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Campylobacter is a major cause of acute human diarrheal illness. Broiler chickens constitute a primary reservoir for C. jejuni leading to human infection. Consequently, there is a need for developing novel intervention methods. Antimicrobial peptides (AMP) are small proteins which have evolved in most lifeforms to provide defense against microbial infections. To date, over 3000 AMP have been discovered; however, few of them have been analyzed specifically for ability to kill campylobacters. We selected and evaluated a set of 11 unique chemically synthesized AMP for ability to inhibit growth of C. jejuni. Six of the AMP we tested produced zones of inhibition on lawns of C. jejuni. These AMP included: NRC-13, RL-37, Temporin L, Cecropin–Magainin, Dermaseptin, and C12K-2β12. In addition, MIC were determined for Cecropin–Magainin, RL-37 and C12K-2β12 against 15 isolates of Campylobacter representing the three most common pathogenic strains. MIC for campylobacters were approximately 3.1 µg/mL for AMP RL-37 and C12K-2β12. MIC were slightly higher for the Cecropin–Magainin AMP in the range of 12.5 to 100 µg/mL. These AMP are attractive subjects for future study and potential in vivo delivery to poultry to reduce Campylobacter spp. populations.

Here, we announce the draft genome sequences of two Clostridium strains, C8-1-8 and C2-6-12, isolated from the cecal contents of commercial broiler chickens (in Athens, GA). These strains may represent potentially novel species within the genus Clostridium, and these draft genomes allow further investigation into potential probiotics for poultry.

Here, we announce the draft genome sequences of two Clostridium strains, C8-1-8 and C2-6-12, isolated from the cecal contents of commercial broiler chickens (in Athens, GA). These strains may represent potentially novel species within the genus Clostridium , and these draft genomes allow further investigation into potential probiotics for poultry.

Avian metapneumoviruses (aMPVs), which have been reported in many countries, cause acute upper respiratory tract disease in chickens and turkeys. Using next-generation sequencing, we report here the complete genome sequence of an aMPV subtype B strain that was isolated from a turkey in Hungary in 1989.

Avian metapneumoviruses (aMPVs), which have been reported in many countries, cause acute upper respiratory tract disease in chickens and turkeys. Using next-generation sequencing, we report here the complete genome sequence of an aMPV subtype B strain that was isolated from a turkey in Hungary in 1989.

Here, we present the draft genome sequences of two Paenibacillus strains, An7 and USDA918EY, isolated from goose feces (Bend, OR, USA) and chicken ceca (Pomona, CA, USA), respectively. These data may assist with analyses of microorganisms associated with free-ranging and commercial avian species.

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.

Increasingly, molecular methods have become important in identification and confirmation of bacteria at the species level. Rapid molecular methods provide sensitivity and specificity while reducing cost and resources. The primary goal of this study was to develop a real-time PCR assay for identification of Escherichia coli from an agar plate. GadE (gadE) directly regulates the glutamate-dependent acid response system (GDAR) in E. coli and is responsible for survival of at pH 2. Based on gene sequence data, a real-time PCR assay targeting gadE was developed for this purpose. Seventy bacterial isolates recovered from ground beef enrichments and 714 isolates from caecal contents were identified biochemically and tested with the real-time PCR assay developed in this study. The PCR assay and the biochemical identification had 100% agreement on the tested isolates. The gadE real-time PCR assay was demonstrated in this study to be an inexpensive, reliable method for confirming E. coli colonies within 1.5 h from an agar plate, thereby saving on final identification time.

Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal, and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates. A predicted N-acetylmuramoyl-L-alanine amidase or MurNAc-LAA (also known as peptidoglycan aminohydrolase, NAMLA amidase, NAMLAA, amidase 3, and peptidoglycan amidase; EC 3.5.1.28) was identified that would hydrolyze the amide bond between N-acetylmuramoyl and L-amino acids in certain cell wall glycopeptides. The gene encoding this protein was subsequently cloned from genomic DNA of a C. perfringens isolate by polymerase chain reaction, and the gene product (PlyCpAmi) was expressed to determine if it could be utilized as an antimicrobial to control the bacterium. By spot assay, lytic zones were observed for the purified amidase and the E. coli expression host cellular lysate containing the amidase gene. Turbidity reduction and plate counts of C. perfringens cultures were significantly reduced by the expressed protein and observed morphologies for cells treated with the amidase appeared vacuolated, non-intact, and injured compared to the untreated cells. Among a variety of C. perfringens strains, there was little gene sequence heterogeneity that varied from 1 to 21 nucleotide differences. The results further demonstrate that it is possible to discover lytic proteins encoded in the genomes of bacteria that could be utilized to control bacterial pathogens.

