Prevalence of Campylobacter spp. in cattle in Finland and antimicrobial susceptibilities of bovine Campylobacter jejuni strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n=952) and carcass surface samples (n=948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n=730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Prevalence and Antimicrobial Resistance of Thermophilic Campylobacter spp. from Cattle Farms in Washington State

The prevalence of thermophilic Campylobacter spp. was investigated in cattle on Washington State farms. A total of 350 thermophilic Campylobacter isolates were isolated from 686 cattle sampled on 15 farms (eight dairies, two calf rearer farms, two feedlots, and three beef cow-calf ranches). Isolate species were identified with a combination of phenotypic tests, hipO colony blot hybridization, and multiplex lpxA PCR. Breakpoint resistance to four antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, and doxycycline) was determined by agar dilution. Campylobacter jejuni was the most frequent species isolated (34.1%), followed by Campylobacter coli (7.7%) and other thermophilic campylobacters (1.5%). The most frequently detected resistance was to doxycycline (42.3% of 350 isolates). Isolates from calf rearer facilities were more frequently doxycycline resistant than isolates from other farm types. C. jejuni was most frequently susceptible to all four of the antimicrobial drugs studied (58.8% of 272 isolates). C. coli isolates were more frequently resistant than C. jejuni, including resistance to quinolone antimicrobials (89.3% of isolates obtained from calves on calf rearer farms) and to erythromycin (72.2% of isolates obtained from feedlot cattle). Multiple drug resistance was more frequent in C. coli (51.5%) than in C. jejuni (5.1%). The results of this study demonstrate that C. jejuni is widely distributed among Washington cattle farms, while C. coli is more narrowly distributed but significantly more resistant.     

Effects of Subtherapeutic Administration of Antimicrobial Agents to Beef Cattle on the Prevalence of Antimicrobial Resistance in Campylobacter jejuni and Campylobacter hyointestinalis

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (≥94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.     

Comparison of Prevalence and Antimicrobial Susceptibilities of Campylobacter spp. Isolates from Organic and Conventional Dairy Herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.The incidence of human enteric infections caused by campylobacters is highest in the summer months, showing a consistent peak at the end of July in Finland (www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf), as well as in other Nordic countries (16, 33). Almost 70% of campylobacter infections detected in July and August in Finland are domestically acquired, whereas the annual average proportion of domestic cases is about 30%, and most of them are caused by Campylobacter jejuni (30). The prevalence of campylobacters in Finnish broiler flocks peaks simultaneously with the human cases (7), and similar sero- and genotypes have been reported among human and poultry strains isolated in Finland and in other countries (5, 8, 21-23). Several epidemiological studies have identified the handling and consumption of raw or undercooked poultry meat as a major risk factor for campylobacter enteritis (for example, see references 18, 20, and 41), whereas opposite conclusions about the significance of the consumption of chicken meat were drawn from the Swedish case-control study among young children (2) and an extensive Danish register-based study (6).Data derived from the genotyping studies of C. jejuni isolates from human infections and animals support the current suggestion that poultry is the most important single source of sporadic campylobacteriosis (12, 22, 29). However, several reports on genotype comparisons suggest that poultry may be a less significant source of campylobacters than generally thought, and other animal reservoirs should also be considered notable sources of campylobacters pathogenic to humans (3, 8, 17, 27, 31). Studies of the temporal occurrence of campylobacters in human infections and poultry flocks have revealed that the peak in prevalence, as well as some of the overlapping sero- and genotypes, is detected in humans prior to being detected in poultry (21, 28).Although cattle are well-known carriers of campylobacters, the survival of these fragile organisms in beef is poor (39, 42). In recent years, some authors (1, 4, 10) have raised the question of an indirect association between cattle and human cases. In a Finnish study combining data from the multilocus sequence typing of campylobacters isolated from production animals and from epidemiological studies of human cases, significant associations emerged between certain sequence-type complexes from human infections and contact with cattle, the consumption of unpasteurized milk, or the tasting or consumption of raw minced meat (23).The aim of this study was to investigate the contributions of poultry and cattle as sources of human C. jejuni infections in Finland by comparing over a limited time frame the macrorestriction profiles obtained from pulsed-field gel electrophoresis (PFGE) analysis of a geographically representative collection of C. jejuni isolates from domestically acquired sporadic human infections, chicken process lots, and cattle.     

