Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.
Carloni Elisa,Rotundo Luca,Brandi Giorgio,Amagliani Giulia
The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.
Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: The effect of pH, dextran sulfate and probe concentration.
Rocha Rui,Santos Rita S,Madureira Pedro,Almeida Carina,Azevedo Nuno F
Journal of biotechnology
Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.
[Molecular beacon based PNA-FISH method combined with fluorescence scanning for rapid detection of Listeria monocytogenes].
Wu Shan,Zhang Xiaofeng,Shuai Jiangbing,Li Ke,Yu Huizhen,Jin Chenchen
Wei sheng wu xue bao = Acta microbiologica Sinica
Objective:To simplify the PNA-FISH (Peptide nucleic acid-fluorescence in situ hybridization) test, molecular beacon based PNA probe combined with fluorescence scanning detection technology was applied to replace the original microscope observation to detect Listeria monocytogenes Methods:The 5′ end and 3′ end of the L. monocytogenes specific PNA probes were labeled with the fluorescent group and the quenching group respectively, to form a molecular beacon based PNA probe. Results:When PNA probe used for fluorescence scanning and N1 treatment as the control, the false positive rate was 11.4%, and the false negative rate was 0; when N2 treatment as the control, the false positive rate decreased to 4.3%, but the false negative rate rose to 18.6%. When beacon based PNA probe used for fluorescence scanning, taken N1 treatment as blank control, the false positive rate was 8.6%, and the false negative rate was 1.4%; taken N2 treatment as blank control, the false positive rate was 5.7%, and the false negative rate was 1.4%. Compared with PNA probe, molecular beacon based PNA probe can effectively reduce false positives and false negatives. The success rates of hybridization of the two PNA probes were 83.3% and 95.2% respectively; and the rates of the two beacon based PNA probes were 91.7% and 90.5% respectively, which indicated that labeling the both ends of the PNA probe dose not decrease the hybridization rate with the target bacteria. Conclusions:The combination of liquid phase PNA-FISH and fluorescence scanning method, can significantly improve the detection efficiency.
[New insight into bacterial zoonotic pathogens posing health hazards to humans].
Ciszewski Marcin,Czekaj Tomasz,Szewczyk Eligia Maria
This article presents the problem of evolutionary changes of zoonotic pathogens responsible for human diseases. Everyone is exposed to the risk of zoonotic infection, particularly employees having direct contact with animals, i.e. veterinarians, breeders, butchers and workers of animal products' processing industry. The article focuses on pathogens monitored by the European Centre for Disease Prevention and Control (ECDC), which has been collecting statistical data on zoonoses from all European Union countries for 19 years and publishing collected data in annual epidemiological reports. Currently, the most important 11 pathogens responsible for causing human zoonotic diseases are being monitored, of which seven are bacteria: Salmonella spp., Campylobacter spp., Listeria monocytogenes, Mycobacterium bovis, Brucella spp., Coxiella burnetti and Verotoxin-producing E. coli (VTEC)/Shiga-like toxin producing E. coli (STEC). As particularly important are considered foodborne pathogens. The article also includes new emerging zoonotic bacteria, which are not currently monitored by ECDC but might pose a serious epidemiological problem in a foreseeable future: Streptococcus iniae, S. suis, S. dysgalactiae and staphylococci: Staphylococcus intermedius, S. pseudintermedius. Those species have just crossed the animal-human interspecies barrier. The exact mechanism of this phenomenon remains unknown, it is connected, however, with genetic variability, capability to survive in changing environment. These abilities derive from DNA rearrangement and horizontal gene transfer between bacterial cells. Substantial increase in the number of scientific publications on this subject, observed over the last few years, illustrates the importance of the problem.
Molecular Methods for the Detection of Oocysts in Fresh Produce: An Extensive Review.
Slana Iva,Bier Nadja,Bartosova Barbora,Marucci Gianluca,Possenti Alessia,Mayer-Scholl Anne,Jokelainen Pikka,Lalle Marco
Human infection with the important zoonotic foodborne pathogen has been associated with unwashed raw fresh produce consumption. The lack of a standardised detection method limits the estimation of fresh produce as an infection source. To support method development and standardisation, an extensive literature review and a multi-attribute assessment were performed to analyse the key aspects of published methods for the detection of oocyst contamination in fresh produce. Seventy-seven published studies were included, with 14 focusing on fresh produce. Information gathered from expert laboratories via an online questionnaire were also included. Our findings show that procedures for oocyst recovery from fresh produce mostly involved sample washing and pelleting of the washing eluate by centrifugation, although washing procedures and buffers varied. DNA extraction procedures including mechanical or thermal shocks were identified as necessary steps to break the robust oocyst wall. The most suitable DNA detection protocols rely on qPCR, mostly targeting the gene or the bp repetitive element. When reported, validation data for the different detection methods were not comparable and none of the methods were supported by an interlaboratory comparative study. The results of this review will pave the way for an ongoing development of a widely applicable standard operating procedure.
Vaccinia virus detection in dairy products made with milk from experimentally infected cows.
de Oliveira T M L,Guedes M I M C,Rehfeld I S,Matos A C D,Rivetti Júnior A V,da Cunha A F,Cerqueira M M O P,Abrahão J S,Lobato Z I P
Transboundary and emerging diseases
Vaccinia virus (VACV) is the agent of bovine vaccinia (BV), an emerging zoonosis that causes exanthematic lesions on the teats of dairy cows and on the hands of milkers. The virus has been detected in the milk of naturally infected cows. The objective of this study was to investigate and quantify VACV DNA as well as the presence of infectious virus particles in samples of cheese curd, cheese whey and pasteurized milk produced using milk from cows experimentally inoculated with VACV-GP2, a Brazilian isolate of VACV (VACV-BR). VACV DNA was detected in samples of cheese and pasteurized milk at different time points, even after the resolution of the typical lesions caused by VACV, which occurred after 22 days post-infection (dpi), on average. Moreover, it was possible to detect infectious viral particles in cheese samples on alternate days until 27 dpi. The presence of both VACV DNA and infectious viral particles in cheese samples throughout the clinical course of BV and even after the disappearance of the typical clinical signs of disease draws attention to the risk associated with consumption of the cheese. Furthermore, VACV-contaminated milk and cheese may represent an occupational risk to cheesemakers who often manipulate milk and cheese curd without wearing gloves.
Foodborne zoonoses due to meat: a quantitative approach for a comparative risk assessment applied to pig slaughtering in Europe.
Fosse Julien,Seegers Henri,Magras Catherine
Foodborne zoonoses have a major health impact in industrialised countries. New European food safety regulations were issued to apply risk analysis to the food chain. The severity of foodborne zoonoses and the exposure of humans to biological hazards transmitted by food must be assessed. For meat, inspection at the slaughterhouse is historically the main means of control to protect consumers. However, the levels of detection of biological hazards during meat inspection have not been established in quantitative terms yet. Pork is the most frequently consumed meat in Europe. The aim of this study was to provide elements for quantifying levels of risk for pork consumers and lack of detection by meat inspection. Information concerning hazard identification and characterisation was obtained by the compilation and statistical analysis of data from 440 literature references. The incidence and severity of human cases due to pork consumption in Europe were assessed in order to calculate risk scores. A ratio of non-control was calculated for each biological hazard identified as currently established in Europe, i.e. the incidence of human cases divided by the prevalence of hazards on pork. Salmonella enterica, Yersinia enterocolitica and Campylobacter spp. were characterised by high incidence rates. Listeria monocytogenes, Clostridium botulinum and Mycobacterium spp. showed the highest severity scores. The three main high risk hazards involved in foodborne infections, Y. enterocolitica, S. enterica and Campylobacter spp. are characterised by high non-control ratios and cannot be detected by macroscopic examination of carcasses. New means of hazard control are needed to complement the classical macroscopic examination.
