Effect of the major histocompatibility complex on the inhibition of induced cellulitis development in a broiler chicken model.

The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance or susceptibility to several bacterial, parasitic, and viral diseases, the most notable of which is Marek's disease. In Marek's disease certain MHC haplotypes have been shown to confer relative resistance (B21), whereas other haplotypes are susceptible (B13). Relatively little work has been performed looking at the association of the MHC with bacterial diseases. One such disease is cellulitis, which is caused by several different bacteria but most notably by Escherichia coli. In this report, a commercial broiler chicken line known to contain standard B13 and B21, as well as the unique MHC types BA9 and BA12, was examined in a challenge model for cellulitis. The MHC-defined birds were challenged with a cellulitis-causing E. coli isolate and the frequency of lesion development and severity was quantified. In conclusion, B21 had the highest incidence of cellulitis development, B13 had the lowest incidence, and BA9 and BA12 had intermediate results. Results concerning the lesion severity showed that it was independent of the birds' MHC type.

Development of a polymerase chain reaction assay for specific identification of Clostridium colinum.

Clostridium colinum is the causative agent of ulcerative enteritis, a serious disease of the bobwhite quail (Colinus virginianus) and sporadically of young chickens. The aim of the present study was to develop a polymerase chain reaction (PCR) assay specific for C. colinum identification. The 16S rDNA sequence of C. colinum was analysed and two species-specific primers were designed. The specificity of these primers was tested with closely related Clostridium species and the expected amplified product (935 base pairs) was observed only with DNA from samples containing C. colinum. Results from performing PCR assays on faecal samples from quails spiked with different concentrations of C. colinum, showed that the detection limit of the assay was 1.6 x 10(4) colony-forming units per gram of faecal material. This PCR assay can be used in diagnostic laboratories to confirm the presence of C. colinum in pure cultures and could be used to screen enriched samples or faecal samples for the presence of this pathogen.

Evaluation of different plate media for direct cultivation of Campylobacter species from live broilers.

Accurate identification and optimal culturing procedures for Campylobacter spp. from live broilers are needed for epidemiological studies. Because there is no standardized protocol, we designed and conducted studies to evaluate different selective media for the culturing and isolation of Campylobacter spp. from cecal and fecal samples obtained from battery-reared and commercial broilers. Five media selective for Campylobacter were evaluated: Campylobacter agar base, Campylobacter, Campy-Line, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar. With contaminated broilers reared in battery cages, Campylobacter agar base, Campylobacter, modified Campy-Cefex, and modified charcoal cefoperazone deoxycholate agar revealed similar isolation rates (P > 0.05), whereas Campy-Line showed a lower efficacy (P < 0.05). With commercial live broilers, modified Campy-Cefex agar was more consistent for the isolation of Campylobacter from feces, whereas modified Campy-Cefex and modified charcoal cefoperazone deoxycholate agar showed similar isolation rates from cecal samples. Campy-Line agar showed a lower identification rate (P < 0.05) for both fecal and cecal samples. A multiplex PCR assay used for identification showed that Campylobacter jejuni and Campylobacter coli DNA was present in the samples. Pulsed field gel electrophoresis restriction profiles differed among samples collected from different commercial farms but were similar for isolates from the same farm, suggesting clonal differences. No variation was seen in pulsed field gel electrophoresis patterns among isolates cultured on different media. Our data suggest that the choice of plate medium may influence the efficiency of isolating Campylobacter spp. from broiler chickens by direct plating from fecal or cecal samples.

High genetic divergences indicate ancient separation of parthenogenetic lineages of the oribatid mite Platynothrus peltifer (Acari, Oribatida).

Theories on the evolution and maintenance of sex are challenged by the existence of ancient parthenogenetic lineages such as bdelloid rotifers and darwinulid ostracods. It has been proposed that several parthenogenetic and speciose taxa of oribatid mites (Acari) also have an ancient origin. We used nucleotide sequences of the mitochondrial gene cytochrome oxidase I to estimate the age of the parthenogenetic oribatid mite species Platynothrus peltifer. Sixty-five specimens from 16 sites in North America, Europe and Asia were analysed. Seven major clades were identified. Within-clade genetic distances were below 2 % similar to the total intraspecific genetic diversity of most organisms. However, distances between clades averaged 56 % with a maximum of 125 %. We conclude that P. peltifer, as it is currently conceived, has existed for perhaps 100 million years, has an extant distribution that results from continental drift rather than dispersal and was subject to several cryptic speciations.