Antibiotics are one of the most important medical discoveries of the 20th century and will remain an essential tool for treating animal and human diseases in the 21st century. However, antibiotic resistance among bacterial pathogens and concerns over their extensive use in food animals has garnered global interest in limiting antibiotic use in animal agriculture. Yet, limiting the availability of medical interventions to prevent and control animal diseases on the farm will directly impact global food security and safety as well as animal and human health. Insufficient attention has been given to the scientific breakthroughs and novel technologies that provide alternatives to antibiotics. The objectives of the symposium ‘Alternatives to Antibiotics’ were to highlight promising research results and novel technologies that could potentially lead to alternatives to conventional antibiotics, and assess challenges associated with their commercialization, and provide actionable strategies to support development of alternative antimicrobials. The symposium focused on the latest scientific breakthroughs and technologies that could provide new options and alternative strategies for preventing and treating diseases of animals. Some of these new technologies have direct applications as medical interventions for human health, but the focus of the symposium was animal production, animal health and food safety during food-animal production. Five subject areas were explored in detail through scientific presentations and expert panel discussions, including: (1) alternatives to antibiotics, lessons from nature; (2) immune modulation approaches to enhance disease resistance and to treat animal diseases; (3) gut microbiome and immune development, health and diseases; (4) alternatives to antibiotics for animal production; and (5) regulatory pathways to enable the licensure of alternatives to antibiotics.

Campylobacter jejuni, a flagellated, spiral-rod Gram-negative bacterium, is the leading etiologic agent of human acute bacterial gastroenteritis worldwide. The source of this microorganism for human infection has been implicated as consumption and handling of poultry meat where this microorganism is a commensal in the gut. Because the genomes of many C. jejuni isolates have been sequenced, our ultimate goal is to develop protein arrays for exploring this microorganism and host interactions. In this communication, we report cloning, expression and purification of C. jejuni flagellar proteins in a bacterial expression system. Twelve recombinant proteins were purified, which were confirmed by SDS-PAGE analysis and a His tag detection kit. The FlgE1, FlgG, FlgK, FliE, FlgH/FliH and FlaA recombinant proteins were further confirmed by LC-ESI-MS/MS. The purified recombinant proteins were tested whether they were immunogenic using antibodies from several sources. BacTrace anti-Campylobacter species antibody reacted to the FlaA recombinant protein, but not others. Rabbit anti-MOMP1 peptide antibody reacted strongly to FliE and weakly to FlaA, but not others. Rabbit anti-MOMP2 peptide antibody reacted strongly to the FlaA, FliG, FliE, FlhF, FlgG, FlgE1 and FliD recombinant proteins, less to FlgK and FlgH/FliH, and did not react to the FliY, FliS and FliH recombinant proteins. These antibody studies suggest that these recombinant flagellar proteins have potential for novel targets for vaccine development. It is also anticipated that these recombinant proteins provide us a very useful tool for investigating host immune response to C. jejuni.

There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as non-foodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl- l -alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.

Listeria monocytogenes is a Gram-positive, non-spore forming, catalase-positive rod that is a major bacterial food-borne disease agent associated with uncooked meats, including poultry, uncooked vegetables, soft cheeses, and unpasteurized milk. The bacterium may be carried by animals without signs of disease, can replicate at refrigeration temperatures, and is frequently associated with biofilms. There is a need to discover innovative pathogen intervention technologies for this bacterium. Consequently, bioinformatic analyses were used to identify genes encoding lytic protein sequences in the genomes of L. monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of a putative N-acetylmuramoyl-L-alanine amidase gene from L. monocytogenes strain 4b. The resultant amplification product was cloned into an expression vector, propagated in Escherichia coli Rosetta strains, and the recombinant protein was purified to homogeneity. Gene and protein sequencing confirmed that the predicted and chemically determined amino acid sequence of the recombinant protein designated PlyLM was a putative N-acetylmuramoyl-L-alanine amidase. The recombinant lytic protein was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot and MIC/MIB assays, but was not active against other bacteria beyond the genus. A microtiter plate assay was utilized to assay for the ability of the recombinant lysin protein to potentially aid with digestion of a L. monocytogenes biofilm. Protease or lysozyme digestion alone did not significantly reduce the L. monocytogenes biofilm. Although the recombinant protein alone reduced the biofilm by only 20%, complete digestion of the bacterial monolayer was accomplished in conjunction with a protease.