Nanoplasmonic sensor for foodborne pathogens detection. Towards development of ISO-SERS methodology for taxonomic affiliation of Campylobacter spp.
Witkowska Evelin,Niciński Krzysztof,Korsak Dorota,Dominiak Bartłomiej,Waluk Jacek,Kamińska Agnieszka
Journal of biophotonics
According to EU summary report on zoonoses, zoonotic agents and food-borne outbreaks in 2017, Campylobacter was the most commonly reported gastrointestinal bacterial pathogen in humans in the EU. Unfortunately, the standard methods for the detection of thermotolerant Campylobacter spp. in foods are time-consuming. Additionally, the qualified staff is obligatory. For this reason, new methods of pathogens detection are needed. The present work demonstrates that surface-enhanced Raman scattering (SERS) is a reliable and fast method for detection of Campylobacter spp. in food samples. The proposed method combines the SERS measurements performed on an Ag/Si substrate with two initial steps of the ISO standard procedure. Finally, the principal component analysis (PCA) allows for statistical classification of the studied bacteria. By applying the proposed ISO-SERS-PCA method in the case of Campylobacter bacteria the total detection time may be reduced from 7 to 8 days required by ISO method to 3 to 4 days in the case of SERS-based approach.
Isolation and characterization of a new Staphylococcus epidermidis broad-spectrum bacteriophage.
Melo Luís D R,Sillankorva Sanna,Ackermann Hans-Wolfgang,Kropinski Andrew M,Azeredo Joana,Cerca Nuno
The Journal of general virology
Staphylococcus epidermidis is considered an important nosocomial pathogen, being very tolerant to the host immune system and antibiotherapy, particularly when in biofilms. Due to its high resistance, alternative antimicrobial strategies are under development. The use of bacteriophages is seen as an important strategy to combat pathogenic organisms. In this study, a S. epidermidis myovirus, SEP1, was isolated and characterized. The genome of this phage was sequenced and shown to be related peripherally to the genus Twortlikevirus. However, when compared with other phages of this genus, it showed DNA sequence identities no greater than 58.2 %. As opposed to other polyvalent viruses of the genus Twortlikevirus, SEP1 is highly specific to S. epidermidis strains. The good infectivity shown by this phage as well as its high lytic spectrum suggested that it might be a good candidate for therapeutic studies.
Survey of Canadian retail pork chops and pork livers for detection of hepatitis E virus, norovirus, and rotavirus using real time RT-PCR.
Wilhelm Barbara,Leblanc Danielle,Houde Alain,Brassard Julie,Gagné Marie-Josée,Plante Daniel,Bellon-Gagnon Pascale,Jones Tineke H,Muehlhauser Victoria,Janecko Nicol,Avery Brent,Rajić Andrijana,McEwen Scott A
International journal of food microbiology
Over the past 15 years, hepatitis E virus (HEV), norovirus (NoV), and rotavirus (RV) have been hypothesized to be potentially zoonotic; swine and pork have been suggested as possible human infection sources for all 3 viruses. Our objective was to estimate HEV, NoV, and RV prevalence and load on Canadian retail pork chops and livers. Using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) sampling platform, pork livers (n=283) and chops (n=599) were collected, processed, and assayed for the 3 viruses by four collaborating federal laboratories using validated real time reverse transcriptase polymerase chain reactions (qRT-PCR). Follow-up qRT-PCR estimating viral load in genomic copies/g was followed by nested classical RT-PCR and isolate sequencing of a partial segment of the ORF2 gene. Local alignments were performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation); a phylogenetic tree was created. Twenty-five livers and 6 chops were classified 'positive' (thresholds for viral RNA detected in both replicates of the assay) or 'suspect' (thresholds detected in one of two replicates) for HEV. Follow-up qRT-PCR detected HEV on 16 livers, 0 chops, and nested classical RT-PCR, on 14 livers and 0 chops. Initial qRT-PCR classified 12 chops 'suspect' for NoV. Follow-up qRT-PCR detected viral RNA on only one sample with thresholds greater than 40 in both replicates. No amplicon was yielded, and therefore no isolate was sequenced from this sample. Partial ORF2 genes from 14 HEV isolates were sequenced, and compared via sequence identity and phylogenetic analysis with selected human case isolates listed in NCBI-GenBank. Overall, HEV prevalence on retail pork was comparable with other published reports.
Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.
Wang Yunzhou,Tao Geru,Cui Yujuan,Lv Qiyao,Xie Li,Li Yuan,Suo Xun,Qin Yinghe,Xiao Lihua,Liu Xianyong
PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists.
Molecular epidemiology and identification of a Staphylococcus aureus clone causing food poisoning outbreaks in Japan.
Sato'o Yusuke,Omoe Katsuhiko,Naito Ikunori,Ono Hisaya K,Nakane Akio,Sugai Motoyuki,Yamagishi Norio,Hu Dong-Liang
Journal of clinical microbiology
Molecular characterization of isolates from staphylococcal food poisoning (SFP) outbreaks in Japan showed that the dominant lineage causing SFP outbreaks is clonal complex 81 (CC81), a single-locus variant of sequence type 1, coagulase type VII, positive for sea and/or seb, and positive for seh. Among various CC lineages producing staphylococcal enterotoxin A, CC81 showed the highest toxin productivity.
Identification of the first transcriptional activator of an archaellum operon in a euryarchaeon.
Ding Yan,Nash John,Berezuk Alison,Khursigara Cezar M,Langelaan David N,Smith Steven P,Jarrell Ken F
The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are regulated under different growth conditions. In this study, we identify MMP1718 as the first transcriptional activator that directly regulates the fla operon in M. maripaludis. Mutants carrying an in-frame deletion in mmp1718 did not express FlaB2 detected by western blotting. Quantitative reverse transcription PCR analysis of purified RNA from the Δmmp1718 mutant showed that transcription of flaB2 was negligible compared to wildtype cells. In addition, no archaella were observed on the cell surface of the Δmmp1718 mutant. FlaB2 expression and archaellation were restored when the Δmmp1718 mutant was complemented with mmp1718 in trans. Electrophoretic motility shift assay and isothermal titration calorimetry results demonstrated the specific binding of purified MMP1718 to DNA fragments upstream of the fla promoter. Four 6 bp consensus sequences were found immediately upstream of the fla promoter and are considered the putative MMP1718-binding sites. Herein, we designate MMP1718 as EarA, the first euryarchaeal archaellum regulator.
Detection of antibodies against hepatitis E virus in pet veterinarians and pet dogs in South Korea.
Lyoo Kwang-Soo,Yang Soo-Jin,Na Woonsung,Song Daesub
Irish veterinary journal
Hepatitis E virus (HEV) is a zoonotic pathogen commonly considered an important foodborne virus. Pet dogs are important reservoirs of zoonotic agents. In the present study, the seroprevalence of HEV in pet dogs and pet veterinarians were found to be 28.2 and 5.0%, respectively. It remains unclear whether pet veterinarians are at higher risk of HEV transmission. However, pet animals and individuals who have contact with infected animals must be continually monitored for public health concerns.
Detection and Molecular Characterization of Staphylococci from Eggs of Household Chickens.
