Prevalence of Salmonella spp. in oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

Prevalence of Salmonella spp. in Oysters in the United States

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.     

PCR detection of Salmonella spp. using primers targeting the quorum sensing gene sdiA

Bacteria communicate with one another and with their host using chemical signalling molecules. This phenomenon is generally described as quorum sensing. A set of primers for PCR detection of Salmonella spp. has been designed using as target the sdiA gene which encodes a signal receptor of the LuxR family. The PCR product (274 bp) was confirmed by sequencing. A number of 81 non-Salmonella strains (representing 24 different species) were tested and gave negative results, while a total of 101 different serotypes of Salmonella (155 strains) tested positive for the presence of the sdiA gene. The sensitivity and specificity of the sdiA-based PCR assay were also checked in artificially contaminated human faecal samples. In this study, we demonstrate that quorum sensing genes can be successfully exploited as diagnostic markers.     

Use of species- and strain-specific PCR primers for identification of conifer root-associated Bacillus spp.

Abstract A polymerase chain reaction amplification of 23S rDNA was developed to identify Bacillus spp. recovered from roots, mycorrhizae, and rhizosphere soil of conifers. The polymerase chain reaction incorporated a conserved 23S rDNA forward primer in combination with a reverse primer designed to hybridize exclusively to nucleotide sequences of either B. polymyxa or B. mycoides . The amplification provided a rapid and simple means of identifying DNA from isolates of Bacillus , and could be used directly on whole Bacillus cells or mixed populations. The reaction was used to detect and differentiate these Gram-positive species from agar plates inoculated with samples from various conifer samples. A strain-specific primer was also synthesized and used to identify Bacillus which were established within conifer roots 4 weeks after inoculation.     

Multiplex PCR with 16S rRNA gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.     

Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.     

Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States

Vancomycin-resistant Enterococcus spp. (VRE) were isolated from sewage and chicken feces but not from other animal fecal sources (dog, cow, and pig) or from surface waters tested. VRE from hospital wastewater were resistant to > or =20 microg of vancomycin/ml and possessed the vanA gene. VRE from residential wastewater and chicken feces were resistant to 3 to 5 microg of vancomycin/ml and possessed the vanC gene.     

Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms

The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.     

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.     

A survey of fluoroquinolone resistance in Escherichia coli and thermophilic Campylobacter spp. on poultry and pig farms in Great Britain

Aims: To estimate the proportions of farms on which broilers, turkeys and pigs were shedding fluoroquinolone (FQ)-resistant Escherichia coli or Campylobacter spp. near to slaughter. Methods and Results: Freshly voided faeces were collected on 89 poultry and 108 pig farms and cultured with media containing 1·0 mg l⁻¹ ciprofloxacin. Studies demonstrated the specificity of this sensitive method, and both poultry and pig sampling yielded FQ-resistant E. coli on 60% of farms. FQ-resistant Campylobacter spp. were found on around 22% of poultry and 75% of pig farms. The majority of resistant isolates of Campylobacter (89%) and E. coli (96%) tested had minimum inhibitory concentrations for ciprofloxacin of ≥8 mg l⁻¹. The proportion of resistant E. coli and Campylobacter organisms within samples varied widely. Conclusions: FQ resistance is commonly present among two enteric bacterial genera prevalent on pig and poultry farms, although the low proportion of resistant organisms in many cases requires a sensitive detection technique. Significance and Impact of the Study: FQ-resistant bacteria with zoonotic potential appear to be present on a high proportion of UK pig and poultry farms. The risk this poses to consumers relative to other causes of FQ-resistant human infections remains to be clarified.     

Prevalence and distribution of Sarcocystis spp. among white-tailed deer of the southeastern United States

Sarcocysts were found by light microscopic examination of muscle in 199 (51%) of 390 white-tailed deer (Odocoileus virginianus) from the southeastern United States. Sarcocystis infections were detected more frequently in histologic sections of tongue (45%) than of heart (9%). Sarcocysts were significantly more prevalent in adult deer (54%) than fawns (26%) (P less than .01). Statistically significant differences in prevalence were not found in deer from different physiographic provinces or between sexes. Artificial digestion was more sensitive in detecting Sarcocystis infections than examination of histologic sections when both techniques were used to examine tongues of 35 deer. Three different size sporocysts, possibly representing at least two species of Sarcocystis, were recovered during feeding trials. Seven dogs (Canis familiaris) shed sporocysts 9 to 12 days after eating infected venison. Sporocysts measured 13.4-16.8 x 9.0-12.3 micrometers with an average measurement of 15.2 x 10.9 micrometers (N = 195). One of three cats (Felis catus) and one of two red foxes (Vulpes vulpes) first shed sporocysts of Sarcocystis 10 days after eating infected venison. Sporocysts from the cat measured 11.2-13.4 x 6.72-8.96 micrometers (avg 12.0 x 8.7 micrometers, N = 18), and those from the fox measured 11.2-15.7 x 9.0-11.2 micrometers (avg 13.6 x 10.2 micrometers, N = 7).     

The incidence of Campylobacter spp. on processed turkey from processing plants in the midwestern United States

AIM: The primary aim of this study was to determine the incidence of Campylobacter spp. on turkey, presented for processing at participating production plants located in the midwest region of the United States. METHODS AND RESULTS: The two participating plants were visited on a monthly basis for a period of 1 year. Sampling of carcasses was carried out using a surface swab technique. Swabs were obtained from carcasses at two points on the production line - prechill and postchill. In addition, samples of chill water were also obtained for examination. Isolation and detection of Campylobacter was carried out using enrichment in Preston broth with recovery of the organism on blood free Campylobacter selective agar (CCDA). Isolates recovered were screened and identified using the API Campy identification system. The study found that 34.9% of all samples tested were positive for Campylobacter spp. The overall, contamination rates observed for both plants were relatively similar (39.2% for plant A and 30.6% for plant B). Differences were observed in the incidence of Campylobacter spp. on prechill vs postchill carcasses (i.e. 40.8% prechill vs 37.6% postchill for plant A and 41.8% prechill vs 19.8% postchill for plant B). Campylobacter species most often isolated included Camp. jejuni and Camp. coli. Other species recovered were Camp. fetus fetus, Camp. upsaliensis and Camp. lari. CONCLUSIONS: The incidence of Campylobacter spp. on processed poultry was relatively common. Factors such as the processing plant examined, season and the farms presenting birds for processing influenced the incidence of the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences were observed in the prevalence of Campylobacter spp. isolated from the two plants examined. The study suggests a seasonal prevalence of Campylobacter in the cooler months with processing conditions also influencing the overall occurrence of the organism. The incidence, isolation and detection of Campylobacter spp. from processed poultry are discussed.     

Development of a multiplex PCR detection of Salmonella spp. and Shigella spp. in mussels

Multiplex PCR amplification of invA and virA genes was developed enabling simultaneous detection in mussels of Salmonella spp. and Shigella spp., respectively. Simultaneous amplification of products of 215 and 275 bp was obtained either by using mixtures of individual strains of Sh. dysenteriae and Salm. typhimurium or spiked contaminated mussels with both bacteria. In the case of the mussels, 10-100 cells of Salmonella spp. and Shigella per millilitre of homogenate were detected by the multiplex PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of cultivable bacteria. Also, the sensitivity and specificity of this method was evaluated. Multiplex PCR amplification was shown to be an effective, sensitive and rapid method for the simultaneous detection of pathogens in mussels.     

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.     

Variation in biological parameters of Trichogramma spp. purchased from commercial suppliers in the United States

The efficacy of biological controlobtained through releases of commerciallysupplied Trichogramma is influenced by bothabiotic conditions in the field during andfollowing release and an array of biologicalattributes of the released Trichogramma. Thisstudy was undertaken to assess the degree ofvariation that existed among commerciallysupplied Trichogramma in an array of biologicalparameters that potentially may influence thelevel of control that can be expected in aTrichogramma release program. Shipments ofTrichogramma were obtained from 12 commercialsuppliers of beneficial insects over a 2-yearperiod and the following parameters weremeasured: percentage of emergence duringshipping, total percentage of emergence,percentage of females, percentage ofbrachypterous females, percentage ofbrachypterous males, adult female longevity,and species composition. In addition, weestimated the percentage of macropterousfemales that had not emerged prior to the timethe shipment was received, based on the firstfour parameters. Results indicate a high levelof variation in each of these parameters amongsuppliers of Trichogramma and among shipmentsby individual suppliers. The proportion ofnon-emerged macropterous female wasps was lowfor all suppliers (range 2.5–27.7%). Thiswas primarily attributable to the highincidence of brachyptery among female wasps. However, variation among suppliers in theincidence of emergence during shipping and theproportion of female wasps in a shipment alsocontributed to the low proportion ofnon-emerged macropterous female wasps. Inaddition, in 11 of 24 shipments received duringthe 2-year study, the Trichogramma speciesdiffered from that specified by the shipper orthe shipment contained a mixture ofTrichogramma species.     

Results of a survey to detect the mosquito Aedes albopictus in the French Riviera.

A programme of surveillance was initiated in 1992, in the French Riviera, to detect a possible introduction of Aedes albopictus from Italy and to prevent nuisances caused by mosquitoes in the touristic localities of the C?te d'Azur. In five years, numerous mosquito breeding places have been located. Nine species have been collected: Anopheles claviger, An. plumbeus, Aedes geniculatus, Ae. vittatus, Culex hortensis, Cx. impudicus, Cx. pipiens, Culiseta fumipennis, Cs. longiareolata but no Ae. albopictus was found. Nuisances were mainly due to hypogean populations of Cx. pipiens. Breeding places in urban sites have been controlled or suppressed. The discovery of an important larval population of An. plumbeus in an urban area might further stress the importance of this species already suspected to transmit indigenous malaria in cities.     

Use of sulfonated primers to detect and type papillomavirus in cell cultures and cervical biopsies.

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.