Rapid construction of Campylobacter jejuni deletion mutants.

AIMS: To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. METHODS AND RESULTS: We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. CONCLUSIONS: This method can be used for rapid construction of Camp. jejuni deletion mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this method should facilitate functional characterization of various Camp. jejuni genes.

Effect of experimental chlorate product administration in the drinking water on Salmonella typhimurium contamination of broilers.

The crop is a known source of Salmonella and Campylobacter contamination. Previously, we evaluated lactic acid in the drinking water during a simulated pretransport feed withdrawal (FW) and reported 0.44% lactic acid significantly (P < 0.05) reduced the number of Salmonella recovered in market-age broiler crops. However, total consumption of the organic acid-treated drinking water was reduced. Presently, we evaluated the effect of experimental chlorate product (ECP; 1x ECP is equivalent to a 15 mM chlorate ion concentration) during a 10-h pretransport FW. Market-age broilers were obtained from a commercial processing plant and randomly assigned to ECP-treated or control (nontreated) groups. Broilers were challenged by crop gavage with 10(8) Salmonella Typhimurium (ST) immediately upon arrival and 1 d prior to termination of the experiment. One day later, broilers were killed for ST enumeration (cfu) in the crop and ceca. Broilers provided ECP 24 h prior to slaughter consumed slightly more ECP water than broilers provided distilled water. Treatment with ECP caused a significant decrease (P < 0.05) in the incidence of ST in crop contents (2%) as compared to the controls (36.7%). Similarly, ECP treatment caused a significant decrease (P < 0.05) in number of ST (0.96 log10 ST/g cecal content) detected in the ceca when compared to controls (2.52 log10 ST). This study suggested that incorporation of ECP in the drinking water 24 to 48 h prior to slaughter could reduce Salmonella contamination in broilers.

Role of Campylobacter jejuni potential virulence genes in cecal colonization.

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.

Comparison of GN Hajna and tetrathionate as initial enrichment for salmonellae recovery from swine lymph nodes and cecal contents collected at slaughter.

An epidemiologic survey was conducted to determine the prevalence of salmonellae in swine from 5 farms of an integrated swine operation. The purpose of this study was to evaluate the recovery efficiencies for salmonellae from swine lymph nodes and cecal contents when GN Hajna and tetrathionate were compared as initial enrichments. Salmonellae were isolated from 61% of 645 pigs at slaughter; 324 positive cultures were from lymph nodes, and 224 were from cecal contents. Frequently, pigs had salmonellae isolated from both the lymph nodes and cecal contents. Total isolations, regardless of source, were similar for GN Hajna (247) and tetrathionate (301). There was no difference (P > 0.05) in the number of isolations from lymph nodes when GN Hajna enrichment was compared with tetrathionate enrichment (174 vs. 150). However, there was a significant (P < 0.05) advantage of utilizing tetrathionate when compared with GN Hajna for isolations from cecal contents (151 vs. 73).

Effects of feed withdrawal and transport on cecal environment and Campylobacter concentrations in a swine surgical model.

The objective of the present study was to evaluate how feed withdrawal and transportation influenced the cecal environment and cecal populations of Campylobacter in swine. Four miniature Yucatan gilts (8.8 kg), naturally infected with Campylobacter jejuni, were surgically implanted with cecal cannulas. The gilts were fasted for 48 h. Samples of cecal contents were collected for 7 days prior to and for 7 days after the fast, and mean values were determined for pH, volatile fatty acids (VFA), and CFU enumeration of C. jejuni. This was replicated three times. In another trial, gilts (full-fed) were transported in a livestock trailer for 4 h and cecal samples were collected before and after transport and analyzed for pH, VFA, and CFU. Following a 48-h fast, cecal pH increased (P < 0.05) by 1 unit; acetic and propionic acids decreased (P < 0.05) by 61% and 71%, respectively; and there was a twofold log10 increase (P < 0.05) in CFU/g cecal content of C. jejuni. Values of pH, VFA, and CFU of C. jejuni did not change in cecal samples from gilts following transportation. These data are important for food safety considerations because feed withdrawal, commonly associated with shipping and slaughter, can increase Campylobacter concentrations in the pig intestinal tract.

Inhibition of in vitro Salmonella Typhimurium colonization in porcine cecal bacteria continuous-flow competitive exclusion culturest.

Continuous-flow (CF) chemostate cultures were used as models to determine the potential usefulness of undefined porcine cecal bacteria as competitive exclusion (CE) cultures against colonization by Salmonella Typhimurium. One culture, pCF1, was derived from cecal bacteria of an animal maintained on antibiotic-free feed, while the other culture, pCF4, was derived from cecal bacteria of an animal maintained on feed containing chlortetracycline. The effectiveness against a chlortetracycline-resistant Salmonella Typhimurium was examined in CF cultures maintained in the absence (pCF1 and pCF4) and presence (cpCFl and cpCF4) of chlortetracycline. CF cultures were inoculated with each of 10(2), 10(4), and 10(6) Salmonella Typhimurium CFU/ml. Chemostat inocula of 10(2) Salmonella CFU/ml resulted in no Salmonella Typhimurium being detected at 2 and 3 days postinoculation in pCF1 and pCF4, respectively, and after 2 days in both cpCF1 and cpCF4. Inoculations of 10(4) Salmonella Typhimurium CFU/ml resulted in clearance from pCF1 and pCF4 within 4 days and within 3 days from cpCF1 and cpCF4. Following inoculation with 10(6) CFU/ml, no Salmonella Typhimurium were detected in all CF cultures by 6 days postinoculation. The results indicated that in vitro CF cultures of porcine cecal bacteria were able to inhibit the growth of Salmonella Typhimurium. The ability to limit Salmonella Typhimurium growth was not restricted by prior exposure of the cecal bacteria to the feed additive chlortetracycline. The present study demonstrates the potential application of CF cultures as models to aid in the identification of CE cultures against salmonellosis in pigs.

Dose response and organ invasion of day-of-hatch Leghorn chicks by different isolates of Campylobacter jejuni.

Colonization of the ceca and organ invasion by different isolates of Campylobacter jejuni were investigated in day-of-hatch leghorn chicks. This model of Campylobacter colonization of the ceca demonstrates that 1) day-of-hatch birds do not naturally contain cecal Campylobacter, 2) ceca can be colonized with C. jejuni by oral gavage and not by cloacal inoculation; 3) C. jejuni can be recovered from the ceca up until at least 7 days postinoculation, 4) cecal colonization occurs when as little as 10(2) colony-forming units is orally inoculated into chicks, and 5) different C. jejuni isolates vary both in their ability to colonize the ceca and in their ability to invade the liver. These studies demonstrate that we have a working animal model for Campylobacter colonization for day-of-hatch chicks. This animal model is being used to examine intervention strategies such as vaccines by which Campylobacter can be reduced or removed from the food animal.

The absence of cecal colonization of chicks by a mutant of Campylobacter jejuni not expressing bacterial fibronectin-binding protein.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.

Prevalence of Campylobacter, Salmonella, and Arcobacter species at slaughter in market age pigs.

A survey was conducted to determine the prevalence of Campylobacter, Salmonella, and Arcobacter species in market age pigs from an integrated swine operation in Texas. Our findings indicate that farms from this commercial operation were heavily contaminated with Campylobacter and Salmonella, that the isolation rates of C. jejuni were higher than predicted, and that there was a low prevalence of Arcobacter.

Prevalence of Campylobacter spp isolated from the intestinal tract of pigs raised in an integrated swine production system.

OBJECTIVE: To enumerate the prevalence of Campylobacter isolates in the intestinal tract of market-weight swine raised in an integrated swine operation in Texas. SAMPLE POPULATION: Samples of cecal contents were collected from 595 pigs (mean body weight, 110 kg [242 lb]) at time of slaughter. Pigs were off-spring of Yorkshire-Landrace sows and Duroc or Hampshire boars. Pigs originated from 4 farrow-to-finish farms. PROCEDURE: During a 9-month period, visits were made to a slaughter plant to remove cecal contents from market-weight hogs. Samples were obtained from 50 pigs/visit from designated farms so that samples were obtained 3 times from pigs of each of 4 farms. Isolation of Campylobacter spp was accomplished by use of enrichment broth and restrictive media, using microaerophilic conditions. RESULTS: Campylobacter spp were isolated from 70 to 100% of the pigs, depending on the farm and the date the samples were collected. Campylobacter coli was isolated from 20 to 100% (mean, 60%) of samples, and C jejuni was isolated from 0 to 76% (mean, 31%) of samples. Campylobacter lari was isolated from 2 pigs. Concentrations of C coli or C jejuni ranged from 10(3) to 10(7) colony-forming units/g of cecal content. CONCLUSIONS AND CLINICAL RELEVANCE: Campylobacter coli generally is accepted as a common inhabitant of the intestinal tract of swine. However, analysis of results of this study suggests that a relatively high prevalence of C jejuni may be found in pigs raised on specific farms.

Reduction of caecal Listeria monocytogenes in Leghorn chicks following treatment with a competitive exclusion culture (PREEMPT)

Day-of-hatch Leghorn chicks were treated by oral gavage with PREEMPT, a continuous-flow competitive exclusion culture containing broiler caecal bacteria, followed by an oral challenge with Listeria monocytogenes, to determine the effects of PREEMPT on L. monocytogenes caecal colonization. Increased (P < 0.001) concentrations of caecal propionic acid in control chicks compared with PREEMPT-treated chicks at 3 days of age were indicative of the establishment of the PREEMPT bacteria. Caeca from control chicks at 7 days after the oral challenge with L. monocytogenes contained mean 3.4 +/- 1.4 log cfu g-1 of caecal content, while caeca from PREEMPT-treated chicks contained no detectable Listeria. Enrichment for L. monocytogenes resulted in 100% of caeca from control chicks testing culture-positive for L. monocytogenes, while none of the caeca from PREEMPT-treated chicks were culture-positive. The results indicated that prophylactic treatment of newly hatched chicks with PREEMPT significantly reduced caecal colonization by L. monocytogenes.

Characterization of the thermal stress response of Campylobacter jejuni.

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.

Heterophil response to intraperitoneal challenge with invasion-deficient salmonella enteritidis and Salmonella-immune lymphokines.

The present work compared the accumulation of intraperitoneal heterophils in day-of-hatch chicks following treatment with Salmonella enteritidis-immune lymphokine (ILK) and challenge with various strains of Salmonella enteritidis (SE). Day-of-hatch chicks received ILK by intraperitoneal injection and were challenged 1 hr later by intraperitoneal inoculation with one of the following SE strains: a wild-type, SE 890034-3; a delta cya-12 delta cyp-11 avirulent vaccine strain, chi 4357; and an invasion-deficient strain, InvA::kan, chi 4420. Four hours after challenge heterophils were recovered from the peritoneal cavity by lavage. The concentration of heterophils in the recovered lavage fluid was determined. Heterophil concentrations increased in response to challenge with each SE strain but there was a lower response to the invasion-deficient strain. The difference was statistically significant. This diminished heterophil response to challenge with invasion-deficient salmonellae supports existing evidence that the initial defensive reaction occurs at the earliest stages of the Salmonella-host interaction.

Efficacy of two avian Salmonella-immune lymphokines against liver invasion in chickens by Salmonella serovars with different O-group antigens.

Newly hatched chicks are susceptible to organ invasion by various serovars of Salmonella. We have previously shown that intraperitoneal administration of Salmonella enteritidis-immune lymphokine (SEILK) increased resistance to organ invasion by S. enteritidis (SE) in day-of-hatch chicks. In the present study, we determined that Salmonella-immune lymphokines can provide protection against organ invasion by serovars that are unrelated to the original serovar used as an immunogen. Immune lymphokines were produced from the splenic T cells of mature hens immunized with either viable SE or Salmonella anatum (SA). Day-of-hatch chicks were treated with either SA-immune lymphokine (SAILK) or SEILK and challenged per os with either Salmonella typhimurium (ST) or SE 1 hr later. Liver samples were aseptically collected 20 hr later and were cultured to detect salmonella. We found that SAILK conferred resistance against invasion by either SE, a serogroup D organism, or ST, a serogroup B organism. Furthermore, SEILK reduced organ invasion by ST. These observations together reveal that the protection against organ invasion conferred by Salmonella-immune lymphokines is unrelated to the serovar-specific antigenic characteristics of the immunizing bacteria.

Efficacy of Salmonella enteritidis (SE)-immune lymphokines from chickens and turkeys on SE liver invasion in one-day-old chicks and turkey poults.

We have shown previously that increased resistance to Salmonella enteritidis (SE) organ infectivity in 1-day-old chicks was conferred by the immunoprophylactic administration of SE-immune lymphokines (SEILK). These lymphokines have been found to be present in the cell culture media of concanavalin A-stimulated splenic lymphocytes obtained from SE-immunized chickens. In the present study we evaluated whether turkeys also produced SEILK and whether these lymphokines could protect 1-day-old chicks and turkey poults against SE liver invasion. In addition, we tested the ability of our original chicken SEILK to reduce SE liver invasion in turkey poults. Day-of-hatch chicks and turkey poults were injected intraperitoneally with immune lymphokines of either chicken or turkey origin. One hour later the birds were challenged per os with SE, and 20 hours later their livers were examined by bacteriological methods for the presence of SE. We found that SEILK induced from the splenic lymphocytes of SE-immunized turkeys reduced SE liver invasion in both chicks and turkey poults. Conversely, we also determined that SEILK produced by chicken splenic lymphocytes conferred protection against invasion by SE in turkey poults. This research is the first report of the production of SEILK in turkeys and also the first report on the cross-species activity of these effector molecules in chickens and turkeys.

Effect of ochratoxin A on Salmonella-challenged broiler chicks.

Poultry products represent a significant reservoir of Salmonella typhimurium. Ochratoxin A, a mycotoxin and natural contaminant of poultry feedstuffs, produces detrimental effects on the immune and other systems of the broiler chick. Because poultry products are possible sources of S. typhimurium contamination that can potentially infect humans, there is a need to know whether ochratoxin A can alter the growth of Salmonella in poultry. We investigated the pathological alterations of young male broiler chicks by S. typhimurium in the presence (3.0 mg/kg) or absence of ochratoxin A in the diet. Ochratoxin A alone in the diet decreased the body weight and increased the relative organ weights of the liver, kidney, gizzard, spleen, pancreas, and proventriculus. It did not affect the heart and bursa of Fabricius. The mycotoxin altered the serum concentrations of proteins, enzymes, calcium and phosphate salts, normal tissue constituents, and catabolic metabolites in a pattern that would suggest damage to skeletal muscle, liver, kidney, pancreas, and bone. Birds fed diets containing ochratoxin A had microcytic and hypochromic erythrocytes and a decrease in phytohemagglutin- and concanavalin A-stimulated blastogenesis. Salmonella typhimurium alone had no affect on the variables measured except for a decrease in body weight. With the exception of an increase in mortality (13.2%, a significant synergistic interaction) and decrease in body weight, Salmonella in combination with ochratoxin A did not alter the values of the remaining variables measured from those measured in the ochratoxin A diet alone. Cecal colony count of S. typhimurium was not affected by treatment with ochratoxin A.

Gastrointestinal uptake and vascular transport of 2,4'-dichlorobiphenyl.

Absorption from the gastrointestinal tract and subsequent vascular transport of [3H]-2,4'-dichlorobiphenyl (Aroclor 1232; DCB) was investigated in an ovine model system. Rapid uptake of DCB and transport as a component of blood plasma without prior occurrence in thoracic duct lymph indicates that DCB was absorbed directly via the gastric mucosa with water soluble compounds. [3H]-DCB did not circulate associated with plasma lipid fractions in vivo, and did not bind to or sequester within plasma lipids in vitro. HPLC analysis of plasma fractions treated in vitro showed DCB to elute within a molecular weight range consistent with unbound product. Further, [3H]-DCB-derived label was associated with low molecular weight plasma components in vivo. Essentially the same elution profile was seen for [3H]-DCB-derived label found in urine. Metabolism of DCB as a function of time resulted in the apparent formation of a biotransformed product(s) that circulated with a plasma fraction(s) at the low end of the albumin molecular size range. These data suggest that DCB was not absorbed and transported in a manner typical of polychlorinated biphenyls with a higher chlorine content; rather, that it was absorbed, transported within the vascular system, and excreted in a pattern typical of a water soluble compound.

Comparison of probiotics maintained by in vivo passage through laying hens and broilers.

Cecal colonization by salmonellae may be greatly reduced by inoculating chickens with normal cecal microflora, a phenomenon known as competitive exclusion. Unfortunately, it has not been possible to reliably store active cecal microflora over long time periods, and it is difficult to obtain consistent experimental results with different batches of microflora. In order to overcome these problems, the present authors have maintained active cecal flora through a 2-yr period by in vivo passage through both broiler chicks and layers that were fed a diet containing 5% lactose. Competitive exclusion cultures maintained in laying hens worked as well as cultures from broilers. Cecal microflora from either source excluded both nonlactose-fermenting and lactose-fermenting Salmonella strains. Colonization by both types of Salmonella was reduced even when the competitive exclusion organisms were given as late as 3 days after oral challenge inoculation with Salmonella typhimurium.

Control of established Salmonella typhimurium intestinal colonization with in vivo-passaged anaerobes.

Broiler chickens were inoculated orally with 10(6) Salmonella typhimurium on the day of hatch. Twenty-four to 72 hr after challenge, the chicks were inoculated orally with cecal microflora that had been repeatedly passed through lactose-fed broiler chicks. In vivo passage proved to be a convenient and practical method for preserving protective anaerobic flora. These organisms effectively reduced S. typhimurium concentrations in the cecal contents by 4-5 orders of magnitude, even when given 24 to 72 hr after Salmonella challenge inoculation.