Syed Muhammad Ali,Jackson Charlene R,Ramadan Hazem,Afridi Riazuddin,Bano Shehr,Bibi Sumera,Fatima Bushra,Tabassum Sadia,Jamil Bushra,Khan Muhammad Fiaz,Barrett John B,Woodley Tiffanie A
Foodborne pathogens and disease
Eggs are a healthy and nutritious food source, but may be contaminated by bacteria. Previous studies have reported the presence of staphylococci in eggs of farmed chickens, but no study has evaluated the staphylococcal population of eggs from household chickens. In this study, staphylococci from eggs ( = 275) of household chickens collected from November 2016 to March 2017 from different villages of Khyber Pakhtunkhwa province, Pakistan, were characterized. Seven species of staphylococci were identified from 65 eggs, including the predominant species, (49/275; 17.8%). isolates ( = 73) were tested for antimicrobial susceptibility, presence of resistance genes, genetic relatedness, and inhibitory activity against other bacteria. The majority of isolates were resistant to oxacillin (83.6%) and tetracycline (24.7%), but also exhibited resistance to daptomycin and linezolid (5.5% each). Of the 10 resistance genes tested, isolates were only positive for A (35.6%; 26/73), C/C1 (2.7%; 2/73), and (K) (14/73; 19%). Using pulsed-field gel electrophoresis (PFGE), nine clusters had identical PFGE patterns. Isolates produced inhibitory activity against a broad spectrum of bacteria; 20.5%, 19.2%, 17.8%, and 16.4% of were able to inhibit growth of serotype Typhi, methicillin-susceptible , , and methicillin-resistant , respectively. This study demonstrated the presence of genetically related antimicrobial-resistant from eggs from household chickens. Like table eggs, eggs of household chickens also contain staphylococci that may be resistant to antimicrobials used to treat human infections. These data will allow comparison between staphylococci from eggs from different sources and may indicate the relative safety of eggs from household chickens. Further study of these egg types and their microbial composition is warranted.
Identification and Characterization of a Novel Staphylococcal Emetic Toxin.
Ono Hisaya K,Sato'o Yusuke,Narita Kouji,Naito Ikunori,Hirose Shouhei,Hisatsune Junzo,Asano Krisana,Hu Dong-Liang,Omoe Katsuhiko,Sugai Motoyuki,Nakane Akio
Applied and environmental microbiology
Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.
Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin.
Ono Hisaya K,Hachiya Nobuaki,Suzuki Yasunori,Naito Ikunori,Hirose Shouhei,Asano Krisana,Omoe Katsuhiko,Nakane Akio,Hu Dong-Liang
Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in was detected by using the sandwich ELISA and showed that -horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in isolated from food poisoning cases.
Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera.
Ma Zhe,Yang Xinyi,Fang Yizhen,Tong Zexin,Lin Huixing,Fan Hongjie
Foodborne pathogens and disease
The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.
Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China.
Li Jingjiao,Xue Feng,Yang Zhenquan,Zhang Xiaoping,Zeng Dexin,Chao Guoxiang,Jiang Yuan,Li Baoguang
Frontiers in microbiology
Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these "environmental" pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.
Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.
Zhang Xuehan,Li Meng,Zhang Bicheng,Chen Kangming,He Kongwang
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.
Whole genome sequencing as a tool to investigate a cluster of seven cases of listeriosis in Austria and Germany, 2011-2013.
Schmid D,Allerberger F,Huhulescu S,Pietzka A,Amar C,Kleta S,Prager R,Preußel K,Aichinger E,Mellmann A
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases
A cluster of seven human cases of listeriosis occurred in Austria and in Germany between April 2011 and July 2013. The Listeria monocytogenes serovar (SV) 1/2b isolates shared pulsed-field gel electrophoresis (PFGE) and fluorescent amplified fragment length polymorphism (fAFLP) patterns indistinguishable from those from five food producers. The seven human isolates, a control strain with a different PFGE/fAFLP profile and ten food isolates were subjected to whole genome sequencing (WGS) in a blinded fashion. A gene-by-gene comparison (multilocus sequence typing (MLST)+) was performed, and the resulting whole genome allelic profiles were compared using SeqSphere(+) software version 1.0. On analysis of 2298 genes, the four human outbreak isolates from 2012 to 2013 had different alleles at ≤6 genes, i.e. differed by ≤6 genes from each other; the dendrogram placed these isolates in between five Austrian unaged soft cheese isolates from producer A (≤19-gene difference from the human cluster) and two Austrian ready-to-eat meat isolates from producer B (≤8-gene difference from the human cluster). Both food products appeared on grocery bills prospectively collected by these outbreak cases after hospital discharge. Epidemiological results on food consumption and MLST+ clearly separated the three cases in 2011 from the four 2012-2013 outbreak cases (≥48 different genes). We showed that WGS is capable of discriminating L. monocytogenes SV1/2b clones not distinguishable by PFGE and fAFLP. The listeriosis outbreak described clearly underlines the potential of sequence-based typing methods to offer enhanced resolution and comparability of typing systems for public health applications.
Virulence profiling of Shiga toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico.
Amézquita-López Bianca A,Quiñones Beatriz,Lee Bertram G,Chaidez Cristóbal
Frontiers in cellular and infection microbiology
Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen that causes human gastrointestinal illnesses. The present study characterized the virulence profiles of O157 and non-O157 STEC strains, recovered from domestic animals in small rural farms within the agricultural Culiacan Valley in Mexico. Virulence genes coding for adhesins, cytotoxins, proteases, subtypes of Shiga toxin (Stx), and other effectors were identified in the STEC strains by PCR. The genotyping analysis revealed the presence of the effectors nleA, nleB, nleE, and nleH1-2, espK, and espN in the O157:H7 and O111:H8 STEC strains. Furthermore, the genes encoding the autoagglutinating adhesin (Saa) and subtilase (SubA) were exclusively identified in the O8:H19 eae-negative strains. The adhesin (iha) and the silent hemolysin (sheA) genes were detected in 79% of the O157 and non-O157 strains. To examine the relative toxicities of the STEC strains, a fluorescent Vero cell line, Vero-d2EGFPs, was employed to measure the inhibition of protein synthesis by Stx. Analysis of culture supernatants from serotype O8:H19 strains with the stx gene profile stx 1a, stx 2a, and stx 2c and serotypes O75:H8 and O146:H8 strains with the stx gene profile stx 1a, stx 1c, and stx 2b, resulted in a significant reduction in the Vero-d2EGFP fluorescent signal. These observations suggest that these non-O157 strains may have an enhanced ability to inhibit protein synthesis in Vero cells. Interestingly, analysis of the stx 2c-positive O157:H7 strains resulted in a high fluorescent signal, indicating a reduced toxicity in the Vero-d2EGFP cells. These findings indicate that the O157 and non-O157 STEC strains, recovered in the Culiacan Valley, display distinct virulence profiles and relative toxicities in mammalian cells and have provided information for evaluating risks associated with zoonotic STEC in this agricultural region in Mexico.
Reference gene analysis and its use for kinase expression profiling in Fasciola hepatica.
Houhou Hicham,Puckelwaldt Oliver,Strube Christina,Haeberlein Simone
The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.
Detection of hepatitis E virus in pork liver sausages.
Di Bartolo Ilaria,Angeloni Giorgia,Ponterio Eleonora,Ostanello Fabio,Ruggeri Franco Maria
International journal of food microbiology
Hepatitis E infection is regarded as an emerging public-health concern. The disease is normally self-limiting (mortality rate 1%), but chronic infections have recently been observed in transplanted patients. The etiological agent HEV is a small RNA virus infecting both humans and animals. In humans, the disease may be food-borne and pig is a main reservoir for zoonotic strains. In the present study, we evaluated the presence of HEV and swine fecal cross-contamination in pork liver sausages sold at a grocery store in Italy. HEV genome detection was performed by RT-qPCR, using harmonized protocols that included a process control (murine norovirus) and an internal amplification control. Swine fecal cross-contamination was assessed by determination of the ubiquitous porcine adenovirus. Overall, HEV genome belonging to genotype 3 was detected in both raw (10 out of 45 slices, 250 mg each, 22.2%) and dry (1 of 23 slices, 4.3%) liver sausages, but infectivity of the virus was not demonstrated. This pilot study fosters more investigations on HEV presence in pork-derived food, to assess the possible risk for the consumers.
Across intra-mammalian stages of the liver f luke Fasciola hepatica: a proteomic study.