Increasing Prevalence of Campylobacter jejuni in Feedlot Cattle through the Feeding Period

The prevalence of Campylobacter jejuni in commercial feedlot cattle was monitored throughout the feeding period by repeated bacteriologic culture of feces. Fecal pats (n = 10) in 20 feedlot pens were sampled at 2-weeks interval beginning at entry into the feedlot and continuing until slaughter. The least-squares mean C. jejuni prevalence increased from 1.6% at the first sampling to 61.3% at the final sampling just prior to slaughter. Diverse C. jejuni pulsed-field gel electrophoresis macrorestriction profiles (MRP) were identified among the cattle isolates, but five prevalent MRP and minor variants accounted for >80% of all typed isolates. Chlorination of the water supplied to the water troughs of half of the pens did not affect C. jejuni prevalence in the cattle. Overall, the least-squares mean C. jejuni prevalences were 45.6 and 43.6% in chlorinated and nonchlorinated feedlot pens, respectively. The results of this study demonstrate apparent transmission of C. jejuni among feedlot cattle during the feeding period, unaffected by water chlorination, resulting in a high prevalence of C. jejuni excretion by cattle approaching slaughter.     

Bovine Campylobacter jejuni Strains Differ from Human and Chicken Strains in an Analysis of Certain Molecular Genetic Markers

The association of four new genetic markers with a chicken, bovine, or human host was studied among 645 Campylobacter jejuni isolates. The γ-glutamate transpeptidase gene and dmsA were common in human and chicken isolates but uncommon among bovine isolates. In the t test, bovine isolates differed significantly (P < 0.05) from human and chicken isolates.     

Colonization of Cattle Intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Prevalence and survival of Campylobacter jejuni in unpasteurized milk.

Campylobacter jejuni was isolated from 1 to 108 (0.9%) milk samples obtained from the bulk tanks of nine grade A dairy farms and from 50 of 78 (64%) cows producing grade A milk. Survival of eight Campylobacter strains in unpasteurized milk (4 degrees C) varied greatly: the most tolerant strain showed a less than 2-log10 decrease in viable cells after 14 days, and the most sensitive strain showed a greater than 6-log10 decrease after 7 days. One strain was still recoverable 21 days after the inoculation of milk. Inactivation of the different strains corresponded with an increase in milk aerobic plate count and a decrease in milk pH; however, no absolute correlation could be made between the rates of change of these parameters and the rates of campylobacter inactivation. When held at 4 degrees C, C. jejuni was most stable in brucella broth, died most rapidly in unpasteurized milk, and was inactivated at an intermediate rate in sterile milk. Our results indicate the presence and possible persistence of C. jejuni in raw grade A milk and reaffirm the need for pasteurization of milk.     

Antimicrobial Susceptibilities of Aeromonas spp. Isolated from Environmental Sources

Aeromonas spp. are ubiquitous aquatic bacteria that cause serious infections in both poikilothermic and endothermic animals, including humans. Clinical isolates have shown an increasing incidence of antibiotic and antimicrobial drug resistance since the widespread use of antibiotics began. A total of 282 Aeromonas pure cultures were isolated from both urban and rural playa lakes in the vicinity of Lubbock, Texas, and several rivers in West Texas and New Mexico. Of these, at least 104 were subsequently confirmed to be independent isolates. The 104 isolates were identified by Biolog and belonged to 11 different species. The MICs of six metals, one metalloid, five antibiotics, and two antimicrobial drugs were determined. All aeromonads were sensitive to chromate, cobalt, copper, nickel, zinc, cefuroxime, kanamycin, nalidixic acid, ofloxacin, tetracycline, and sulfamethoxazole. Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Dual resistances were found in 5 of the 104 Aeromonas isolates. Greater numbers of resistant isolates were obtained from samples taken in March versus July 2002 and from sediment versus water. Plasmids were isolated from selected strains of the arsenite- and mercury-resistant organisms and were transformed into Escherichia coli XL1-Blue MRF′. Acquisition of the resistance phenotypes by the new host showed that these resistance genes were carried on the plasmids. Mercury resistance was found to be encoded on a conjugative plasmid. Despite the low incidence of resistant isolates, the six playa lakes and three rivers that were sampled in this study can be considered a reservoir for antimicrobial resistance genes.     

