Kerry K. Cooper

Necrotic enteritis in chickens: A paradigm of enteric infection by Clostridium perfringens type A

Withdrawal of antimicrobial growth promoters and ionophore coccidiostats has been accompanied by a resurgence in incidence of necrotic enteritis (NE), a severe Clostridium perfringens-induced disease which some consider the most clinically dramatic bacterial enteric disease of poultry. Lesions, in jejunum and ileum, are focal-to-confluent, often with a tightly adhered pseudomembrane, and hemorrhage is uncommon. The key risk factor for development of NE is an intestinal environment that favors growth of the organism. Birds on high energy, protein-rich, wheat- or barley-based diets experience NE at a rate up to ten times greater than do birds on maize-based diets. Specific strains of type A cause NE, although only a few specific virulence attributes are known. The role of alpha toxin (CPA) has been called into question by the finding that an engineered CPA mutant retained full virulence in vivo, although the counterpoint to this is the finding that immunization with CPA toxoids provides substantial protection against NE. A recently described toxin, NetB, seems likely to be involved in pathogenesis of infection by most NE strains. Immunization with CPA, NetB, or other proteins, delivered by conventional means or vectored by recombinant attenuated Salmonella vectors may help the industry deal with NE. Future progress may be based in large part on genomic and proteomic analyses.

Immunization with recombinant alpha toxin partially protects broiler chicks against experimental challenge with Clostridium perfringens.

Necrotic enteritis (NE) in poultry has re-emerged as a concern for poultry producers, due in part to banning, by many countries, of the use of antimicrobial growth promoters in feeds. This re-emergence has led to a search for alternative methods for control of the disease, particularly vaccination. The objective of this work was to determine if vaccination of broiler chicks with recombinant alpha toxin protected against experimental challenge. Broiler chicks were vaccinated subcutaneously at 5 and 15 days of age, followed 10 days later by challenge with Clostridium perfringens. Birds were challenged twice daily on 4 consecutive days by mixing C. perfringens cultures with feed (three parts culture: four parts feed). Non-vaccinated birds challenged with C. perfringens developed NE at the rate of 87.8%, while only 54.9% of vaccinated birds developed lesions. In addition, non-vaccinated birds had lesion scores averaging 2.37, while average scores in vaccinated birds were 1.35. Vaccination produced an antibody response, with post-vaccination anti-alpha toxin IgG (IgY) titers in vaccinated birds more than 5-fold greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers>15-fold higher than those in non-vaccinated birds. These results suggest that alpha toxin may serve as an effective immunogen, and, as such, may play a role in pathogenesis.

Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis.

Poultry necrotic enteritis (NE) has, over recent decades, been prevented and treated by addition of antimicrobials to poultry feed. Recent bans of antimicrobial growth promoters in feed, as well as other factors, have led to a slow, worldwide re-emergence of NE. Understanding of pathogenesis of NE has been hampered by lack of a consistent and effective experimental model in which virulence of strains can be reliably evaluated, with an endpoint yielding lesions comparable to those seen in acute NE in the field. The overall objective of this work was to develop an experimental approach that would allow consistent production of a full range of clinical signs and lesions of the disease, and to do so without use of coccidia as inciting agents. In addition, we assessed the virulence of strains of Clostridium perfringens from field cases of NE. Broiler chicks fed a commercial chick starter for 7 days post-hatch were switched to a high protein feed mixed 50:50 with fishmeal for an additional 7 days. On day 14, feed was withheld for 20 h, and birds were then offered feed mixed with C. perfringens (3 parts culture to 4 parts feed) twice daily on 4 consecutive days. On average, >75% of challenged birds developed typical gross lesions when inoculated with type A strains from field cases of NE. In addition, in vivo passage apparently increases strain virulence. Virulence varies from strain-to-strain; NetB-producing strains were virulent, as were some NetB non-producing strains.

TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks.