Di Maggio Lucía Sánchez,Tirloni Lucas,Pinto Antonio F M,Diedrich Jolene K,Yates Iii John R,Benavides Uruguaysito,Carmona Carlos,da Silva Vaz Itabajara,Berasain Patricia
Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite.
Identification and Control of Sources of Infection - the Attempts To Eradicate the Parasite.
Samorek-Pieróg Małgorzata,Karamon Jacek,Cencek Tomasz
Journal of veterinary research
is a parasite causing porcine cysticercosis and human taeniosis and cysticercosis, parasitic zoonoses with a serious public health and economic influence. It has been globally ranked as the top foodborne parasite by the Food and Agriculture Organisation of the United Nations (FAO) and the World Health Organisation (WHO). This parasite is transmitted mainly in countryside regions where animals are free roaming, having access to human faeces, and infected pork is widely available. More developed countries eliminated cysticercosis; nonetheless, there are insufficient data about the current endemicity status of . , due to increased human migration from endemic areas. Formally submitted statistics on cysticercosis in pigs are extremely inadequate. This is the result of not reporting all cases of the disease by some countries and lack of molecular verification during identification of the parasite. There is a need to develop diagnostic tests with increased sensitivity and specificity. The purpose of the present review is to summarise current knowledge about diagnostic and control methods concerning . infection. The article does not address the diagnostics of human cysticercosis, since there is a distinct medical field which should be discussed separately. The paper focuses mainly on identifying the sources of . infection, presenting the methods to detect and control porcine cysticercosis and taeniosis in humans.
Factors Affecting Detection of Hepatitis E Virus on Canadian Retail Pork Chops and Pork Livers Assayed Using Real-Time RT-PCR.
Wilhelm B J,Leblanc D,Avery B,Pearl D L,Houde A,Rajić A,McEwen S A
Zoonoses and public health
We collected 599 Canadian retail pork chops and 283 pork livers routinely (usually weekly) from April 2011 to March 2012 using the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) retail sampling platform. Samples were assayed using validated real-time (q) reverse transcriptase polymerase chain reaction (RT-PCR) and nested classical RT-PCR for the detection of hepatitis E virus (HEV), porcine enteric calicivirus (PEC) and rotavirus (RV). The presence of Escherichia coli, Salmonella spp. and Campylobacter spp. was measured on a subset of our samples. Exact logistic regression models were fitted for predictors for HEV detection, for each assay. For both assays, sample type (pork chop versus liver) was a significant predictor for HEV RNA detection. For nested classical RT-PCR but not qRT-PCR, region of sample collection was a significant predictor (P = 0.008) of HEV detection. Odds of HEV detection were greatest in spring relative to other seasons. E. coli was a significant predictor for HEV RNA detection using the qRT-PCR (P = 0.03). Overall, the prevalence of E. coli, Salmonella spp. and Campylobacter spp. was significantly greater than HEV, PEC or RV on our retail pork samples. Our sparse data set for the detection of PEC and RV precluded modelling of risk factors for the detection of these viruses.
Bovine leukaemia virus DNA in fresh milk and raw beef for human consumption.
Epidemiology and infection
Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leucosis, which has been reported worldwide. BLV has been found recently in human tissue and it could have a significant impact on human health. A possible hypothesis regarding viral entry to humans is through the consumption of infected foodstuffs. This study was aimed at detecting the presence of BLV DNA in raw beef and fresh milk for human consumption. Nested PCR directed at the BLV gag gene (272 bp) was used as a diagnostic test. PCR products were confirmed by Sanger sequencing. Forty-nine per cent of the samples proved positive for the presence of proviral DNA. This is the first study highlighting the presence of the BLV gag gene in meat products for human consumption and confirms the presence of the viral DNA in raw milk, as in previous reports. The presence of viral DNA in food products could suggest that viral particles may also be found. Further studies are needed to confirm the presence of infected viral particles, even though the present findings could represent a first approach to BLV transmission to humans through foodstuff consumption.
Helicobacter canis colonization in sheep: a Zoonotic link.
Swennes Alton G,Turk Michelle L,Trowel Elise M,Cullin Cassandra,Shen Zeli,Pang Jassia,Petersson Katherine H,Dewhirst Floyd E,Fox James G
BACKGROUND:Helicobacter canis has been associated with hepatobiliary and gastrointestinal disease in dogs, cats, and humans. Infection has not been documented in other species. MATERIALS AND METHODS:Sheep feces subjected to microaerobic culture. Isolates were characterized by genus-specific PCR, restriction fragment length polymorphism, biochemical profiling, and 16S rRNA sequence analysis. RESULTS:Helicobacter canis was isolated from sheep feces and confirmed by the above methods. These isolates are distinct from other sheep-origin enterohepatic Helicobacter species previously isolated. CONCLUSIONS:This study identifies sheep as H. canis reservoirs potentially important in zoonotic or foodborne transmission.
Animals as sources of food-borne pathogens: A review.
Heredia Norma,García Santos
Animal nutrition (Zhongguo xu mu shou yi xue hui)
Food-producing animals are the major reservoirs for many foodborne pathogens such as species, non-Typhi serotypes of , Shiga toxin-producing strains of , and . The zoonotic potential of foodborne pathogens and their ability to produce toxins causing diseases or even death are sufficient to recognize the seriousness of the situation. This manuscript reviews the evidence that links animals as vehicles of the foodborne pathogens , Shiga toxigenic , and , their impact, and their current status. We conclude that these pathogenic bacteria will continue causing outbreaks and deaths throughout the world, because no effective interventions have eliminated them from animals and food.
Detection of Echinococcus multilocularis DNA in fruit, vegetable, and mushroom samples collected in the non-endemic territory of the Pomerania province and comparison of the results with data from rural areas of the neighbouring highly endemic Warmia-Masuria province, Poland.
Lass Anna,Szostakowska Beata,Myjak Przemyslaw,Korzeniewski Krzysztof
Echinococcus multilocularis is a tapeworm that may cause alveolar echinococcosis (AE), one of the most dangerous parasitic zoonoses. As in the case of some foodborne diseases, unwashed fruits and vegetables contaminated with eggs of E. multilocularis may serve as an important transmission route for this parasite. The aim of this study was to investigate the presence of E. multilocularis DNA in fruit, vegetables, and mushrooms in rural areas of the Pomerania province, Poland (non-endemic territory). In total, 104 environmental fruit, vegetable, and mushroom samples collected in forests, plantations, and kitchen gardens were analysed using nested PCR based on the mitochondrial 12S rRNA gene. E. multilocularis DNA was detected in 6.7 % of the samples tested, which indicated that the environment of the Pomerania province is contaminated with this parasite, creating a potential risk for humans. Therefore, fresh fruit, vegetables, and mushrooms should be washed before consumption. Additionally, the results showed that the level of contamination is significantly lower than in the highly endemic Warmia-Masuria province. The differences in the occurrence of E. multilocularis in the environment of these neighbouring provinces appears to be connected with the general epidemiological situation of these two regions, but further study is required for an exact explanation.
Clostridium difficile in Food and Animals: A Comprehensive Review.
Rodriguez C,Taminiau B,Van Broeck J,Delmée M,Daube G
Advances in experimental medicine and biology
Zoonoses are infections or diseases that can be transmitted between animals and humans through direct contact, close proximity or the environment. Clostridium difficile is ubiquitous in the environment, and the bacterium is able to colonise the intestinal tract of both animals and humans. Since domestic and food animals frequently test positive for toxigenic C. difficile, even without showing any signs of disease, it seems plausible that C. difficile could be zoonotic. Therefore, animals could play an essential role as carriers of the bacterium. In addition, the presence of the spores in different meats, fish, fruits and vegetables suggests a risk of foodborne transmission. This review summarises the current available data on C. difficile in animals and foods, from when the bacterium was first described up to the present.
A novel visual-mixed-dye for LAMP and its application in the detection of foodborne pathogens.
Pang Bo,Yao Shuo,Xu Kun,Wang Juan,Song Xiuling,Mu Ying,Zhao Chao,Li Juan
Loop-mediated isothermal amplification (LAMP) has been widely applied for the detection of foodborne pathogens. Obtaining a simple, accurate readout of LAMP reaction results is crucial. Herein, a visual-mixed-dye (VMD) containing calcein (precombined with MnCl) and hydroxynaphthol blue (HNB) for LAMP end-point detection was developed. The optimal final concentrations of these components in VMD were 25 and 300 μM, respectively. Due to the formation of HNBMn during the LAMP reaction, the VMD-loaded assay exhibited superior visual properties, changing from light gray (negative) to dark blue (positive) under natural light. Additionally, compared with traditional single calcein or HNB dye, a weakly positive result with the VMD was purple, making it easier to distinguish by the naked eye. The visual sensitivity reached down to 20.9 copies/μL, which was comparable to that based on fluorescence. In food-contaminated samples, the practicality of VMD was verified by Vibrio parahaemolyticus and Staphylococcus aureus detection with excellent specificity. Moreover, the VMD was stable over a period of 3 months. Collectively, these findings establish the VMD as a novel dye for LAMP.
Salmonella Enteritidis ST183: emerging and endemic biotypes affecting western European hedgehogs (Erinaceus europaeus) and people in Great Britain.
Lawson Becki,Franklinos Lydia H V,Rodriguez-Ramos Fernandez Julia,Wend-Hansen Clare,Nair Satheesh,Macgregor Shaheed K,John Shinto K,Pizzi Romain,Núñez Alejandro,Ashton Philip M,Cunningham Andrew A,M de Pinna Elizabeth
The impacts of hedgehog (Erinaceus europaeus) Salmonella infection on public health and on animal welfare and conservation are unknown. We isolated Salmonella Enteritidis multi-locus sequence-type (ST)183 from 46/170 (27%) hedgehog carcasses (27 S. Enteritidis phage type (PT)11, 18 of a novel PT66 biotype and one with co-infection of these PTs) and from 6/208 (3%) hedgehog faecal samples (4 PT11, 2 PT66) from across Great Britain, 2012-2015. Whole genome phylogenetic analysis of the hedgehog isolates and ST183 from people in England and Wales found that PT11 and PT66 form two divergent clades. Hedgehog and human isolates were interspersed throughout the phylogeny indicating that infections in both species originate from a common population. PT11 was recovered from hedgehogs across England and Scotland, consistent with endemic infection. PT66 was isolated from Scotland only, possibly indicating a recent emergence event. People infected with ST183 were four times more likely to be aged 0-4 years than people infected by the more common ST11 S. Enteritidis. Evidence for human ST183 infection being non-foodborne included stronger correlation between geographic and genetic distance, and significantly increased likelihood of infection in rural areas, than for ST11. These results are consistent with hedgehogs acting as a source of zoonotic infection.
Identification of a Plasmid-Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples.
Cummings K J,Rodriguez-Rivera L D,Norman K N,Ohta N,Scott H M
Zoonoses and public health
A recent increase in plasmid-mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti-microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole-genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid-mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.
Emergence of a plasmid-borne multidrug resistance gene cfr(C) in foodborne pathogen Campylobacter.
Tang Yizhi,Dai Lei,Sahin Orhan,Wu Zuowei,Liu Mingyuan,Zhang Qijing
The Journal of antimicrobial chemotherapy
Objectives:To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen. Methods:WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST. Results:The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion. Conclusions:These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens.
Salmonella Vaccination in Pigs: A Review.
Wales A D,Davies R H
Zoonoses and public health
The control of Salmonella enterica in pig production is necessary for both public and animal health. The persistent and frequently asymptomatic nature of porcine Salmonella infection and the organism's abilities to colonize other animal species and to survive in the environment mean that effective control generally requires multiple measures. Vaccination is one such measure, and the present review considers its role and its future, drawing on studies in pigs from the 1950s to the present day. Once established in the body as an intracellular infectious agent, Salmonella can evade humoral immunity, which goes some way to explaining the often disappointing performance of inactivated Salmonella vaccines. More recent approaches, using mucosal presentation of antigens, live vaccines and adjuvants to enhance cell-mediated immunity, have met with more success. Vaccination strategies that involve stimulating both passive immunity from the dam plus active immunity in offspring appear to be most efficacious, although either approach alone can yield significant control of Salmonella. Problems that remain include relatively poor control of Salmonella serovars that are dissimilar to the vaccine antigen mix, and difficulties in measuring and predicting the performance of candidate vaccines in ways that are highly relevant to their likely use in commercial production.
Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen .
Yang Yong,Tong Mingwei,Bai Xue,Liu Xiaolei,Cai Xuepeng,Luo Xuenong,Zhang Peihao,Cai Wei,Vallée Isabelle,Zhou Yonghua,Liu Mingyuan
Frontiers in microbiology
Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in . In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes. Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.
Arcobacter: an emerging food-borne zoonotic pathogen, its public health concerns and advances in diagnosis and control - a comprehensive review.
Ramees Thadiyam Puram,Dhama Kuldeep,Karthik Kumaragurubaran,Rathore Ramswaroop Singh,Kumar Ashok,Saminathan Mani,Tiwari Ruchi,Malik Yashpal Singh,Singh Raj Kumar
The veterinary quarterly
Arcobacter has emerged as an important food-borne zoonotic pathogen, causing sometimes serious infections in humans and animals. Newer species of Arcobacter are being incessantly emerging (presently 25 species have been identified) with novel information on the evolutionary mechanisms and genetic diversity among different Arcobacter species. These have been reported from chickens, domestic animals (cattle, pigs, sheep, horses, dogs), reptiles (lizards, snakes and chelonians), meat (poultry, pork, goat, lamb, beef, rabbit), vegetables and from humans in different countries. Arcobacters are implicated as causative agents of diarrhea, mastitis and abortion in animals, while causing bacteremia, endocarditis, peritonitis, gastroenteritis and diarrhea in humans. Three species including A. butzleri, A. cryaerophilus and A. skirrowii are predominantly associated with clinical conditions. Arcobacters are primarily transmitted through contaminated food and water sources. Identification of Arcobacter by biochemical tests is difficult and isolation remains the gold standard method. Current diagnostic advances have provided various molecular methods for efficient detection and differentiation of the Arcobacters at genus and species level. To overcome the emerging antibiotic resistance problem there is an essential need to explore the potential of novel and alternative therapies. Strengthening of the diagnostic aspects is also suggested as in most cases Arcobacters goes unnoticed and hence the exact epidemiological status remains uncertain. This review updates the current knowledge and many aspects of this important food-borne pathogen, namely etiology, evolution and emergence, genetic diversity, epidemiology, the disease in animals and humans, public health concerns, and advances in its diagnosis, prevention and control.
Risk ranking of foodborne parasites: State of the art.