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in1 h. Pyrosequencing results show the assay to be extremely (99%) sensitive, and spike-back experiments demonstrated a detection threshold of10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (0.04%) of sequences present in the feces, but 88-97% of sequences from each media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes.

Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents and ΦCPV1, a virulent bacteriophage, was classified in the family Podoviridae . The purified virus had an icosahedral head and collar of approximately 42 nm and 23 nm in diameter, respectively, with a structurally complex tail of 37 nm lengthwise and a basal plate of 30 nm. The ΦCPV1 double-stranded DNA genome was 16,747 base pairs with a GC composition of 30.5%. Twenty-two open reading frames (ORFs) coding for putative peptides containing 30 or more amino acid residues were identified and analyzed in the genome. Amino acid sequences of the predicted proteins from the ΦCPV1 genome ORFs were compared with those from the NCBI database and potential functions of 12 proteins were predicted by sequence homology. Three putative proteins were similar to hypothetical proteins with unknown functions, whereas seven proteins did not have similarity with any known bacteriophage or bacterial proteins. Identified ORFs formed at least four genomic clusters that accounted for predicted proteins involved with replication of the viral DNA, its folding, production of structural components and lytic properties. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl- l -alanine amidases and a second structural lysin was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens .

Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.

Campylobacter spp. readily colonize the intestinal tracts of both human and avian species. While most often commensal organisms in birds, campylobacters remain the leading cause of bacterial gastroenteritis in humans. The association of campylobacters with poultry is well established as a primary route for human exposure. The difference in normal core body temperature between chickens (42 degrees C) and humans (37 degrees C) has been suggested to trigger potential colonization or virulence factors and investigators have demonstrated differential gene expression at the two temperatures. Campylobacter spp. exhibit unique nutritional requirements and have been thought to only utilize amino acids and Kreb cycle intermediates as carbon sources for growth. We evaluated the ability of the genome-sequenced strain of Campylobacter jejuni 11168 (GS) to oxidize 190 different substrates as sole carbon sources at 37 degrees C and 42 degrees C using phenotype microarray (PM) technology. Results indicate that the expected amino acids, l-serine, l-aspartic acid, l-asparagine, and l-glutamic acid were utilized in addition to a number of organic acids. In general, oxidation of the substrates was greater at 42 degrees C than at 37 degrees C with a few exceptions. By employing the PM method, we observed a number of potential false-positive reactions for substrates including the triose, dihydroxyacetone; and the pentose sugars, d-xylose, d-ribose, l-lyxose, and d- and l-arabinose. The presence of genes possibly responsible for utilization of pentose sugars is supported by the genomic sequence data, but actual utilization as sole carbon sources for active respiration has not been observed. A better understanding of the metabolic pathways and nutritional requirements of campylobacters could lead to improvements in culture media for detection and isolation of the pathogen and to future intervention methods to reduce human exposure.

The antimicrobial spectra of previously published bacteriocin E 50-52 (39 a.a.; 3,932 Da; pI = 8.5) and bacteriocin B 602 (29 a.a.; 3,864 Da; pI = 7.2) were determined. Named peptides were related to class IIa (pediocin-like) bacteriocins. Minimal inhibitory concentrations (MICs) of bacteriocins have been determined for bacterial isolates that were causative agents of nosocomial infections collected from Russian hospitals in 2003-2007, namely methicillin-resistant Staphylococcus aureus (MRSA) (n = 10); Acinetobacter baumannii (n = 11); Citrobacter freundii (n = 8); Escherichia coli (n = 9); Klebsiella pneumoniae (n = 10); Proteus spp. (n = 6); and Pseudomonas aeruginosa (n = 10). The majority of these tested isolates have been shown to be multidrug resistant and carry genetic determinants of antimicrobial resistance that were detected using polymerase chain reaction (PCR). The MICs of bacteriocin B 602 ranged from ≤0.025-1.56 μg/ml, and for bacteriocin E 50-52 from 0.05 to 6.25 μg/ml for all of 64 bacterial clinical isolates tested. Interestingly, the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E 50-52 and B 602 show good activity against nosocomial bacterial agents resistant to many classes of modern antibacterials used in clinical practice. These bacteriocins should be examined as an alternative in treating infections caused by such agents.

Campylobacter spp. are a significant contributor to the bacterial etiology of acute gastroenteritis in humans. Epidemiological evidence implicates poultry as a major source of the organism for human illness. However, the factors involved in colonization of poultry with Campylobacter spp. remain unclear. Determining colonization-associated factors at the proteome level should facilitate our understanding of Campylobacter spp. contamination of poultry. Therefore, proteomic analyses were utilized to identify expression differences between two Campylobacter jejuni isolates, a robust colonizer A74/C and a poor colonizing strain of the chicken gastrointestinal system designated NCTC 11168-PMSRU. Proteomic analyses by two-dimensional gel electrophoresis revealed the specific expression of an outer membrane-fibronectin binding protein, serine protease, and a putative aminopeptidase in the soluble portion of the robust colonizer A74C. Several proteins including a cysteine synthase and aconitate hydratase were detected specifically in the poor colonizer C. jejuni NCTC 11168-PMSRU isolate. Variation in the amino acid sequences resulting in different isoelectric points and relative mobility of the flagellin and C. jejuni major outer membrane (MOMP) protein were also detected between the two isolates. Western blotting of the bacterial proteins revealed the presence of two flagellin proteins in the poor colonizer versus one in the robust colonizing isolate, but no differences in MOMP. The results demonstrated that proteomics is useful for characterizing phenotypic variation among Campylobacter spp. isolates. Interestingly, different gene products potentially involved in robust colonization of chickens by Campylobacter spp. appear to conform to recently identified expression patterns in Biofilm or agar-adapted isolates.

Production and processing samples were collected from eight commercial poultry flocks in the southeastern United States and examined for the presence of Campylobacter spp. In an effort to determine relatedness, recovered isolates were typed using flaA short variable region (SVR) DNA sequence analysis. Six of the eight production flocks tested were Campylobacter positive. In general, multiple Campylobacter flaA SVR types were present within a flock. Additionally, types found within a flock were also recovered from the final processed carcass. However, in two cases, the population of Campylobacter flaA SVR types on the processed carcass differed from those recovered from the production samples. Comparison of subtypes among flocks reared on different farms and during different seasons revealed that subtypes of Campylobacter spp. persisted throughout the year and in different locations. Environmental samples from seven of the eight farms tested were also Campylobacter positive. In one flock, a drag swab of the rearing facility was Campylobacter spp. positive while the flock and the final product were both negative. For the remaining sampling periods, environmental samples were positive for Campylobacter spp. concomitant with recovery of Campylobacter spp. from the chickens. In the remaining six flocks, the majority of environmental isolates recovered possessed flaA SVR types identical to isolates recovered from the birds, while in only one case, a recovered environmental isolate possessed a flaA SVR type that was not related to isolates obtained from the flock. Interpretation of these data suggest that the external environment and the poultry production environment share common subtypes of Campylobacter spp. and that these subtypes can contribute to contamination of the final commercial product.

The usefulness of reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of 2 NDV isolates: a low virulent virus (LaSota) and a virulent virus (from the 2002–2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just before euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By seminested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-μm sections and to those of virus isolation from swabs. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens that were no longer shedding virus detectable by virus isolation. The RT-PCR was an effective and sensitive method to detect NDV in FFPE tissues. To the authorsn' knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.

There is a great deal of interest in characterizing the complex microbial communities in the poultry gut, and in understanding the effects of these dynamic communities on poultry performance, disease status, animal welfare, and microbes with human health significance. Investigations characterizing the poultry enteric virome have identified novel poultry viruses, but the roles these viruses play in disease and performance problems have yet to be fully characterized. The complex bacterial community present in the poultry gut influences gut development, immune status, and animal health, each of which can be an indicator of overall performance. The present metagenomic investigation was undertaken to provide insight into the colonization of specific pathogen free chickens by enteric microorganisms under field conditions and to compare the pre-contact intestinal microbiome with the altered microbiome following contact with poultry raised in the field. Analysis of the intestinal virome from contact birds (“sentinels”) placed on farms revealed colonization by members of the Picornaviridae, Picobirnaviridae, Reoviridae, and Astroviridae that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus families and genera. Members of the avian enteric Reoviridae and Astroviridae have been well-characterized and have historically been implicated in poultry enteric disease; members of the Picobirnaviridae and Picornaviridae have only relatively recently been described in the poultry and avian gut, and their roles in the recognized disease syndromes and in poultry performance in general have not been determined. This metagenomic analysis has provided insight into the colonization of the poultry gut by enteric microbes circulating in commercial broiler flocks, and has identified enteric viruses and virus communities that warrant further study in order to understand their role(s) in avian gut health and disease.

Aims: The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology. Methods and Results: Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. Conclusions: The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups. Significance and Impact of the Study: These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.

We evaluated anti- Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90°C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni . Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

Background Poultry remains a major source of foodborne bacterial infections. A variety of additives with presumed anti-microbial and/or growth-promoting effects are commonly added to poultry feed during commercial grow-out, yet the effects of these additives on the gastrointestinal microbial community (the GI microbiome) as the bird matures remain largely unknown. Here we compared temporal changes in the cecal microbiome to the effects of formic acid, propionic acid, and medium-chain fatty acids (MCFA) added to feed and/or drinking water. Results Cecal bacterial communities at day of hatch (n = 5 birds), 7d (n = 32), 21d (n = 27), and 42d (n = 36) post-hatch were surveyed using direct 454 sequencing of 16S rRNA gene amplicons from each bird in combination with cultivation-based recovery of a Salmonella Typhimurium marker strain and quantitative-PCR targeting Clostridium perfringens. Treatment effects on specific pathogens were generally non-significant. S. Typhimurium introduced by oral gavage at day of hatch was recovered by cultivation from nearly all birds sampled across treatments at 7d and 21d, but by 42d, S. Typhimurium was only recovered from ca. 25% of birds, regardless of treatment. Sequencing data also revealed non-significant treatment effects on genera containing known pathogens and on the cecal microbiome as a whole. In contrast, temporal changes in the cecal microbiome were dramatic, highly significant, and consistent across treatments. At 7d, the cecal community was dominated by three genera (Flavonifractor, Pseudoflavonifractor, and a Lachnospiracea sequence type) that accounted for more than half of sequences. By 21d post-hatch, a single genus (Faecalibacterium) accounted for 23-55% of sequences, and the number of Clostridium 16S rRNA gene copies detected by quantitative-PCR reached a maximum. Conclusions Over the 42 d experiment, the cecal bacterial community changed significantly as measured by a variety of ecological metrics and increases in the complexity of co-occurrence networks. Management of poultry to improve animal health, nutrition, or food safety may need to consider the interactive effects of any treatments with the dramatic temporal shifts in the taxonomic composition of the cecal microbiome as described here. Electronic supplementary material The online version of this article (doi:10.1186/s12917-014-0282-8) contains supplementary material, which is available to authorized users.

Avian paramyxovirus type 1, commonly referred to as Newcastle disease virus (NDV), is a serious pathogen of significant economic importance to the industry. To investigate the role of the fusion (F), hemagglutinin-neuraminidase (HN), and (P) phosphoprotein gene sequences in virulence, six strains of Newcastle disease virus (NDV) representing all pathotypes and seven recombinant strains created by reverse genetics were inoculated into 9-day-old chicken embryos. Tissues and chorioallantoic membranes (CAM) were harvested at 24-hour intervals post-inoculation. Riboprobe in situ hybridization and immunohistochemistry highlighted distinct tissue tropisms among the viruses. Presence of F and/or HN from virulent viruses inserted into lentogenic backbones caused dissemination of virus in a manner similar to wild type virulent viruses. Disruption of P gene decreased dissemination of velogeinic infectious clones. It is concluded that each of these genes contributes to pathogenicity.