A longitudinal study of Salmonella and Campylobacter jejuni isolates from day of hatch through processing by automated ribotyping.

Comparisons of bacterial populations over long periods of time allow researchers to identify clonal populations, perhaps those responsible for contamination of farms or humans. Salmonella and Campylobacter can cause human illness, and our objective was to use a library typing system to track strains that persist in the poultry house and through the processing plant. Two farms, over four consecutive flocks, were studied. Multiple samples were taken of the poultry house environment, feed mill, transport crates, and carcasses in the processing plant. Sample collection on the farm took place on chick placement day, midgrowout, and the day of harvest. This study found that 80.3% of isolates belonged to a single strain of Salmonella Kentucky that persisted in several environmental samples for all flocks at both farms, from chick placement day to the final product at the plant. Surgical shoe covers produced most isolates (n = 26), and processing day yielded the highest recovery (n = 68). Additional serotypes were recovered, but the Salmonella Kentucky-positive eggshells and chick mortality appeared to be the source of the organism for both farms. All Campylobacter isolates recovered were identified as C. jejuni. Most Campylobacter isolates (90.1%) belonged to one of three core strains. C. jejuni was not recovered on chick placement day. Cecal droppings yielded all nine strains. Most isolates (98.2%) were from one farm. Cluster analysis grouped C. jejuni and Salmonella isolates into four and six distinct clusters, respectively, on the basis of a similarity level of 80%.

No evidence for the 'Meselson effect' in parthenogenetic oribatid mites (Oribatida, Acari).

It has been hypothesized that in ancient apomictic, nonrecombining lineages the two alleles of a single copy gene will become highly divergent as a result of the independent accumulation of mutations (Meselson effect). We used a partial sequence of the elongation factor-1alpha (ef-1alpha) and the heat shock protein 82 (hsp82) genes to test this hypothesis for putative ancient parthenogenetic oribatid mite lineages. In addition, we tested if the hsp82 gene is fully transcribed by sequencing the cDNA and we also tested if there is evidence for recombination and gene conversion in sexual and parthenogenetic oribatid mite species. The average maximum intra-specific divergence in the ef-1alpha was 2.7% in three parthenogenetic species and 8.6% in three sexual species; the average maximum intra-individual genetic divergence was 0.9% in the parthenogenetic and 6.0% in the sexual species. In the hsp82 gene the average maximum intra-individual genetic divergence in the sexual species Steganacarus magnus and in the parthenogenetic species Platynothrus peltifer was 1.1% and 1.2%, respectively. None of the differences were statistically significant. The cDNA data indicated that the hsp82 sequence is transcribed and intron-free. Likelihood permutation tests indicate that ef-1alpha has undergone recombination in all three studied sexual species and gene conversion in two of the sexual species, but neither process has occurred in any of the parthenogenetic species. No evidence for recombination or gene conversion was found for sexual or parthenogenetic oribatid mite species in the hsp 82 gene. There appears to be no Meselson effect in parthenogenetic oribatid mite species. Presumably, their low genetic divergence is due to automixis, other homogenizing mechanisms or strong selection to keep both the ef-1alpha and the hsp82 gene functioning.

Food security issues--a potential comprehensive plan.

The need for a comprehensive plan to protect the food production system has emerged as a critical issue over the last several years. To address this need, a comprehensive food security plan has been developed at Auburn University. The proposed program, entitled the Consolidated American Network for Agriculture Resource Intelligence (CANARI) system is one of several systems being proposed to deal with potential agricultural bioterrorism or agroterrorism events. Unlike other systems, which hastily emerged in many agencies after the tragedy of September 11, 2001, the system has been planned over the last 5 yr with the input of the agricultural industries, is comprehensive in its conception, and is designed to coordinate all components (existing and planned) necessary to prevent, detect, and respond to potential agroterrorism events. The plan uses the principle that the first line of defense must be within the states and agricultural companies for the detection of agroterrorism incidents to be rapid and the response effective, organized, and timely. CANARI is designed to integrate the previously disparate elements by fostering a cooperative network of local, state, and federal agencies as well as commodity entities and interested non-governmental organizations. Using a market-driven approach, the system encourages commodity membership and cooperation through positive incentives rather than regulatory duress. A centralized command structure is envisioned, which would be provided through the creation of a National Agroterrorism Defense Center. The responsibility of this Center would be to coordinate all of the activities presently available in components at the local, state, and federal levels and develop and manage new and emerging activities provided by the stakeholders. CANARI offers a new paradigm by which all of its constituent members act collectively and cooperatively to lessen the risk of an attack and better ensure the continued availability of a safe, abundant, and economical food supply.

