Shedding of Campylobacter spp. in Finnish cattle on dairy farms

Aims:  The aim of this study was to determine variation of prevalence throughout a year, colonization levels and genotypes of Campylobacter jejuni in Finnish dairy cattle herds.
Methods and Results:  Faecal samples and tank milk samples from three dairy cattle herds were taken five times, and swab samples from drinking troughs once during a 1-year sampling period. The samples were enriched in Bolton broth and subsequently spread on mCCDA. Isolates were then subtyped by pulsed-field gel electrophoresis using SmaI. Campylobacter jejuni was detected in 169 of the 340 faecal samples and in one drinking trough sample. Prevalences between herds and sampling times varied widely. The faecal levels of C. jejuni were mainly low. Between one and four SmaI subtypes were identified from each herd per sampling. Two SmaI subtypes persisted in two of the herds throughout the study.
Conclusions:  Dairy cattle can be a long-term reservoir of C. jejuni subtypes similar to clinical isolates. Differences in the colonization potential among C. jejuni strains as well as in the resistance to campylobacter colonization among animals are possible.
Significance and Impact of the Study:  The study provides data on contamination dynamics, colonization levels and the persistence of C. jejuni in dairy cattle.     

Prevalence of antimicrobial resistance among serotypes of Campylobacter jejuni isolates from cattle and poultry in Japan

Penner serotypes of C. jejuni in a total of 601 isolates from apparently healthy cattle, layer and broiler chickens in Japan were examined between 2001 and 2006. Predominant serotypes were B (O: 2, 19.1%), D (O: 4, 13.5%), Y (O: 37, 7.3%) and G (O: 8, 5.8%), whereas the remaining serotypes made up less than 5% of the total isolates. The frequency of ampicillin resistance in serotype G (65.6%) was significantly higher than in serotypes D (12.5%), B (11.2%), and Y (0%). Our results suggest that serotype is one factor contributing to the prevalence of ampicillin resistance in C. jejuni isolates.     

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix)

Aim:  To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges ( Perdix perdix ) in southern Italy.
Methods and Results:  Cloacal swabs of partridges ( n  =   240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni . In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence ( P  <   0·05) than younger ones.
Conclusion:  These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species.
Significance and Impact of the Study:  Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Evaluation of cytotoxic activity in fecal filtrates from patients with Campylobacter jejuni or Campylobacter coli enteritis

We sought to determine the prevalence of cytotoxic activity in fecal filtrates from persons with C. jejuni or C. coli enteritis. Stool specimens were collected from 20 persons with C. jejuni or C. coli enteritis, 20 persons with acute diarrheal illnesses of other causes, and 9 healthy, asymptomatic persons. Fecal filtrates were then incubated with Chinese hamster ovary (CHO) or HeLa cells. The fecal filtrate from 1 of the 20 (5%) persons with Campylobacter enteritis was cytotoxic for HeLa cells at a titer of 1:40, and 10 (50%) were cytotoxic for CHO cells at maximum titers of 1:20. Cytotoxic activity for CHO cells at a median titer of 1:20 was also present in 40% of the fecal filtrates from persons with diarrhea due to causes other than Campylobacter enteritis, and in 33% of filtrates from healthy, asymptomatic persons. The observed low level of cytotoxicity in fecal filtrates from all patient groups studied likely resulted from non-specific factors, unrelated to the pathogenesis of Campylobacter enteritis.     

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.     

139株空肠弯曲菌耐药性分析

空肠弯曲菌(Campylobacter jejuni)是最常见的食源性病原菌之一。本研究采用微量肉汤稀释法对分离得到的139株空肠弯曲菌(117株为禽源样本分离株,22株为人源样本分离株)进行耐药性检测。通过对最小抑菌浓度(MIC)的判定结果得出:120株(86. 33%)空肠弯曲菌分离株对6类9组临床常用的抗生素表现出不同程度的耐药,其中禽源空肠弯曲菌耐药率为83. 76%,22株人源空肠弯曲菌均表现出耐药性。对喹诺酮类抗生素表现出高度耐药(环丙沙星80. 58%,萘啶酸77. 70%);对四环素类表现为中等耐药(四环素53. 24%);对部分大环内酯类、氨基糖苷类、林可酰胺类表现为低耐药(庆大霉素7. 19%,阿奇霉素5. 76%,克林霉素6. 47%);对酰胺醇类、部分大环内酯类表现为敏感(氟苯尼考0%,红霉素0%、泰利霉素0%)。139株空肠弯曲菌共产生14种耐药谱型,以TET-CIP-NAL谱型最多,占比38. 13%,耐三重及以上抗生素的多重耐药菌株占比53. 24%。禽源菌株中多重耐药占比46. 15%,人源菌株中多重耐药占比90. 91%。研究结果显示空肠弯曲菌耐药现状不容乐观,尤其对喹诺酮类与四环素类抗生素耐药性较为突出,且过半数菌株为多重耐药。本研究为食源性空肠弯曲菌的防控及临床用药提供参考。     