Devleesschauwer Brecht,Bouwknegt Martijn,Dorny Pierre,Gabriël Sarah,Havelaar Arie H,Quoilin Sophie,Robertson Lucy J,Speybroeck Niko,Torgerson Paul R,van der Giessen Joke W B,Trevisan Chiara
Food and waterborne parasitology
In a time of increasing threats and decreasing financial resources, monitoring and controlling all possible foodborne hazards at the same time and to the same extent has become more challenging than ever. Therefore, attention is increasingly being paid to the so-called "risk ranking" methods that enable decision makers to focus on the most important foodborne hazards - even when time is limited and knowledge incomplete. In this review paper, we provide an overview of the most common quantitative methods and metrics used for ranking the risks associated with foodborne parasites and present the state of the art on risk ranking exercises for foodborne parasites. A number of risk ranking metrics and methods are available, ranging from simple approaches that can be used to assess the health or economic impact of a foodborne parasitic disease, to more complicated but more comprehensive multi-criteria assessments. For health impact assessment, measures of population health such as disease occurrence and number of deaths; Disability-Adjusted Life Years (DALYs) measuring the healthy life years lost; and Quality-Adjusted Life Years (QALYs) measuring the number of life years lived in optimal health, are described. For economic impact assessment, applied approaches that measure the cost-of-illness from a societal perspective and stated preference methods are outlined. Finally, Multi-Criteria Decision Analysis (MCDA), which can be used to integrate multiple metrics and criteria into a single ranking, is described. These risk ranking methods for foodborne parasites are increasingly performed to aid priority setting at global, regional, and national levels. As different stakeholders have their own prioritization objectives and beliefs, the outcome of such exercises is necessarily context-dependent. Therefore, when designing a risk ranking exercise for foodborne parasites, it is important to choose the metrics and methods, as well as what to rank, in the light of the predefined context of the question being addressed and the target audience.
Nematode infection among ruminants in monsoon climate (Ban-Lahanam, Lao PDR) and its role as food-borne zoonosis.
Sato Marcello Otake,Sato Megumi,Chaisiri Kittipong,Maipanich Wanna,Yoonuan Tippayarat,Sanguankiat Surapol,Pongvongsa Tiengkham,Boupha Boungnong,Moji Kazuhiko,Waikagul Jitra
Revista brasileira de parasitologia veterinaria = Brazilian journal of veterinary parasitology : Orgao Oficial do Colegio Brasileiro de Parasitologia Veterinaria
Trichostrongylids infection has gained significant public health importance since Trichostrongylus spp. infections have been reported in humans in Lao PDR. In this study, gastrointestinal nematodes were identified and the intensity of infections was determined in goats and cattle, which are animals greatly used for meat production in Lahanam Village, Lao PDR. The total number of goats and bovines was 23 and 29, respectively, pertaining to 32 households surveyed in the area. Feacal samples were randomly collected from 14 goats and 11 bovines. Ninety three percent (13/14) of goats and 36% (3/11) of cattle were infected, with an average of 1,728 and 86 eggs per gram of faeces (EPG), respectively. Coproculture showed Trichostrongylus spp. (goats 16%; bovines 48%), Haemonchus spp. (goats 69%; bovines 37%), Cooperia spp. (bovines 8%) and Oesophagostomum spp. (goats 15%; bovines 6%). After performing the necropsy on an adult goat, Trichuris spp. was also found. We confirmed the presence of Oesophagostomum spp., H. contortus and T. colubriformis by morphology and DNA sequencing analysis of the ITS region of rDNA. Due to interactions between humans and goats in Lahanam Village and high EPG results, the diagnosis of species and the intensity of gastrointestinal nematode infection in these animals are important public-health issues. Other ruminant parasites, such as Oesophagostomum and Haemonchus, found in caprines and bovines, are reported to be causes of zoonosis and their presence in humans should be investigated in future field surveys in this area.
from microbiology to prevention.
Facciolà A,Riso R,Avventuroso E,Visalli G,Delia S A,Laganà P
Journal of preventive medicine and hygiene
In last years, Campylobacter spp has become one of the most important foodborne pathogens even in high-income countries. Particularly, in Europe, Campylobacteriosis is, since 2005, the foodborne disease most frequently notified and the second in USA, preceded by the infection due to Salmonella spp. Campylobacter spp is a commensal microorganism of the gastrointestinal tract of many wild animals (birds such as ducks and gulls), farm animals (cattle and pigs) and companion animals (such as dogs and cats) and it is responsible for zoonoses. The transmission occurs via the fecal-oral route through ingestion of contaminated food and water. The disease varied from a watery diarrhea to a severe inflammatory diarrhea with abdominal pain and fever and can be burdened by some complications. The main recognized sequelae are Guillain-Barré Syndrome (GBS), the Reactive Arthritis (REA) and irritable bowel syndrome (IBS). Recently, many cases of Campylobacter spp isolated from human infections, showed an important resistance to various antibiotics such as tetracyclines and fluoroquinolones. For these reasons, the prevention of this infection plays an essential role. Many preventive measures exist to limit the transmission of the pathogens and the subsequent disease such as the health surveillance, the vaccination of the poultry and the correct food hygiene throughout the entire production chain. A global surveillance of Campylobacteriosis is desirable and should include data from all countries, including notifications of cases and the microbiological data typing of strains isolated from both human and animal cases.
Progress on the development of rapid diagnostic tests for foodborne neglected zoonotic helminthiases: A systematic review.
Mubanga Chishimba,Mwape Kabemba E,Phiri Isaac K,Trevisan Chiara,Zulu Gideon,Chabala Chishala,van Damme Inge,Schmidt Veronika,Dorny Pierre,Gabriël Sarah
BACKGROUND:Foodborne Neglected Zoonotic Helminths (FNZH) are parasites of both economic and public health importance. They include Taenia solium, Echinococcus granulosus sensu lato, Echinococcus multilocularis and Foodborne trematodes (FBT). FNZH are earmarked for major interventions for control, elimination and eradication. This systematic review highlights the progress towards development of rapid tests for the diagnosis of FNZH since 2010 when they were listed as neglected tropical diseases. METHODOLOGY:A systematic search was conducted in three databases, World of Science, Embase and PubMed using the same search phrase. The search produced 480 hits. Three studies from back referencing were included. Only 22 of these met the inclusion criteria. Data was extracted from these and presented qualitatively. RESULTS:Twenty-five rapid diagnostic tests were found to have been developed since 2010, eight for diagnosis of T. solium infections, eight for echinococcosis and nine for FBT infections. The rapid tests for diagnosing T. solium infections included six antibody detecting and two antigen detecting tests. They constitute a combination among them, with some tests providing qualitative, others quantitative results. Similarly, seven out of the eight rapid tests developed for Echinococcus infections were antibody detecting tests save for one loop mediated isothermal amplification test. All of them were qualitative tests. For FBT infections, nine rapid tests were described; two antibody and one nucleic acid detecting test for diagnosis of Fascioliasis; three nucleic acid detecting tests for Opisthorchiasis; one antibody detecting test for Paragonimiasis; and for Clonorchiasis, one antibody and one nucleic acid detecting test. The FBT infection rapid tests were all qualitative in nature. Most of these tests have not undergone field evaluation in endemic areas where they will be used most. CONCLUSION:This review describes the development and evaluation of rapid diagnostic tests, while highlighting the need for in depth validations of the tools to determine how well they can perform in endemic areas.
Dracunculiasis: water-borne anthroponosis vs. food-borne zoonosis.
Galán-Puchades M T
Journal of helminthology
Dracunculiasis is the first parasitic disease set for eradication. However, recent events related to the Dracunculus medinensis epidemiology in certain African countries are apparently posing new challenges to its eradication. Two novel facts have emerged: the existence of animal reservoirs (mainly dogs but also cats and baboons), and possibly a new food-borne route of transmission by the ingestion of paratenic (frogs) or transport (fish) hosts. Therefore, instead of being exclusively a water-borne anthroponosis, dracunculiasis would also be a food-borne zoonosis. The existence of a large number of infected dogs, mainly in Chad, and the low number of infected humans, have given rise to this potential food-borne transmission. This novel route would concern not only reservoirs, but also humans. However, only animals seem to be affected. Dracunculus medinensis is on the verge of eradication due to the control measures which, classically, have been exclusively aimed at the water-borne route. Therefore, food-borne transmission is probably of secondary importance, at least in humans. In Chad, reservoirs would become infected through the water-borne route, mainly in the dry season when rivers recede, and smaller accessible ponds, with a lower water level containing the infected copepods, appear, whilst humans drink filtered water and, thus, avoid infection. The total absence of control measures aimed at dogs (or at other potential reservoirs) up until the last years, added to the stimulating reward in cash given to those who find parasitized dogs, have presumably given rise to the current dracunculiasis scenario in Chad.
Hepatitis E Virus: A New Foodborne Zoonotic Concern.
Rodríguez-Lázaro David,Hernandez Marta,Cook Nigel
Advances in food and nutrition research
Hepatitis E virus (HEV) is an enteric nonenveloped single-stranded RNA virus. Among the mammalian lineages, four genotypes are associated to human infection: genogroups 1 and 2 infect only humans and are mainly found in developing countries, while genogroups 3 and 4 are zoonotic, being found in a variety of animal species including pigs, and are autochthonous in developed countries. HEV infection can result in liver damage and with genotypes 1 and 2 symptoms can be particularly severe in pregnant women, with a high lethality ratio. Several cases of foodborne transmission of hepatitis E have been reported, often involving consumption of meat, especially raw or undercooked. Information is lacking on the exact extent of foodborne transmission of HEV.
Multi-laboratory evaluation of the rapid genoserotyping array (SGSA) for the identification of Salmonella serovars.
Yoshida Catherine,Lingohr Erika J,Trognitz Friederike,MacLaren Nikki,Rosano Andrea,Murphy Stephanie A,Villegas Andre,Polt Marlies,Franklin Kristyn,Kostic Tanja, , ,Kropinski Andrew M,Card Roderick M
Diagnostic microbiology and infectious disease
Salmonella serotyping is an essential first step for identification of isolates associated with disease outbreaks. The Salmonella genoserotyping array (SGSA) is a microarray-based alternative to standard serotyping designed to rapidly identify 57 of the most commonly reported serovars through detection of the genes encoding surface O and H antigens and reporting the corresponding serovar in accordance with the existing White-Kaufmann-Le Minor serotyping scheme. In this study, we evaluated the SGSA at 4 laboratories in 3 countries by testing 1874 isolates from human and non-human sources. The SGSA correctly identified 96.7% of isolates from the target 57 serovars. For the prevalent and clinically important Salmonella serovars Enteritidis and Typhimurium, test specificity and sensitivity were greater than 98% and 99%, respectively. Due to its high-throughput nature, the SGSA is a rapid and cost-effective alternative to standard serotyping for identifying the most prevalent serovars of Salmonella.
Comparison between RT droplet digital PCR and RT real-time PCR for quantification of noroviruses in oysters.
Persson Sofia,Eriksson Ronnie,Lowther James,Ellström Patrik,Simonsson Magnus
International journal of food microbiology
Oysters are frequently associated with norovirus outbreaks, but the presence of norovirus RNA in oysters does not necessarily imply a health risk to humans. There is a close link between human illness and consumption of oysters with high levels of norovirus RNA, but oysters with low levels of norovirus RNA are more unlikely to be associated with illness. Reliable and precise quantification methods are therefore important for outbreak investigations and risk assessments. This study optimised and validated RT droplet digital PCR (RT-ddPCR) assays for quantification of norovirus genogroups I and II in artificially contaminated oysters, and compared them with the standard method, RT real-time PCR (RT-qPCR). The two methods had comparable 95% limits of detection, but RT-ddPCR generally showed greater precision in quantification. Differences between fluorometric measurements and quantification with RT-ddPCR were determined on in vitro transcribed RNA with targets for norovirus genogroups I and II. Quantification by RT-ddPCR was on average 100 times lower than the fluorometric value for norovirus GI and 15.8 times lower than the fluorometric value for norovirus GII. The large inter-assay difference observed highlights the need for monitoring the RT efficiency in RT-ddPCR, especially when results from different assays are compared. Overall, this study suggests that RT-ddPCR can be a suitable method for precise quantification of norovirus genogroups I and II in oysters.
Culture versus PCR for Salmonella Species Identification in Some Dairy Products and Dairy Handlers with Special Concern to Its Zoonotic Importance.
Gwida Mayada M,Al-Ashmawy Maha A M
Veterinary medicine international
A total of 200 samples of milk and dairy products as well as 120 samples of dairy handlers were randomly collected from different dairy farms and supermarkets in Dakahlia Governorate, Egypt. The conventional cultural and serotyping methods for detection of Salmonella in dairy products were applied and the results were compared with those obtained by molecular screening assay using (ttr sequence). The obtained results revealed that 21% of milk and dairy products (42/200) were positive for Salmonella species using enrichment culture-based PCR method, while 12% of different dairy samples (24/200) were found to be positive for Salmonella species by using the conventional culture methods. Two stool specimens out of 40 apparently healthy dairy handlers were positive by the PCR method. Serotyping of Salmonella isolates revealed that 58.3% (14/24) from different dairy products were contaminated with Salmonella Typhimurium. We conclude that the enrichment culture-based PCR assay has high sensitivity and specificity for detection of Salmonella species in dairy products and handlers. High incidence of Salmonella Typhimurium in the examined dairy samples highlights the important role played by milk and dairy products as a vehicle in disease prevalence. Great effort should be applied for reducing foodborne risk for consumers.
Quantification of E. coli O157 and STEC in feces of farm animals using direct multiplex real time PCR (qPCR) and a modified most probable number assay comprised of immunomagnetic bead separation and qPCR detection.
Guy Rebecca A,Tremblay Donald,Beausoleil Louise,Harel Josée,Champagne Marie-Josée
Journal of microbiological methods
To better understand Escherichia coli O157:H7 on-farm transmission dynamics requires sensitive methods for quantification of a broad range of concentrations of target organisms. For this purpose, a multiplex real time PCR (qPCR) assay was developed for quantification of O157 E. coli from 1g fecal samples of cattle and other animal species, targeting the Shiga toxin genes (stx1 and stx2) and the O157 somatic antigen gene, per. The multiplex qPCR assay provided specific detection across a broad range of bacterial concentrations with a lower limit of detection (LOD) of 10(1) genome copies which is equivalent to 10(1) bacteria. However, the LOD, when direct qPCR was applied to quantification of the targets in the feces of dairy cattle, was 10(3) genome copies per gram of feces. Enumeration below the threshold for direct qPCR was performed using a modified most probable number (mMPN) method whereby E. coli O157 in enriched samples was isolated using immunomagnetic bead separation (IMS) and detected using qPCR, thus reducing the time and logistic constraints of biochemical/serological/gel analysis. Application of the mMPN (IMS/qPCR) assay to samples that were negative when tested using direct qPCR alone permitted quantification of low levels of E. coli O157 below levels detectable with direct qPCR. The direct qPCR and mMPN (IMS/qPCR) assays were applied to fecal samples from dairy, beef, swine and poultry feces. This approach can be employed to gain a better understanding of the patterns of infection in animals for analysis of on-farm transmission dynamics, for evaluating the effects of on-farm control strategies and for risk assessment in public health.
Genome-Wide Survey of Genes Under Positive Selection in Avian Pathogenic Escherichia coli Strains.
