Paul J. Plummer

Emergence of a Tetracycline-Resistant Campylobacter jejuni Clone Associated with Outbreaks of Ovine Abortion in the United States

ABSTRACT Campylobacter infection is one of the major causes of ovine abortions worldwide. Historically, Campylobacter fetus subsp. fetus was the major cause of Campylobacter-associated abortion in sheep; however, Campylobacter jejuni is increasingly associated with sheep abortions. We examined the species distribution, genotypes, and antimicrobial susceptibilities of abortion-associated Campylobacter isolates obtained from multiple lambing seasons on different farms in Iowa, Idaho, South Dakota, and California. We found that C. jejuni has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in the United States. Most strikingly, the vast majority (66 of 71) of the C. jejuni isolates associated with sheep abortion belong to a single genetic clone, as determined by pulsed-field gel electrophoresis, multilocus sequence typing, and cmp gene (encoding the major outer membrane protein) sequence typing. The in vitro antimicrobial susceptibilities of these isolates to the antibiotics that are routinely used in food animal production were determined using the agar dilution test. All of the 74 isolates were susceptible to tilmicosin, florfenicol, tulathromycin, and enrofloxacin, and 97% were sensitive to tylosin. However, all were resistant to tetracyclines, the only antibiotics currently approved in the United States for the treatment of Campylobacter abortion in sheep. This finding suggests that feeding tetracycline for the prevention of Campylobacter abortions is ineffective and that other antibiotics should be used for the treatment of sheep abortions in the United States. Together, these results indicate that a single tetracycline-resistant C. jejuni clone has emerged as the major cause of Campylobacter-associated sheep abortion in the United States.

Molecular Evidence for Zoonotic Transmission of an Emergent Highly Pathogenic Campylobacter jejuni Clone in the United States

ABSTRACT Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.

Bacterial Community Profiling of Milk Samples as a Means to Understand Culture-Negative Bovine Clinical Mastitis

Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease.

Deep Sequencing Analysis Reveals Temporal Microbiota Changes Associated with Development of Bovine Digital Dermatitis

ABSTRACT Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive etiologic agent associated with the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study details the deep sequencing-based metagenomic evaluation of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression. Over 175 million sequences were evaluated by utilizing both shotgun and 16S metagenomic techniques. Based on the shotgun sequencing results, there was no evidence of a fungal or DNA viral etiology. The bacterial microbiota of biopsy specimens progresses through a systematic series of changes that correlate with the novel morphological lesion scoring system developed as part of this project. This scoring system was validated, as the microbiota of each stage was statistically significantly different from those of other stages (P < 0.001). The microbiota of control biopsy specimens were the most diverse and became less diverse as lesions developed. Although Treponema spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The consortium of Treponema spp. identified at the onset of disease changes considerably as the lesions progress through the morphological stages identified. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development.

Pathogenicity of an emergent, ovine abortifacient Campylobacter jejuni clone orally inoculated into pregnant guinea pigs

OBJECTIVE To compare pathogenicity of an emergent abortifacient Campylobacter jejuni (IA 3902) with that of reference strains after oral inoculation in pregnant guinea pigs. ANIMALS 58 pregnant guinea pigs. PROCEDURES 12 animals were challenged IP with C jejuni IA 3902 along with 5 sham-inoculated control animals to confirm abortifacient potential. Once pathogenicity was confirmed, challenge via oral inoculation was performed whereby 12 guinea pigs received IA 3902, 12 received C jejuni isolated from ovine feces (OF48), 12 received a fully sequenced human C jejuni isolate (NCTC 11168), and 5 were sham-inoculated control animals. After abortions, guinea pigs were euthanized; samples were collected for microbial culture, histologic examination, and immunohistochemical analysis. RESULTS C jejuni IA 3902 induced abortion in all 12 animals following IP inoculation and 6 of 10 animals challenged orally. All 3 isolates colonized the intestines after oral inoculation, but only IA 3902 induced abortion. Evidence of infection existed for both IA 3902 and NCTC 11168; however, C jejuni was only recovered from fetoplacental units of animals inoculated with IA 3902. Immunohistochemical analysis localized C jejuni IA 3902 infection to subplacental trophoblasts, perivascular tissues, and phagocytes in the placental transitional zone. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that C jejuni IA 3902 was a unique, highly abortifacient strain with the ability to colonize the intestines, induce systemic infection, and cause abortion because of its affinity for the fetoplacental unit. Guinea pigs could be effectively used in the study of septic abortion after oral inoculation with this Campylobacter strain.