Avian paramyxovirus 1 (APMV-1), also referred to as Newcastle disease virus (NDV), variants of low virulence were isolated from chickens, ducks and other unidentified species found in live-bird markets of the northeastern United States. These isolates were characterized as APMV-1 by the hemagglutination-inhibition (HI) assay utilizing NDV-specific polyclonal antisera. However, the isolates failed to react with a monoclonal antibody that has specificity for a wide variety of APMV-1 isolates. Although only highly virulent isolates require reporting to international regulatory agencies, the ability to correctly identify APMV-1 types is important for control and regulatory purposes. Protein gel patterns of the purified isolates resembled previously reported APMV-1 and anti-NDV polyclonal sera recognized the viral proteins. For three isolates oligonucleotide primers specific for the nucleoprotein, fusion protein and polymerase genes of NDV were utilized to synthesize cDNA using viral RNA as a template. Approximately 12kb of the genome was subsequently sequenced for the three isolates that included the nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase protein genes and a 5' portion of the polymerase gene. The isolates had an F protein cleavage site sequence of ERQER/LVG indicating low-virulence viruses that phylogenetically separated with other unique NDV isolates designated as a lineage 6 genotype. Additionally, a four amino acid insert was detected in the predicted phosphoprotein which complies with the "rule of six" among paramyxoviruses. These APMV-1 genotypes have not been previously reported in North America and further substantiate the heterogeneous genetic nature of these commercially important pathogens found worldwide.

Respiratory tract infections are a leading cause of morbidity and mortality worldwide. Human metapneumovirus (HMPV) is a recently discovered respiratory pathogen of the Paramyxovirus family in the Metapneumovirus genus. HMPV was first isolated from young children in The Netherlands with respiratory illness similar to human respiratory syncytial virus (RSV) infection. Epidemiological data indicates that HMPV co-circulates with RSV in the community. Few immunological tools are available to study the virological features of HMPV infection, thus current studies rely on reverse-transcription (RT) polymerase chain reaction (PCR) for detection. In this study, we examine serological cross-reactivity of RSV, HMPV and other Metapneumovirus members, i.e. avian metapneumovirus (AMPV), and show that polyclonal and monoclonal antibodies reactive to a conserved region in AMPV nucleoprotein (N) cross-react with HMPV N protein, but not with RSV N protein by ELISA, Western blot and immunohistochemical assays. In addition, we show that HMPV infection in the lungs of BALB/c mice can be detected using anti-N protein antibody. These reagents provide new tools and methods for investigating HMPV infection, for differentiating HMPV from RSV infection, and may be useful for characterizing potential links between HMPV with other respiratory complications.

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.

Avian metapneumovirus (aMPV) subtype B (aMPV/B) nucleotide sequences were obtained for the phosphoprotein (P), second matrix protein (M2), and small hydrophobic protein (SH) genes. By comparison with sequences from other metapneumoviruses, aMPV/B was most similar to subtype A aMPV (aMPV/A) relative to the US subtype C isolates (aMPV/C) and human metapneumovirus (hMPV). Strictly conserved residues common to all members of the Pneumovirinae were identified in the predicted amino acid sequences of the P and M2 protein-predicted amino acid sequences. The Cys 3 -His 1 motif, thought to be important for binding zinc, was also present in the aMPV M2 predicted protein sequences. For both the P and M2-1 protein-predicted amino acid sequences, aMPV/B was most similar to aMPV/A (72 and 89% identity, respectively), having only approximately 52 and 70% identity, respectively, relative to aMPV/C and hMPV. Differences were more marked in the M2-2 proteins, subtype B having 64% identity with subtype A but ≤25% identity with subtype C and hMPV. The A and B subtypes of aMPV had predicted amino acid sequence identities for the SH protein of 47%, and less than 20% with that of hMPV. An SH gene was not detected in the aMPV/C. Phylogenetically, aMPV/B clustered with aMPV/A, while aMPV/C grouped with hMPV.

The matrix protein gene was cloned and sequenced for several recent isolates of avian paramyxovirus type 1 (APMV-1). Specifically, isolates from pigeons and doves, members of the Columbidae family were examined. APMV-1 is the causative agent of Newcastle disease and the virus is associated with disease among a diverse number of avian species. Newcastle disease virus (NDV) isolates from pigeons have also been classified as pigeon paramyxovirus type 1 (PPMV-1). Matrix protein gene sequences for PPMV-1 isolates clustered together as a group relative to isolates from other species phylogenetically. However, there were also isolates from pigeons or doves that grouped with APMV-1 isolates from other species. This indicates that PPMV-1 may be circulating among Columbidae members as a distinct lineage, but that these avian species may also harbor other NDV strains as well. Of particular interest was a dove isolate from Europe that had an aberrant fusion protein cleavage site and was an outlying member phylogenetically between the two major groups of APMV-1 isolates.