Prevalence of Campylobacter jejuni in Chicken Wings

Campylobacter jejuni was found in 82.9% of 94 chicken wing packages analyzed on the day of arrival at supermarkets and in 15.5% of 45 packages obtained from the supermarket shelves a few days later. The number of bacterial cells ranged from 10² to 10^3.9 per wing. The prevalence of C. jejuni in the wings varied with the brand, the day of sampling, and the age of the product.     

Prevalence of Campylobacter jejuni in chicken wings

Campylobacter jejuni was found in 82.9% of 94 chicken wing packages analyzed on the day of arrival at supermarkets and in 15.5% of 45 packages obtained from the supermarket shelves a few days later. The number of bacterial cells ranged from 10(2) to 10(3.9) per wing. The prevalence of C. jejuni in the wings varied with the brand, the day of sampling, and the age of the product.     

Prevalence and Antimicrobial Resistance of Campylobacter spp. and Salmonella Serovars in Organic Chickens from Maryland Retail Stores

Retail organic (n = 198) and conventional (n = 61) chickens were analyzed. Most organic (76%) and conventional (74%) chickens were contaminated with campylobacters. Salmonellae were recovered from 61% of organic and 44% of conventional chickens. All Salmonella enterica serovar Typhimurium isolates from conventional chickens were resistant to five or more antimicrobials, whereas most S. enterica serovar Typhimurium isolates (79%) from organic chickens were susceptible to 17 antimicrobials tested.     

Prevalence of Campylobacter jejuni in two California chicken processing plants.

Two federally inspected California chicken processing plants participated in Campylobacter jejuni prevalence studies. Twelve sampling sites were included in each of four groups. Groups were based on bird age, scald water temperature, and plant sampled. Scald water temperatures of 60 degrees C (140 degrees F) did not contribute to a lower prevalence of C. jejuni in edible parts, as did temperatures of 53 degrees C (127 degrees F) and 49 degrees C (120 degrees F). The feather picker and chilling tank were areas of major cross-contamination. C. jejuni was isolated from 68% of the ready-for-market products. The organism was recovered from 60 to 100% of the ceca in the four groups, and some numbers in the fecal material exceeded 10(6)/g. The level of C. jejuni in intestinal tracts seemed to correlate with the presence of the organism in the edible parts.     

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains

Campylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock on C. jejuni conjugation efficiency and the underlying mechanisms were examined. With a modified Escherichia coli donor strain, different C. jejuni recipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10⁻⁸ to 6.0 × 10⁻³ CFU per recipient cell. Despite reduced viability, heat shock of standard C. jejuni NCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhance C. jejuni conjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation in C. jejuni recipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treated C. jejuni cells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation in C. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standard C. jejuni strains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer in C. jejuni.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Effect of Conventional and Organic Production Practices on the Prevalence and Antimicrobial Resistance of Campylobacter spp. in Poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR

Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 10³ CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.     

Avian wildlife reservoir of Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. in Norway

Cloacal swabs from 540 wild-living birds were cultured for Campylobacter fetus subsp. jejuni, Yersinia spp., and Salmonella spp. The carrier rates detected were as follows: C. fetus subsp. jejuni, 28.4%; Yersinia spp., 1.2%; and Salmonella spp., 0.8%. All birds were apparently healthy when captured. C. fetus subsp. jejuni was isolated from 11 of the 40 bird species examined. Among birds inhabiting the city of Oslo, the highest isolation rate was found in crows (Corvus corone cornix) (89.8%), followed by gulls (Larus spp.) (50.0%) and domestic pigeons (Columba livia domesticus) (4.2%). The gulls and crows scavenge on refuse dumps. High carrier rates were also detected among the following birds from nonurban, coastal areas: puffin (Fratercula arctica) (51.3%), common tern (Sterna hirundo) (5.6%), common gull (Larus canus) (18.9%), black-headed gull (Larus ridibundus) (13.2%), and herring gull (Larus argentatus) (4.2%). The list of species harboring C. fetus subsp. jejuni also includes the Ural owl (Strix uralensis), goldeneye (Bucephala clangula), and reed bunting (Emberiza schoeniclus). The following five Yersinia strains were isolated: Y. kristensenii (two strains), Y. intermedia (two strains), and "Yersinia X2" (one strain). Four strains belonging to the genus Salmonella were isolated from three different species of gulls. These isolates were identified as S. typhimurium, S. indiana, and S. djugu. The results indicate that campylobacters are a normal component of the intestinal flora in several bird species, whereas Salmonella and Yersinia carriers are more sporadic.