Campylobacter in chicken livers and their destruction by pan frying

AIM: To enumerate Campylobacter spp. on the external surface and internal portions of chicken livers, and to assess the cooking required to inactivate naturally present cells. METHODS AND RESULTS: Of 30 livers tested all yielded Campylobacter spp. on their surfaces and 90% were found to contain the organism in internal tissue. Four (13%) livers contained >10(4) MPN campylobacters, and an additional seven (23%) contained >10(3) MPN campylobacters per liver. The internal temperature of pan-fried livers under the conditions used reached a maximum of 70-80 degrees C, and maintaining this temperature for 2-3 min was necessary to inactivate naturally occurring Campylobacter spp. All isolates identified were either C. jejuni or C. coli. CONCLUSIONS: Chicken livers represent a potential source of human campylobacteriosis as they contained >10(4) MPN per liver in 13% of the samples tested. Pan-frying can produce an acceptable product that is safe to eat. SIGNIFICANCE AND IMPACT OF THIS STUDY: The data provided can be used in exposure assessments of Campylobacter in poultry products in terms of both quantitative data and assessing pan-frying and its ability to destroy campylobacters.     

Enterotoxigenicity and frequency of Campylobacter jejuni, C. coli and C. laridis in human and animal stool isolates from different countries

Campylobacter jejuni and C. coli strains were collected during three different years from adult patients with enterocolitis in Sweden (n = 372) from 49 patients in Kuwait, and Campylobacter strains from hens from Mexico, Pakistan and Sweden (n = 107) and Swedish pigs (n = 47). C. jejuni was the predominant species in human and hen isolates, and C. coli in pigs C. coli was significantly more common in human isolates from Sweden, and more common in hen isolates from Pakistan, than in hens from Sweden and Mexico. C. laridis was only isolated from pigs (17%) and was in no case enterotoxigenic. Both in human and hen isolates, C. jejuni strains were more enterotoxigenic than C. coli strains. C. jejuni strains from Swedish hens were less enterotoxigenic than those from Pakistan and Mexico (P less than 0.001), and strains from pigs were less enterotoxigenic than those from hens (P less than 0.001). We conclude that C. jejuni are more often enterotoxigenic and possibly more virulent than c. coli and C. laridis. The relative frequency of C. jejuni and C. coli in humans and animals differs from one country to another.     

Development of a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection and identification of Campylobacter jejuni, Campylobacter coli and Campylobacter fetus

A cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for the detection of cdtA, cdtB or cdtC gene of Campylobacter jejuni, Campylobacter coli or Campylobacter fetus, respectively, was developed and evaluated with 76 Campylobacter strains belonging to seven different species and 131 other bacterial strains of eight different genera. The cdtA, cdtB or cdtC gene of C. jejuni, C. coli or C. fetus, respectively, could be successfully amplified using the corresponding set of primers in a highly species-specific manner. Furthermore, the specific primer set for the cdtA, cdtB or cdtC gene of a particular species could amplify the desired gene from a mixture of DNA templates of any of two or all three species. The detection limit of C. jejuni, C. coli or C. fetus was 10-100 CFU tube(-1) by the multiplex PCR assay on the basis of the presence of the cdtA, cdtB or cdtC gene. These data indicate that the cdt gene-based multiplex PCR assay may be useful for rapid and accurate detection as well as identification of Campylobacter strains in a species-specific manner.     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

In vitro efficacy of the novel peptide CECT7121 against bacteria isolated from mastitic dairy cattle

Aims:  To evaluate the in vitro bactericidal activity of the novel antimicrobial peptide (AP) CECT7121 against Gram-positive bacteria from mastitic dairy cattle.
Methods and Results:  A total of 15 Staphylococcus aureus , 10 Streptococcus dysgalactiae , 7 Strep. uberis , 1 Strep. agalactiae strains were isolated from 33 different mastitic dairy cattle, sourced from two dairies in Tandil-Argentina. Isolates from each of the bacterial species screened which developed the lowest inhibition zones in response to the peptide, were further evaluated in a series of time-killing curve studies. No survivors were detected in whole strains (from the three Streptococcal species isolated) within 120 min of incubation in presence of the peptide. The Staph. aureus isolates were less sensitive but, nevertheless, a drop in viable counts to below the detection limit was achieved for all the test strains by the final postincubation sampling point at 180 min.
Conclusions:  The study demonstrated the in vitro efficacy of the AP-CECT7121 against a variety strains of Gram positives isolated from mastitic dairy cattle.
Significance and Impact of the Study:  There is urgent global interest in the development of natural alternatives for the control and prevention of mastitis. Confirmation of the in vitro activity of the novel AP-CECT7121 against Gram-positive isolates encourages further research.     

Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test®

Aims: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. Methods and Results: Adhesion assays were conducted in 96‐well, polystyrene microtiter plates using the BioFilm Ring Test^® method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test^® and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food‐processing and clinical cases. Conclusions: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. Significance and Impact of the Study: The adhesion capability of strains could partly explain the cross‐contamination or re‐contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.     

Routine identification of Campylobacter jejuni and Campylobacter coli from human stool samples

Correct identification of Campylobacter jejuni and Campylobacter coli isolates to the species or subspecies level is a cumbersome but nevertheless important task for a routine diagnostic laboratory. The widely used biochemical tests might be often misleading while more sophisticated phenotypic or genotypic methods are not generally available. This investigation was performed to assess the performance of common biochemical identification in comparison with species-specific PCR and gas liquid chromatography of whole cell fatty acid extracts (GLC). A total of 150 consecutive isolates from human stool samples were investigated (134 C. jejuni ssp. jejuni, 14 C. coli, two Helicobacter pullorum). From these 144, 145 and 149 isolates were correctly identified by biochemistry, GLC and PCR, respectively. Biochemical identification of all C. jejuni isolates was confirmed by PCR. GLC detected both H. pullorum strains but misidentified two C. coli strains as C. jejuni and one C. jejuni strain as C. coli. No single method can be defined as 'gold standard' for identification of C. jejuni and C. coli but a combination of techniques is needed. Therefore a stepwise identification scheme starting with biochemical reactions is suggested. All results other than C. jejuni should be confirmed by further methods. For indoxyl acetate-positive isolates species-specific PCR is recommended while GLC seems to be advantageous in indoxyl acetate-negative isolates.     

Prevalence and antimicrobial susceptibility of Salmonella spp. isolates from US cattle in feedlots in 1999 and 2000

AIMS: Faecal samples from cattle in US feedlots were evaluated for the presence of Salmonella. When Salmonella isolates were recovered the antimicrobial resistance patterns were determined. METHODS AND RESULTS: Faecal samples were collected from pen floors in 73 feedlots in 12 states during the period from October 1999 to September 2000. Pens of cattle selected for sampling were those that had been in the feedlot for the shortest period of time, the longest period of time and a randomly selected pen from the remaining pens. Faecal samples were cultured for Salmonella spp. and all Salmonella isolates were categorized by serotype. The susceptibilities of all isolates were determined using a panel of 17 antimicrobials. Overall, 6.3% (654/10,417) of the samples cultured positive for Salmonella spp. and 22.2% (94/422) of pens and 50.7% (37/73) of feedlots had one or more positive samples. There was little difference in the proportion of positive samples from short-fed (6.1%, 212/3482), random (6.4%, 217/3400) and long-fed (6.4%, 224/3485) pens of cattle. One of two pens of cattle that could not be attributed to a pen type had a single positive sample (2.0%, 1/50). Samples collected during the period of April to June (6.8%, 209/3054) and July to September (11.4%, 286/2500) were more likely to be positive than those collected during October to December (4.0%, 73/1838) and January to March (2.8%, 86/3025). The most common serotypes of Salmonella were dissimilar from those that are typically seen in human illness and cattle illness. The majority of isolates (62.8%, 441/702) were sensitive to all of the antimicrobials tested. Resistance was most frequently observed to tetracycline (35.9%, 252/702) followed by streptomycin (11.1%, 78/702), ampicillin (10.4%, 73/702) and chloramphenicol (10.4%, 73/702). Multiple resistance (resistance to > or =2 antimicrobials) was observed for 11.7% (82/702) of the isolates. CONCLUSIONS: Salmonella was isolated at low frequency from faeces of feedlot cattle and the serotypes were not those commonly associated with human illness. In addition most of the Salmonella isolates were sensitive to all the antimicrobials tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to understanding the ecology of Salmonella in cattle feedlots and the prevalence of resistance among potential food-borne pathogens.     

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

Aims: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. Methods and Results: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which – in combination with MLST – increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37·5, 33·1 and 8·1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone‐sensitive human isolates showed overlapping MLST‐flaB types with those of chicken origin, resistant strains showed only 39% of matching types. Conclusion: Mainly quinolone‐sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. Significance and Impact of the Study: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.