The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus 1 (PPMV-1), and two isolates were classified as avian paramyxovirus 1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI. Tissues were examined by histopathology, immunohistochemistry (IHC) for the presence of NDV nucleoprotein, and in situ hybridization (ISH) for the presence of viral mRNA for the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1-infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP. Among all the isolates, the IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC.

The nucleocapsid (N) protein genes from 24 Newcastle disease virus (NDV) isolates representing various pathotypes with different geographical and chronological origins were cloned and sequenced. The N-terminal region of the N protein to residue 401 was highly conserved among isolates with several conservative substitutions occurring that correlated with phylogenetic relationships. Variability of the N protein was detected in the C-terminal portion similar to what has been reported for other members of the Paramyxovirinae. Amino acids previously identified as invariant or highly conserved in N proteins of other paramyxoviruses were also present in the NDV protein. Phylogenetic analysis of N gene coding sequences among NDV isolates again demonstrated the existence of two major groups. One clade contained viruses that included vaccine and virulent strains isolated in the USA prior to 1970 while a second clade included vaccine and virulent viruses isolated worldwide. Comparison of N protein amino acid sequences among members of the Paramyxoviridae resulted in NDV and avian paramyxovirus 6 separating as a cluster distinct from the Rubulavirus genus. This provides further support for avian paramyxoviruses being considered for their own genus among the Paramyxovirinae.

This chapter discusses efforts to exploit Campylobacter-specific bacteriophages to reduce the numbers of C. jejuni and C. coli colonizing poultry and contaminating poultry meat products. All the phages reported by investigators in two studies had icosahedral heads and long contractile tails that were classified as members of the Myoviridae. Two phages with head diameters of 140.6 and 143.8 nm and large genome sizes of 320 kb were classified as group I. Five phages classified into group II had average head diameters of 99 nm and average genome sizes of 184 kb. A fourth phage had an icosahedral head that was classified as morphotype B1 of the Siphoviridae, while a fifth phage had an icosahedral head with a short tail of morphotype C1 in the Podoviridae. Campylobacter phages on the skin of retail chicken portions have been recovered at levels of 2 X 103 PFU/10 cm2 and this study found that Campylobacter phage could be isolated from chicken skin only when detectable levels of their host were also present. The spontaneous production of CampMu bacteriophages after bacteriophage therapy is of concern because Mu bacteriophages are potential agents of mutation. Attempts to utilize bacteriophage, initially for typing purposes and more recently for their biocontrol potential, have led to a greater awareness of the role that phage play in the complex ecology of Campylobacter. It should be noted that bacteriophage can shape the evolution of Campylobacter genomes as they do in other bacterial genera.

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry.

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison ( Bison bison ) throughout North America. No vaccine exists to protect animals from disease. Since OvHV-2 has not been propagated in vitro , one strategy to develop a modified live vaccine is to use a closely related, non-pathogenic member of the malignant catarrhal fever virus family as a vector expressing potentially protective OvHV-2 epitopes. To date, no controlled experimental challenge studies with alcelaphine herpesvirus 2 (AlHV-2) derived from topi ( Damaliscus lunatus jimela ) have been reported The unique or light DNA segment of the AlHV-2 genome was sequenced and annotated and the virus was tested for its ability to infect and induce disease in American bison. Yearling bison were inoculated intranasally ( n = 4) or intramuscularly ( n = 3) with 2 × 10 −4.7 TCID 50 of AlHV-2, and monitored for infection and the development of disease. Six inoculated bison became infected with AlHV-2. Two of the six animals developed clinical signs and had gross and histological lesions consistent with terminal MCF, which differed in distribution from those in bison with MCF due to OvHV-2. One other animal developed minor clinical signs and had gross and histological pulmonary lesions consistent with early (pre-clinical) stages of MCF. Unmodified low cell culture passage AlHV-2 derived from topi is an unsuitable vaccine vector for the prevention of MCF. However, the annotated genome might be useful in identifying genes which could be deleted to potentially attenuate the virus for bison.

Astroviruses are small round viruses that cause enteric disease in the young of several species. Detection and diagnosis of astrovirus infection in non-human hosts relies heavily on electron microscopy and fluorescent antibody tests. Recently, our laboratory isolated and sequenced an avian astrovirus from poult enteritis mortality syndrome affected turkeys. These studies describe the development of RT-PCR methods, which specifically detect regions of the viral capsid and polymerase genes, and demonstrate their use in detecting astrovirus infection in commercial turkey flocks.