LuxS and Quorum-Sensing in Campylobacter

Several intercellular bacterial communication mechanisms have been identified in a broad range of bacterial species. These systems, collectively termed quorum-sensing systems, have been demonstrated to play significant roles in a variety of bacterial processes including motility, biofilm formation, expression of virulence genes, and animal colonization. Campylobacter jejuni is known to possess a LuxS/ autoinducer-2 (AI-2) mediated system that have been partially characterized over the last decade. AI-2 is formed as a byproduct of the activated methyl recycling pathway, specifically by the LuxS enzyme. Previous work in our laboratory and that of others has demonstrated that this gene is involved in a variety of physiologic pathways of C. jejuni including motility, autoagglutination, cytolethal distending toxin (CDT) expression, flagellar expression, oxidative stress, and animal colonization. This review article will summarize the current research associated with LuxS in C. jejuni and will provide insights into the role of this system in the metabolism and intercellular communication of this organism. Additionally, the evidence for other quorum-sensing pathways in Campylobacter will be discussed.

Occurrence and molecular analysis of Campylobacter in wildlife on livestock farms

Wildlife harbor a variety of Campylobacter spp. and may play a significant role in the transmission of Campylobacter to livestock. Although studies have been conducted on wildlife-associated Campylobacter isolates from farms in other countries, there are little data available for livestock farms in the United States. In addition, the critical questions of whether wildlife harbor Campylobacter that is pathogenic to ruminants and/or antibiotic-resistant Campylobacter have yet to be addressed. We captured wild small mammals (n=142) and small birds (n=188) at livestock farms in central Iowa and sampled them for thermophilic Campylobacter during autumn 2009, spring 2010, and autumn 2010. Overall prevalence was 4.79%, with isolates found only in wild birds. Molecular typing revealed four multilocus sequence types (STs), three of which are novel. The remaining ST (ST-806) was found in two house sparrows and is an ST previously associated with ruminant abortion cases. Further analysis of ST-806 wild bird and ruminant abortion isolates by pulsed-field gel electrophoresis, resistance gene location, and antibiotic susceptibility tests indicated that the isolates are nearly identical. This is the first account of isolation of Campylobacter types from wild birds that are known to be pathogenic to ruminants. Furthermore, these same two wild bird isolates are resistant to the antibiotic fluoroquinolone. Our results indicate there is an overall low prevalence of Campylobacter in selected wildlife in Iowa, but suggest that wildlife may play a role in the epidemiology of pathogenic Campylobacter for domestic livestock, and may also serve as a reservoir for antibiotic-resistant Campylobacter.

Critical Role of LuxS in the Virulence of Campylobacter jejuni in a Guinea Pig Model of Abortion

ABSTRACT Previous studies on Campylobacter jejuni have demonstrated the role of LuxS in motility, cytolethal distending toxin production, agglutination, and intestinal colonization; however, its direct involvement in virulence has not been reported. In this study, we demonstrate a direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxS complement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902 luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxS gene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivo model of natural disease.

Assessment and Management of Pain in Small Ruminants and Camelids

Many disease processes and management procedures of small ruminants have the potential to result in painful or noxious stimuli. There are a variety of medications and interventions that can be used to minimize the long-term consequences of pain in these species. The first portion of this article focuses on the commonly used medications available for pain management of small ruminants and discusses the benefits and side effects to their use. The second portion of the article focuses on the management of pain associated with common diseases or procedures of these species.

Identification of a Key Amino Acid of LuxS Involved in AI-2 Production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2− variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ∼100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter.