leading cause of mortality in grower-finisher pigs in the United States (7). Diagnostic submissions to veterinary investigation centers of the Animal and Plant Health Agency (APHA) in 2013 and 2014 recorded the highest annual rates of diagnosis of disease incidents due to H. parasuis in England and Wales since 2002 (8, 9). In the third quarter of 2013, the diagnostic rate reached nearly 8% of diagnosable submissions (8,9). This disease characteristically manifests postweaning and is associated with the loss of maternally derived antibodies and the endemic presence of the bacterium in herds (1, 5).Treatment and prevention of Glässer's disease are implemented via strategic delivery of penicillin-based antimicrobials in feed or water. Ongoing treatment may be...

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“…The complete genome sequence of IA3902 (a clone SA isolate), including the entire CPS locus, was reported previously (19). Similar to other C. jejuni CPS loci studied so far, the capsular locus of IA3902 is organized into three regions in which the variable biosynthetic region is flanked by the conserved regions responsible for capsule transport and assembly (20,22,35,36). The biosynthetic CPS locus of IA3902 (located between kpsC and kpsF, excluding these two genes) is approximately 26.4 kb long and comprises 22 genes of various predicted functions involved in capsule synthesis and modification ( Table 1).…”

Section: Resultsmentioning

confidence: 82%

Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

et al. 2017

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Campylobacter jejuni is a zoonotic pathogen, and a hypervirulent clone, named clone SA, has recently emerged as the predominant cause of ovine abortion in the United States. To induce abortion, orally ingested Campylobacter must translocate across the intestinal epithelium, spread systemically in the circulation, and reach the fetoplacental tissue. Bacterial factors involved in these steps are not well understood. C. jejuni is known to produce capsular polysaccharide (CPS), but the specific role that CPS plays in systemic infection and particularly abortion in animals remains to be determined. In this study, we evaluated the role of CPS in bacteremia using a mouse model and in abortion using a pregnant guinea pig model following oral challenge. Compared with C. jejuni NCTC 11168 and 81-176, a clone SA isolate (IA3902) resulted in significantly higher bacterial counts and a significantly longer duration of bacteremia in mice. The loss of capsule production via gene-specific mutagenesis in IA3902 led to the complete abolishment of bacteremia in mice and abortion in pregnant guinea pigs, while complementation of capsule expression almost fully restored these phenotypes. The capsule mutant strain was also impaired for survival in guinea pig sera and sheep blood. Sequence-based analyses revealed that clone SA possesses a unique CPS locus with a mosaic structure, which has been stably maintained in all clone SA isolates derived from various hosts and times. These findings establish CPS as a key virulence factor for the induction of systemic infection and abortion in pregnant animals and provide a viable candidate for the development of vaccines against hypervirulent C. jejuni. KEYWORDS Campylobacter, capsule, systemic infection, abortion, sheep, bacteremiaA s a zoonotic pathogen, Campylobacter is a leading cause of bacterial foodborne gastroenteritis in humans (1). In addition, Campylobacter species have been recognized as one of the most common causes of ovine abortion in the United States and worldwide, with an overall abortion rate of 5 to 50% in affected flocks (2). As a consequence, Campylobacter poses a significant economic burden on sheep producers and greatly impacts sheep health and welfare. A national study, NAHMS Sheep 2001, conducted by the USDA/APHIS/Veterinary Services in collaboration with the American Sheep Industry Association revealed that Campylobacter species ranked first among all infectious causes of abortion within the last 3 years of the study, with 53.7% of the reported cases being confirmed by a veterinarian or diagnostic laboratory (3).Historically, Campylobacter fetus subsp. fetus accounted for the majority of the Campylobacter spp. associated with ovine abortion; however, we recently discovered a remarkable shift in the etiology of the disease (4, 5). Specifically, a highly virulent

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Discrimination of Major Capsular Types of Campylobacter jejuni by Multiplex PCR

Cited by 52 publications

References 46 publications

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

Key Role of Capsular Polysaccharide in the Induction of Systemic Infection and Abortion by Hypervirulent Campylobacter jejuni

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