Clostridium perfringens type A and type C are causative agents of necrotic enteritis (NE) in poultry. TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. Others have since reported TpeL in type A isolates from NE outbreaks, suggesting that it may contribute to the pathogenesis of NE. The virulence of TpeL-positive and -negative C. perfringens strains from cases of NE was examined by challenge of broiler chicks. Gross lesions typical of NE were observed in all challenged birds, and those inoculated with TpeL(pos) strains had higher average macroscopic lesion scores than those inoculated with a TpeL(neg) strain. Infection with TpeL(pos) strains may yield disease with a more rapid course and higher case fatality rate. Thus, TpeL may potentiate the effect of other virulence attributes of NE strains of C. perfringens. However, TpeL(pos) and Tpel(neg) strains compared here were not isogenic, and definitive results await the production and testing of specific TpeL mutants.

The Campylobacter jejuni Dps Homologue Is Important for In Vitro Biofilm Formation and Cecal Colonization of Poultry and May Serve as a Protective Antigen for Vaccination

ABSTRACT In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 105 CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.

Diagnosing clostridial enteric disease in poultry

The world’s poultry industry has grown into a multibillion-dollar business, the success of which hinges on healthy intestinal tracts, which result in effective feed conversion. Enteric disease in poultry can have devastating economic effects on producers, due to high mortality rates and poor feed efficiency. Clostridia are considered to be among the most important agents of enteric disease in poultry. Diagnosis of enteric diseases produced by clostridia is usually challenging, mainly because many clostridial species can be normal inhabitants of the gut, making it difficult to determine their role in virulence. The most common clostridial enteric disease in poultry is necrotic enteritis, caused by Clostridium perfringens, which typically occurs in broiler chickens but has also been diagnosed in various avian species including turkeys, waterfowl, and ostriches. Diagnosis is based on clinical and pathological findings. Negative culture and toxin detection results may be used to rule out this disease, but isolation of C. perfringens and/or detection of its alpha toxin are of little value to confirm the disease because both are often found in the intestine of healthy birds. Ulcerative enteritis, caused by Clostridium colinum, is the other major clostridial enteric disease of poultry. Diagnosis of ulcerative enteritis is by documentation of typical pathological findings, coupled with isolation of C. colinum from the intestine of affected birds. Other clostridial enteric diseases include infections produced by Clostridium difficile, Clostridium fallax, and Clostridium baratii.

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

BackgroundAlthough serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.ResultsWe determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.ConclusionsOur data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a Bsu BI/Pst I methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.

Complete Genome Sequence of Campylobacter jejuni Strain S3

Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis in the world; however, there is only one complete genome sequence of a poultry strain to date. Here we report the complete genome sequence and annotation of the second poultry strain, C. jejuni strain S3. This strain has been shown to be nonmotile, to be a poor invader in vitro, and to be a poor colonizer of poultry after minimal in vitro passage.

A Campylobacter jejuni Dps Homolog Has a Role in Intracellular Survival and in the Development of Campylobacterosis in Neonate Piglets

Iron acquisition is an absolute requirement by most microorganisms for host survival. In this work, we investigated the Campylobacter jejuni iron binding Dps protein for a potential role in virulence. In vitro assays using J774A.1 macrophage-like cells demonstrated a 2.5 log reduction in C. jejuni survival of the Dps mutant and a reduction of four logs in invasion of HEp-2 epithelial cells compared to the wild-type strain. To examine the role of the dps gene in host pathogenesis, the piglet model was used in C. jejuni challenge studies. In vivo inoculation studies of newborn piglets with wild-type C. jejuni demonstrated an 11-fold upregulation of the dps gene and intestinal lesion production typical of campylobacteriosis in humans. In contrast, piglets inoculated with the dps mutant were not colonized and remained normal throughout the study period. Mucosal lesion production was restored in piglets inoculated with the complemented Dps mutant strain. Based on these results, we conclude that the C. jejuni Dps homolog is a virulence factor in the production of campylobacteriosis, and warrants further investigation.

Complete Genome Sequences of Two Escherichia coli O145:H28 Outbreak Strains of Food Origin

ABSTRACT Escherichia coli O145:H28 strain RM12581 was isolated from bagged romaine lettuce during a 2010 U.S. lettuce-associated outbreak. E. coli O145:H28 strain RM12761 was isolated from ice cream during a 2007 ice cream-associated outbreak in Belgium. Here we report the complete genome sequences and annotation of both strains.