Robert E. Droleskey

Genotypic variation among arcobacter isolates from a farrow-to-finish swine facility.

Arcobacter spp. were isolated from nursing sows and developing pigs on three farms of a farrow-to-finish swine operation and market-age pigs at slaughter. Isolates were identified by polymerase chain reaction and genotypic fragment patterns were examined by pulsed-field gel electrophoresis (PFGE). Incidences of Arcobacter-positive samples increased progressively as the pigs aged, resulting in all of the pens at the end of the growth cycle in the finishing barn containing Arcobacter-positive feces. However, only 10 of 350 cecal samples from slaughtered pigs were positive. There was little similarity between genotypic patterns for Arcobacter collected from the three farms. The level of genotypic variation revealed by PFGE suggested that pigs in this farrow-to-finish operation were colonized by multiple Arcobacter parent genotypes that may have undergone genomic rearrangement, common to members of Campylobacteraceae, during successive passages through the animals. Additionally, the level of genotypic diversity seen among Arcobacter isolates from farms of a single farrow-to-finish swine operation suggests an important role for genotypic phenotyping as a source identification and monitoring tool during outbreaks.

Prevalence of antimicrobial resistance in Salmonellae isolated from market-age swine.

Antimicrobial resistance levels were examined for 365 Salmonella isolates recovered from the lymph nodes (n = 224) and cecal contents (n = 141) of market-age swine at slaughter. Antimicrobial resistance testing was performed by disk diffusion using 13 antibiotics common in the treatment of disease in human and veterinary medicine. Although none of the antibiotics tested were used subtherapeutically within the last 5 years on the farms sampled, resistance to chlortetracycline, penicillin G, streptomycin, and sulfisoxazole was common. Penicillin G resistance was significantly more frequent (P = 0.03) and sulfisoxazole resistance was significantly less frequent (P < 0.01) in lymph node versus cecal isolates. Multidrug resistance was observed among 94.7% of the lymph node isolates and 93.5% of the cecal isolates. The most frequent multidrug resistance pattern included three antibiotics-penicillin G, streptomycin, and chlortetracycline. Isolates in somatic serogroup B, and more specifically, Salmonella Agona and Salmonella Schwarzengrund isolates, were often resistant to a greater number of antibiotics than were isolates in the other serogroups. Streptomycin, sulfisoxazole, ampicillin (lymph node isolates), and nitrofurantoin (cecal isolates) resistance levels differed significantly between somatic serogroups. The prevalence of penicillin G-, streptomycin-, and sulfisoxazole-resistant isolates differed significantly between serovars for both lymph node and cecal isolates. Results of this study suggest that a correlation exists between the somatic serogroup or serovar of a Salmonella isolate and its antimicrobial resistance status, which is specific to the antibiotic of interest and the source of the isolate (lymph node versus cecal contents).

The Acquisition and Internalization of Salmonella by the Lesser Mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae)

In poultry broiler production facilities, it is important to understand the sources and contribution of reservoir populations of pathogens. The lesser mealworm beetle, Alphitobius diaperinus (Panzer), is a common pest in poultry litter that is reported to carry pathogens affecting both human and animal health. This study investigates whether the carriage of a bacterial pathogen occurs by the harboring of bacteria internally by these insects. Beetles were exposed to a marker bacterium, Salmonella enterica serovar Typhimurium-green fluorescent protein (ST-GFP), at concentrations up to 10(7) colony-forming units (cfu)/mL for 0.5 to 12 h, and then subsequently surface disinfected and dissected. The head, gastrointestinal tract and hemolymph were cultured for the presence of ST-GFP. This study definitively demonstrates the internal carriage of Salmonella by this insect and found that the beetles rapidly acquired bacteria from external sources and harbored the bacteria within their alimentary canal after exposure for 30 min at 10(4) cfu/mL and within the hemolymph after exposure for 2 h at 10(6) cfu/mL. Beetles internalized an average of 9.5 × 10(1) and 3.2 × 10(3) after a 2-h exposure to 2 × 10(4) and 2 × 10(6) cfu/mL, respectively. The lesser mealworm is a serious pest within the poultry brooder and laying industry and because of their mobility, voracious feeding habits, and prey potential may represent an active source facilitating the dissemination of Salmonella.

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.

