Production and characterization of monoclonal antibodies against the antibiotic tilmicosin.

Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocalization of tilmicosin.

Toxicity of fumonisin from Fusarium verticillioides culture material and moniliformin from Fusarium fujikuroi culture material when fed singly and in combination to growing barrows.

The effects of fumonisin B1 (FB1) from Fasarium verticillioides culture material and moniliformin from Fusarum fujikuroi culture material on growing barrows were evaluated. Four groups of six barrows (three replicates of two each; mean body weight, 11.1 kg) were fed diets containing 0 mg of FB1 and 0 mg of moniliformin per kg of feed (control), 100 mg of FB1 per kg of feed, 100 mg of moniliformin per kg of feed, and 100 mg of FB1 plus 100 mg of moniliformin per kg of feed. Barrows were fed these diets for 28 days. Body weight gain, feed efficiency, serum biochemical analytes, and hematological values were adversely affected by the FB1 and the FB1-plus-moniliformin diets. The moniliformin diet decreased body weight gain. Two barrows in the moniliformin diet group died, and two barrows in the FB1-plus-moniliformin diet group died. All deaths occurred during the first 6 days of the study. Mild to moderate lesions were observed microscopically in heart and lung tissues of the groups fed moniliformin and FB1 plus moniliformin and in liver tissues of the groups fed FB1 and FB1 plus moniliformin. Except for the acute mortality associated with the two diets containing moniliformin. clinical disease induced by the combined feeding of these two mycotoxins appears to be additive or less than additive and due primarily to the toxic expression of FB1.

Campylobacter prevalence in lactating dairy cows in the United States.

The objective of the present study was to determine the prevalence of intestinal Campylobacter in lactating dairy cows from various regions of the United States. Participating commercial dairy farms were chosen at random and were part of a national survey to determine E. coli O157:H7 and Salmonella prevalence in dairy cows. Farms had no previous history of Campylobacter problems. Fecal samples were collected rectally from 720 cows on farms in the northeast (four farms), in the desert southwest (three farms), and in the Pacific west (two farms). A minimum of 60 fecal samples per visit were collected from each farm. Thirty isolates were analyzed using the RiboPrinter Microbial Characterization System to obtain ribosomal RNA patterns. Twenty isolates were tentatively identified as Campylobacter jejuni, two as Campylobacter coli, three as Campylobacter spp., and five as unknown. Individual single-visit farm prevalence ranged from 0 to 10%. The disk diffusion method, employing 11 antibiotics, was used to test the antibiotic sensitivities of 27 of the isolates. Eight isolates were resistant to two or more antibiotics, 13 isolates were resistant to one antibiotic, and 6 were totally susceptible. Under the conditions of this study, the authors conclude that Campylobacter prevalence in lactating dairy cows in the United States is low, there is no difference in prevalence on the basis of geographical location, the predominant species is C. jejuni, and that the majority of these isolates are sensitive to antibiotics.

Inability of cecal microflora to promote reversion of viable nonculturable Campylobacter jejuni.

Campylobacter jejuni cells are able to enter a viable but nonculturable (VBNC) state when they are suspended in water. In the present experiments we inoculated day-of-hatch leghorn and broiler chicks with normal gut microflora and subsequently challenged these with high doses of VBNC C. jejuni. The objective was to determine if the pre-establishment of a normal gut flora would enable VBNC Campylobacter to recover, revert to the vibrionic form, and colonize the cecum. Day-of-hatch leghorn and broiler chicks were gavaged through the esophagus with 0.75 ml of a continuous-flow culture of normal cecal organisms. Two days after gavage, the same chicks were gavaged with 0.75 ml (greater than 10(9) colony-forming units) of a VBNC suspension of C. jejuni. Seven days later, cecal contents were collected, serially diluted, and examined for the presence of viable culturable C. jejuni. Our results demonstrated that the VBNC C. jejuni cells were unable to revert to a vibrionic culturable form capable of colonizing the cecum.

Cecal colonization of chicks by bovine-derived strains of Campylobacter.

Campylobacter jejuni and Campylobacter coli strains were isolated from feces of dairy cattle at farms with no known problem due to campylobacteria. Farms were located in the northeast, desert southwest, and Pacific west. Twenty isolates were identified by ribotyping with a RiboPrinter. The ability of these bovine isolates to colonize the ceca of chicks was determined by challenge inoculation and reisolation of the challenge strain from the ceca at 1 and 2 wk after challenge. Isolates recovered from chick ceca were examined by ribotyping to assure they matched the challenge strain. One hundred percent of the bovine-derived challenge strains were capable of colonizing chicks. These results indicate that dairy cattle may be asymptomatic Campylobacter carriers and potential sources of campylobacteria contamination of poultry facilities.