Major histocompatibility complex effect on cellulitis among different chicken lines.

The chicken major histocompatibility complex (MHC) has been implicated in conferring resistance/susceptibility to several bacterial, parasitic, and viral diseases. Investigators have shown that the chicken MHC plays a major role in determining the outcome of a Marek's disease infection, in that standard B(13) is susceptible to the virus while B(21) confers resistance to the virus. Previous work with a broiler line has shown that B(21) is susceptible to an Escherichia coli-induced cellulitis infection and that B(13) conferred resistance to the infection. For this experiment, a broiler and a Leghorn chicken line shown to contain standard B(13) and B(21) were examined in a challenge model for cellulitis. The birds were challenged with a cellulitis-causing E. coli isolate. Homozygous B(21) had the highest incidence of cellulitis development compared with either homozygous B(13) or the heterozygous B(13)/B(21) for both the broiler and Leghorn lines. Additionally, cellulitis lesion severity was measured in both lines and shown to be independent of MHC type.

Differences in the pathogenicity of various bacterial isolates used in an induction model for gangrenous dermatitis in broiler chickens.

A gangrenous dermatitis model was developed in broiler chickens, in which birds previously vaccinated at 14 days of age with a bursal disease virus vaccine were challenged at 4 wk of age with various bacterial combinations with the combination of subcutaneous and intramuscular injection. Gangrenous dermatitis lesions were not produced in birds injected with one of the Staphylococcus aureus isolates, either alone or in combination with various Clostridium septicum isolates. Other S. aureus isolates produced significant levels of gangrenous dermatitis either alone or in combination with the same C. septicum isolates. These same C. septicum isolates when given alone did not produce gangrenous lesions. Data from this experiment show the highest level of mortality occurred in birds challenged with a mixture of C. septicum and S. aureus isolates, whereas lower or no mortality was associated with the same isolates given separately. The data clearly demonstrate that the pathogenicity of isolates responsible for gangrenous dermatitis varies widely, indicating that the frequency and severity of lesion production, as well as the occurrence of mortality, are largely dependent upon the specific isolate or isolates with which the birds are challenged.

The effects of early exposure of cellulitis-associated Escherichia coli in 1-day-old broiler chickens.

Two experiments were performed to test the effect of various field strains of Escherichia coli of cellulitis origin. In the first experiment, 1-day-old broiler chicks were challenged with one of two E. coli field strains using inoculation routes including oral gavage, swabbing of the navel and subcutaneous injection. No cellulitis lesions were produced, although the birds experienced high levels of septicemia/toxemia, characteristic of colibacillosis. The birds that received the E. coli by subcutaneous injection experienced the highest rate of mortality, while those that were challenged by gavage and those that had their navels swabbed experienced lesser rates of mortality. Birds in the second experiment were challenged at 1 day of age with one of three field strains of cellulitis-origin E. coli administered alone or in combination (1:1), which were serially diluted prior to subcutaneous injection. No significant differences in body weight, mortality or cellulitis rates were associated with specific isolates given; however, significant differences were seen with mortality and cellulitis rates according to the dilution of bacteria given. A linear effect was also noted with body weight at 3 weeks, again correlating to the dilution of bacteria that the chicks received.

The influence of Escherichia coli strains from different sources and the age of broiler chickens on the development of cellulitis.

In two experiments, broilers were challenged with one of several field strains of Escherichia coli to determine whether the source of the E. coli and age of the bird at time of inoculation affected the development of cellulitis lesions. In the first experiment, birds inoculated at 52 days of age with E. coli of faecal, airsacculitis and cellulitis origin exhibited a cellulitis lesion incidence of 47.5, 25 and 77.5%, respectively. This study confirms earlier observations that E. coli strains isolated from cellulitis lesions express a higher propensity for producing these same lesions than other strains, including those associated with airsacculitis. In the second experiment, birds were inoculated at 4, 7, 10, 16, 28, and 52 days of age with an E. coli strain of cellulitis origin and necropsied 2 days post-infection. The resulting incidence of cellulitis ranged from 20% (day 7) to 95% (days 16 and 28), indicating that cellulitis can develop in any age of bird, although the lesions were frequently associated with other manifestations of colibacillosis (perihepatitis, pericarditis, airsacculitis) in birds challenged from 4 to 16 days of age.