Failure of Viable Nonculturable Campylobacter jejuni to Colonize the Cecum of Newly Hatched Leghorn Chicks

Abstract 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.

In Vitro Cultivation of a Zoonotic Babesia sp. Isolated from Eastern Cottontail Rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts

ABSTRACT A Babesia sp. found in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, is the same organism that caused human babesiosis in Missouri and Kentucky, on the basis of morphology and identical small-subunit rRNA (SSU rRNA) gene sequences. Continuous cultures of the rabbit parasite were established from infected blood samples collected from two cottontail rabbits livetrapped on Nantucket Island. HL-1 medium or minimal essential medium alpha medium supplemented with 20% human serum best supported in vitro propagation of the parasite in human or cottontail erythrocytes, respectively. Parasite growth was not sustained in domestic-rabbit erythrocytes or in medium supplemented with domestic-rabbit serum. The cultured parasites were morphologically indistinguishable from the Kentucky human isolate. Transmission electron microscopy revealed similar fine structures of the parasite regardless of the host erythrocyte utilized in the cultures. Two continuous lines of the zoonotic Babesia sp. were established and confirmed to share identical SSU rRNA gene sequences with each other and with the Missouri and Kentucky human Babesia isolates.

Transmission Electron Microscopy

Transmission electron microscopy has long been an important analytical tool in the field of microbiology. This unit describes preparation techniques for examining particulate samples as well as samples presenting more complex ultrastructural considerations that require analysis in thin sections. Negative staining is a useful technique for routine examination of particulate samples in suspension ranging from bacteria to purified macromolecules. In order to investigate the relationships between microbes and the environments with which they interface, fixed samples can be prepared for imaging in sections of 60‐ to 90‐nm thickness. Due to the many steps in sample preparation for ultrastructural analysis of thin‐sectioned samples, the major steps in the process are divided into fixation and initial processing of samples for thin sectioning, the embedment of samples into a plastic resin for sectioning, ultramicrotomy, and staining of samples. Procedures for immunolocalization of antigens in negatively stained and thin‐sectioned preparations are also considered.

Colonization of Cecal Mucosal Epithelium in Chicks Treated with a Continuous Flow Culture of 29 Characterized Bacteria: Confirmation by Scanning Electron Microscopy

Bacterial colonization of cecal mucosal epithelium in 3-day-old chicks administered a characterized continuous-flow (CF) culture of 29 microorganisms on the day of hatch was evaluated by scanning electron microscopy. Extensive colonization of the mucosa was noted in the ceca of CF-treated chicks, with large colonies of bacteria located predominately within and between crypts. Cecal crypts from control chicks contained only thin strands of mucus with a few bacteria. Individual cells and clumps of bacteria were observed bound to the mucosal epithelium in both CF-treated and control chicks. Colonization by CF culture bacteria was accompanied by an increase in the concentration of volatile fatty acids in the cecal contents and increased resistance to colonization by Salmonella typhimurium .

In vitro growth of Babesia bovis in white-tailed deer (Odocoileus virginianus) erythrocytes.

Babesia bovis cultured in bovine erythrocytes was passaged into white-tailed deer (Odocoileus virginianus) erythrocytes and medium containing either white-tailed deer serum or bovine serum. Deer erythrocytes supported the growth of the parasite only in the presence of bovine serum. Cryopreserved cultures were recovered successfully in white-tailed deer erythrocytes. By light and electron microscopy, B. bovis structure appeared similar in host cells of either species.

Campylobacter coli Pulsed Field Gel Electrophoresis Genotypic Diversity Among Sows and Piglets in a Farrowing Barn

Genotypes of Campylobacter coli isolates from feces of three sows and rectal swabs of 17 piglets were examined by pulsed field gel electrophoresis (PFGE). All of the animals originated from a single farrowing barn of a farrow-to-finish swine operation. Five Campylobacter colonies were picked from a single agar plate for each sample after broth enrichment and growth on Campy-Cefex agar. Genotypes were examined by PFGE after genomic DNA digestion with SmaI and SacII restriction endonucleases. Twenty SmaI genotypes and 12 SacII genotypes were detected among 99 Campylobacter coli isolates. There was no pattern of shared genotypes between sows and their respective piglets, nor between littermates. Results indicate that a high number of Campylobacter genotypes may coexist in related pigs from a single housing facility.