Campylobacter colonization of the crops of newly hatched leghorn chicks.

We have studied colonization of crops in newly hatched leghorn chicks (a layer breed) by wild-type and mutant strains of Campylobacter jejuni. We established that the wild-type parent strain forms a stable population level within the crop and that the mutant strains will do likewise. Concentrations of mutant strains in the crop were usually below that of the wild-type parent strain and ranged from 10(3) to 10(5) colony-forming units. These results differ from results we have previously reported concerning cecal colonization, where these same mutant strains lacked colonizing ability. The present results, therefore, indicate that bacterial factors necessary for colonization of the crop are not the same as those needed for colonization of the cecum.

Failure of viable nonculturable Campylobacter jejuni to colonize the cecum of newly hatched leghorn chicks.

Campylobacter jejuni cells entered the viable but nonculturable (VBNC) state upon suspension in sterile water. Cell viability was determined with tetrazolium violet. VBNC cells suspended in water for 7, 10, or 14 days were given, by gavage, to day-of-hatch leghorn chickens. The ceca of control and challenged birds were examined for the presence of campylobacteria by conventional microbiological methods at 1 wk and 2 wk after challenge inoculation and by polymerase chain reaction methods at 1 wk after challenge. We did not find culturable Campylobacter cells in the ceca. Neither was Campylobacter DNA found in cecal samples. Therefore, VBNC cells did not revert to the culturable colonizing form, nor did VBNC cells persist within the cecal environment.

Cecal colonization of chicks by porcine strains of Campylobacter coli.

Ten genotypically distinct strains of Campylobacter coli were isolated from a swine production facility. These porcine isolates were then orally inoculated into day-of-hatch leghorn chicks and were excellent colonizers of the chick cecum. Campylobacter coli recovered from inoculated chickens were genotypically identical to the challenge strain. The absence of host specificity suggests a possible movement of strains among swine, field animals and birds, and poultry houses.

Antibiotic and disinfectant susceptibility profiles of vancomycin-resistant Enterococcus faecium (VRE) isolated from community wastewater in Texas.

Vancomycin-resistant Enterococcus faecium (VRE) from human wastewater effluents in a nonclinical semiclosed agri-food system in Texas were characterized for susceptibility to antibiotics and disinfectants. The 50 VRE were resistant to eight fluoroquinolones and 10 of 17 antimicrobials typically active against Gram-positive organisms. The VRE were susceptible to quinupristin/dalfopristin and linezolid. Lack of the insertion element IS1251 correlated with VRE susceptibility to streptomycin and gentamicin at p < 0.0001 and p = 0.033, respectively. An association was observed between pulsed-field gel electrophoresis genotypes Ic and II and susceptibility to streptomycin at p = 0.0006. VRE susceptibility for nine disinfectants and five disinfectant components is shown. Ninety-two percent of the isolates had a minimum inhibitory concentration (MIC) for triclosan > or =2 ppm. Triclosan MICs for many of the VRE were well over expected product application levels. No association was observed between antibiotic resistance and disinfectant susceptibility in these VRE. Enterococci multiply-resistant to vancomycin and aminoglycosides were found in a non-hospital environment where one would not expect to find them.

News and notes: An atypical Campylobacter coli exhibiting unusual morphology.

Campylobacter coli cells are characterized by a comma, or spiral shape, and a single polar flagellum. Here we report stable spontaneous changes in morphology to an aflagellated straight rod. The ability to colonize the chick cecum was lost. The atypical cells were compared with the original C. coli 67 culture from which they were derived, with PCR, Riboprinting, and PFGE.

Further characterization of Campylobacter isolated from U.S. dairy cows.

The objective of the present study was to compare polymerase chain reaction (PCR) identification with ribotype results and to use pulsed field electrophoresis (PFGE) to correlate genotypic patterns with antibiotic resistance of Campylobacter isolated from lactating dairy cows in the United States. Thirty isolates were studied. Twenty-seven of the isolates were identified by PCR as Campylobacter jejuni and three were identified as Campylobacter coli. Genotypic patterns of 15 isolates were determined by PFGE, and although isolates originated from geographically separated regions of the United States, some were genotypically identical. In contrast to their genetic similarity, antibiotic sensitivity patterns differed within some genotypes. Under the conditions of our study, we concluded that ribotyping is not as discriminatory as PCR for speciation, and that a phenotypic trait such as antimicrobial resistance cannot always be predicted within the same genotype.

Inoculation of chicks with viable non-colonizing strains of Campylobacter jejuni: evaluation of protection against a colonizing strain.

We have treated chicks with viable non-colonizing mutant strains of Campylobacter jejuni to test these as a possible vaccine. We found that intramuscular inoculation with and without adjuvant, and with or without a concomitant oral dose of non-colonizing strains, failed to provoke protective immunity.