The effect of vitamin E on cellulitis in broiler chickens experiencing scratches in a challenge model.

Two experiments are described; each experiment contained five treatments with each treatment consisting of a specific diet and vitamin E at 8.82 mg, 41.89 mg, 74.96 mg, 108.03 mg, or 141.10 mg vitamin E per kilogram of feed. Birds were raised with continuous feed containing the various levels of vitamin E available throughout the experiment. At 4 wk of age, the birds were scratched on the breast and placed onto avian cellulitis Escherichia coli-seeded litter. One week later, the birds were euthanatized and lesion presence was noted. There appeared to be a positive correlation between vitamin E and the inhibition of cellulitis formation when the birds were fed a diet containing 74.96 mg vitamin E/kg feed. Conflicting results were seen in the two experiments when the birds were fed 41.89 and 108.03 mg vitamin E/kg feed. Both experiments had a high incidence of cellulitis in birds whose diets consisted of 141.10 mg vitamin E/kg feed.

Isoform patterns of chitinase and beta-1,3-glucanase in maturing corn kernels (Zea mays L.) associated with Aspergillus flavus milk stage infection.

Isoform patterns of chitinase and beta-1,3-glucanase of maturing kernels of yellow dent corn (Pioneer 3394) infected with Aspergillus flavus at the milk stage were investigated through polyacrylamide gel electrophoresis (PAGE). Proteins on the sodium dodecyl sulfate (SDS) gel with an apparent molecular mass range of 23-46 kDa were differentially present in the kernels infected with both aflatoxin-producing and non-aflatoxin-producing strains of A. flavus. From in-gel (native PAGE) enzyme activity assays, three bands corresponding to chitinase isoforms and two bands corresponding to beta-1,3-glucanase isoforms were detected in the infected kernels. One chitinase isoform of 29 kDa was present only in the infected kernels, and another one of 28 kDa was present in both infected and noninfected kernels. They were judged to be acidic on the basis of their migration on an acrylamide isoelectric focusing (IEF) gel. For the beta-1,3-glucanase, one isoform of 35 kDa was present in both infected and noninfected kernels, but another one, a 33 kDa isoform, was present only in the infected kernels. Both acidic and basic beta-1,3-glucanase isoforms were detected in the IEF gel. The results of this study are the first to demonstrate patterns of enhanced or inducible proteins in maturing corn kernels in response to A. flavus infection at the milk stage. The results also indicate that only particular isoforms of the two hydrolytic enzymes are involved in the maturing corn kernels infected at the milk stage with A. flavus.

The association of various isolates of Escherichia coli from the United States with induced cellulitis and colibacillosis in young broiler chickens.

An experiment was conducted to observe the effects of 10 different avian Escherichia coli isolates in 3-day post-hatch broiler chicks after subcutaneous administration. Isolates were originally obtained from various avian sources throughout the US. Chicks were injected subcutaneously on the ventral surface and necropsied at 7-day intervals for 3 weeks. Cellulitis was produced in all treatments receiving E. coli of cellulitis origin, with the highest incidence occurring 2 weeks post-infection in birds that received an isolate recovered in a previous challenge experiment. Cellulitis was also observed at week 1 post-infection in a small percentage of the birds in two of the treatments receiving E. coli of enteric origin, although lesions disappeared from the group after week 1 post-infection. Septicaemia was the most frequent sequel to challenge and occurred regardless of which isolate was injected. Chicks exposed to cellulitis origin isolates developed septicaemia more frequently than birds challenged with E. coli of non-cellulitis origin. The data implies that cellulitis is unlikely to occur early in the bird's life, since young birds exposed to E. coli frequently develop septicaemia.

Inhibition of aflatoxin B(1) biosynthesis in Aspergillus flavus by anthocyanidins and related flavonoids.