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.

We report the isolation and characterization of eight polymorphic and five monomorphic microsatellites in North Island brown kiwi (NIBK, Apteryx mantelli), using two polymerase chain reaction (PCR) techniques employing either short-tandem repeat primers (STR method) or random PCR-based isolation of microsatellite arrays (PIMA method). Microsatellite polymorphism was subsequently determined using 65 individuals. There were two to seven alleles for each polymorphic locus with heterozygozity ranging between 0.04 and 0.86. These primers will be used in future studies to determine the level of extra-pair copulation, dispersal patterns, and genetic diversity within and between wild populations of NIBK.

Newcastle disease virus {NDV (avian paramyxovirus type 1 [APMV1])} isolates were recovered from imported exotic birds confiscated following importation into the United States, from waterbirds in the United States, and from poultry. The exotic birds probably originated from Central and South America, Asia, and Africa. The NDV isolates were initially characterized as highly virulent because of a short mean death time in embryonated chicken eggs. The isolates were typed as neurotropic or viscerotropic velogenic by intracloacal inoculation of adult chickens. Intracerebral pathogenicity index values for the virulent NDV isolates ranged from 1.54 to 1.90, compared to a possible maximum value of 2.0. These isolates had a dibasic amino acid motif in the fusion protein cleavage site sequence required for host systemic replication. Sequence differences were detected surrounding the fusion protein cleavage site and the matrix protein nuclear localization signal, indicating evolution of highly virulent NDV. Phylogenetically, these isolates were categorized with other highly virulent NDV strains that caused outbreaks in southern California poultry during 1972 and in cormorants in the north central United States and southern Canada during 1990 and 1992. These isolates are related to NDV that may have the APMV1 strain chicken/Australia/AV/32 or a related virus as a possible progenitor. Recent virulent NDV isolates and those recovered during disease outbreaks since the 1970s are phylogenetically distinct from current vaccine viruses and standard challenge strains.

Vesicular exanthema of swine virus (VESV), the prototype calicivirus, is the etiologic agent of the porcine disease vesicular exanthema of swine (VES). VES is characterized by vesicle formation on the extremities, mouth and snout and causes abortions and stillbirths if infection occurs during pregnancy. VESV is considered an exotic agent in the US, following its eradication in 1956. The single capsid protein gene of VESV serotype A48 was cloned and sequenced. The capsid amino acid sequence was 69% similar to the San Miguel sea lion virus serotype 1 (SMSV 1) and 89% similar to the SMSV serotype 4 (SMSV 4) capsid proteins. The six functional regions (A–F) previously identified in SMSV 1, SMSV 4, feline calicivirus and rabbit hemorrhagic disease virus capsid proteins were present in VESV A48. Two sets of PCR primers were designed which directed amplification of the 5′ end (A region) and the hypervariable (E region) sequences of the capsid protein precursor gene of these viruses, as well as seven additional SMSV serotypes. Alignment and phylogenetic analysis of the N-terminal sequences demonstrated the close relationship of these viruses. Alignment of the hypervariable region amino acid sequences of the ten viruses confirmed that a great variety of sequence exists in this region; however, a consensus sequence (NxT(N/H)F(K/R)GxYI(C/M)GxLx(T/R)) was derived which is also present in the feline calicivirus capsid protein. Comparison of the E region sequences provides further evidence that this area of animal calicivirus capsid protein may contain the major antigenic determinants.

Amino acid sequence of the capsid protein hypervariable region of nine feline calicivirus (FCV) isolates recovered from cats persistently infected after inoculation with the FCV strain 255 parent virus is reported. Capsid proteins from all the isolates were highly cross reactive by Western blot analysis using polyclonal antisera to FCV. Reverse-transcription PCR was used to obtain sequence information of the FCV capsid protein highly variable E region. Amino acid substitutions occurred between residues 426 and 458 of the FCV capsid protein E region. The sequence data and phylogenetic reconstructions based on the sequence information correlated well with antigenic differences among isolates determined by two-way cross neutralization. These results agree with previous reports using divergent isolates of FCV that correlated amino acid differences with serology. This further supports the hypothesis that the FCV capsid protein E region from residues 426 to 458 contains the serotypic determinants of FCV important to antigenic variation.