Anthocyanidins and precursors or related flavonoids were tested at concentrations from 0.3 to 9.7 mM ( approximately 0.1-3.0 mg/mL) for activity against growth and aflatoxin B(1) biosynthesis by Aspergillus flavus Link:Fr. NRRL 3357. Aflatoxin B(1) production was inhibited by all anthocyanidins tested, and 3-hydroxy compounds were more active than 3-deoxy forms. Monoglycosides of cyanidin were 40% less inhibitory than the aglycon, whereas a monoglucoside and a diglucoside of pelargonidin were 80 and 5%, respectively, as active as the aglycon. Of eight flavonoids tested, only kaempferol was moderately active, whereas luteolin and catechin were weakly inhibitory. Binary combinations of delphinidin and three other aflatoxin inhibitors acted independently of each other. Results with an aflatoxin pathway mutant indicated that anthocyanidin inhibition occurred before norsolorinic acid synthesis.

Detection of human herpesvirus 6 by reverse transcription-PCR.

The role of human herpesvirus 6 (HHV-6) in disease beyond primary infection remains unclear. We have developed and validated a new reverse transcription-PCR (RT-PCR) assay for HHV-6 that can determine the presence of HHV-6 in clinical specimens and differentiate between latent and replicating virus. Peripheral blood mononuclear cells from 109 children were evaluated for HHV-6 by RT-PCR, DNA PCR, and viral culture. Of these samples, 106 were suitable for analysis. A total of 20 samples were positive for HHV-6 by culture and DNA PCR, of which 19 were positive by RT-PCR (sensitivity, 95%). All 28 samples from children that were negative by viral culture, but positive by DNA PCR, were negative for viral transcripts by our RT-PCR assay. One positive RT-PCR result was observed in 56 samples that were negative by tissue culture and DNA PCR. This indicates a low rate of false-positive results (1.2%) and a specificity of 98.8%. This RT-PCR assay can reliably differentiate between latent and actively replicating HHV-6 and should allow insight into the pathogenesis of this ubiquitous virus.

Evaluation of scratches as an essential element in the development of avian cellulitis in broiler chickens.

Currently, the published cellulitis models do not adequately address the actual pathogenesis as seen in the commercial broiler industry. In this model, small dermal scratches were made on the skin of broiler chickens, which were then placed on litter seeded with avian cellulitis-associated Escherichia coli. The research confirms scratches are required for the induction of avian cellulitis. The research also confirms that "type I" cellulitis lesions or those previously thought to be due to hatchery-borne infections can be induced with scratches. The described methods provide a realistic model for cellulitis development that will improve the reliability of prophylactic and therapeutic-regimen efficacy testing data, thereby providing information more directly useful to the commercial broiler industry.

An outbreak of histomoniasis in turkeys infected with a moderate level of Ascaridia dissimilis but no Heterakis gallinarum.

Histomoniasis was diagnosed in a commercial turkey flock. All morbidity and mortality occurred in one house. Birds exhibited lesions characteristic for histomoniasis, and the diagnosis was confirmed by histopathologic examination. Affected turkeys were infected with moderate levels of Ascaridia dissimilis but not Heterakis gallinarum. Compression smears of hepatic tissues showed typical histotrophic phase Histomonas meleagridis, whereas cecal smears exhibited large numbers of Trichomonas gallinarum. A challenge experiment was conducted in which turkey poults were placed on contaminated litter. Although histomoniasis was not reproduced in the experiment, the birds did become infected with low numbers of A. dissimilis.

Ascarid-associated hepatic foci in turkeys.

Hepatic foci are a serious economic problem for most turkey-producing areas in the United States. Current estimates indicate that as much as 43% of the flocks sent to slaughter may experience condemnations because of hepatic foci. The present experiments were designed to duplicate naturally occurring lesions with Ascaridia dissimilis. Newly hatched poults were placed on fresh litter and given feed containing either 500 embryonated A. dissimilis ova/bird/day (from day of hatch) or no ova, in three experiments. Hepatic foci were reproduced in exposed poults in all three experiments, indicating that A. dissimilis is directly involved in the etiology of hepatic foci.

Biotransformation of 24 alpha-methylcholesterol and 24 beta-methylcholesterol by yeast mutant GL7.

Incubation of 24 alpha- and 24 beta-methylcholesterols with yeast mutant GL7 afforded their corresponding C-22-desaturated products under the catalysis of sterol delta 22(23)-desaturase. The metabolites were identified to be 22-dehydro-24 alpha-methylcholesterol (2% yield from 24 alpha-methylcholesterol) and 22-dehydro-24 beta-methylcholesterol (51% yield from 24 beta-methylcholesterol) respectively on the basis of their chromatographic and spectral properties. It was concluded that the sterol delta 22(23)-desaturase prefers the 24 beta-methyl sterols and is highly stereospecific.