Rojas Thaís Cabrera Galvão,Lobo Francisco Pereira,Hongo Jorge Augusto,Vicentini Renato,Verma Renu,Maluta Renato Pariz,da Silveira Wanderley Dias
Foodborne pathogens and disease
The ability to obtain bacterial genomes from the same host has allowed for comparative studies that help in the understanding of the molecular evolution of specific pathotypes. Avian pathogenic Escherichia coli (APEC) is a group of extraintestinal strains responsible for causing colibacillosis in birds. APEC is also suggested to possess a role as a zoonotic agent. Despite its importance, APEC pathogenesis still has several cryptic pathogenic processes that need to be better understood. In this work, a genome-wide survey of eight APEC strains for genes with evidence of recombination revealed that ∼14% of the homologous groups evaluated present signs of recombination. Enrichment analyses revealed that nine Gene Ontology (GO) terms were significantly more represented in recombinant genes. Among these GO terms, several were noted to be ATP-related categories. The search for positive selection in these APEC genomes revealed 32 groups of homologous genes with evidence of positive selection. Among these groups, we found several related to cell metabolism, as well as several uncharacterized genes, beyond the well-known virulence factors ompC, lamB, waaW, waaL, and fliC. A GO term enrichment test showed a prevalence of terms related to bacterial cell contact with the external environment (e.g., viral entry into host cell, detection of virus, pore complex, bacterial-type flagellum filament C, and porin activity). Finally, the genes with evidence of positive selection were retrieved from genomes of non-APEC strains and tested as were done for APEC strains. The result revealed that none of the groups of genes presented evidence of positive selection, confirming that the analysis was effective in inferring positive selection for APEC and not for E. coli in general, which means that the study of the genes with evidence of positive selection identified in this study can contribute for the better understanding of APEC pathogenesis processes.
Epidemiology of Campylobacter, Salmonella and antimicrobial resistant Escherichia coli in free-living Canada geese (Branta canadensis) from three sources in southern Ontario.
Vogt Nadine A,Pearl David L,Taboada Eduardo N,Mutschall Steven K,Janecko Nicol,Reid-Smith Richard,Bloomfield Bryan,Jardine Claire M
Zoonoses and public health
Antimicrobial resistant bacteria and zoonotic pathogens have previously been isolated from Canada geese. We examined the prevalence of three enteric bacteria (i.e. Campylobacter, Salmonella, Escherichia coli) among Canada geese from three sampling sources in southern Ontario from 2013 through 2015. Samples were obtained by convenience from hunting groups, diagnostic birds submitted for post-mortem, and fresh faeces from live birds in parks. Escherichia coli isolates were isolated and tested for susceptibility to 15 antimicrobials using the Canadian Integrated Program for Antimicrobial Resistance Surveillance test panel. The prevalences of Salmonella, Campylobacter and E. coli were 0%, 11.2% and 72.6%, respectively. Among E. coli isolates, 7.9% were resistant to ≥1 class of antimicrobials and 5.6% were resistant to ≥2 classes of antimicrobials, with some including resistance to antimicrobials of highest importance in human medicine. A significant association between season and E. coli resistance among samples from live birds was noted; summer samples had no resistant E. coli isolates, whereas spring samples demonstrated the highest prevalence of E. coli resistant to ≥1 class of antimicrobials (20.0%) among all sources. In addition, Campylobacter coli were only isolated from the spring faecal samples. Flock-level clustering was an important statistical consideration, as flock was a significant random effect in all but two of our models. Detection of Campylobacter and antimicrobial resistant E. coli from Canada geese suggests that these birds may play a role in disseminating these organisms within the environment.
A Simple and Rapid Staining Technique for Sex Determination of Larvae Parasites by Confocal Laser Scanning Microscopy.
Gavarane Inese,Kirilova Elena,Rubeniņa Ilze,Mežaraupe Ligita,Osipovs Sergejs,Deksne Gunita,Pučkins Aleksandrs,Kokina Inese,Bulanovs Andrejs,Kirjušina Muza
Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.
Validation according to ISO 16140:2003 of a commercial real-time PCR-based method for detecting Campylobacter jejuni, C. coli, and C. lari in foods.
Vencia W,Nogarol C,Bianchi D M,Gallina S,Zuccon F,Adriano D,Gramaglia M,Decastelli L
International journal of food microbiology
Campylobacteriosis was the most frequently reported zoonosis in the European Union (EU) in 2010, with Campylobacter jejuni, Campylobacter coli, and Campylobacter lari as the most frequently reported species in foodborne outbreaks (FBOs). Relatively sensitive to environmental factors, these species may be present in low numbers. In line with EU policy for food control and FBO detection and in view of the need to reduce response time, we validated an alternative molecular method according to ISO 16140:2003 which establishes the general principle and technical protocol for the validation of alternative methods in the microbiological analysis of food. We used a qualitative real-time PCR commercial kit for the detection of C. jejuni, C. coli, and C. lari in two food categories "fruit and vegetable-based products" and "dairy products". The validation protocol comprises two phases: the first is a method comparison study of the alternative method against the reference method, and the second is an interlaboratory study of each of the two methods. In the first step, ISO 16140:2003 validation examines the following parameters: limit of detection (LOD); relative accuracy, relative specificity and sensitivity; relative detection level (RDL); and inclusivity and exclusivity. Except for LOD, inclusivity and exclusivity, the other steps were performed against the reference method (ISO 10272:2006). The LOD of the real-time PCR method was set at 4CFU/25g or mL for both food categories. Relative accuracy (98.33%), specificity (96.77%), and sensitivity (100%) were recorded for the food category "fruit and vegetable-based products" and 93.3%, 88.24%, 100%, respectively, for "dairy products". The RDL according to Fisher's exact test was p=1 for both food categories, for each level, and each food/strain combination. The interlaboratory study results showed correct identification of all 24 blind samples with both methods by all the participating laboratories. The results show that this commercial kit is suitable for the rapid detection of C. jejuni, C. coli, and C. lari on fruit, vegetables and dairy products and may aid in official controls. In conclusion, the use of alternative methods is recommended for the rapid identification of positive samples and the identification of the possible bacterial source in a FBO within 48 h.
Loop-Mediated Isothermal Amplification of the sefA Gene for Rapid Detection of Salmonella Enteritidis and Salmonella Gallinarum in Chickens.
Gong Jiansen,Zhuang Linlin,Zhu Chunhong,Shi Shourong,Zhang Di,Zhang Linji,Yu Yan,Dou Xinhong,Xu Bu,Wang Chengming
Foodborne pathogens and disease
Salmonella spp. pose a threat to both human and animal health, with more than 2600 serovars having been reported to date. Salmonella serovars are usually identified by slide agglutination tests, which are labor intensive and time consuming. In an attempt to develop a more rapid screening method for the major poultry Salmonella serovars, we developed a loop-mediated isothermal amplification (LAMP) assay, which directly detected the sefA gene, a fimbrial operon gene existing in several specific serovars of Salmonella enterica including the major poultry serovars, namely Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) and Salmonella enterica serovar Gallinarum (Salmonella Gallinarum). With the 177 bacterial strains we tested, positive reactions were only observed with 85 strains of serovar Salmonella Enteritidis and Salmonella Gallinarum. The detection limit of the LAMP assay was 4 CFU/reaction with genomic DNAs of Salmonella Enteritidis (ATCC 13076) from pure culture and 400 CFU/ reaction with DNA extracted from spiked chicken feces. The LAMP assay was more sensitive than conventional culture, especially without enrichment, in detecting Salmonella Enteritidis (CMCC 50041) in the spiked fecal samples. The results show the sefA LAMP method is a rapid, sensitive, specific, and practical method for directly detection of Salmonella Enteritidis and Salmonella Gallinarum in chickens. The sefA LAMP assay can potentially serve as new on-site diagnostics in the poultry industry.
Development of a nest-PCR for detection of Fasciola hepatica DNA in the intermediate snail host, Radix cucunorica, and the prevalence in northwestern China.
Huang Si-Yang,Gong Jing-Zhi,Yang Bin,Fan Yi-Min,Yao Na,Wang Chun-Ren
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
Fasciolosis, a foodborne zoonotic disease, caused by Fasciola species which is considered an important problem for human health and livestock husbandry development. Snails are intermediate hosts of F. hepatica, the epidemiological surveillance of snails can evaluate the transmission risk of this disease in human and livestock. In this study, we developed a nest-polymerase chain reaction (nest-PCR) to detect the DNA of F. hepatica in Radix cucunorica, a prevalent intermediate host of this parasite in northwestern China. The nest-PCR was used to amplify a 208 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica with two pairs of primers. The method was able to detect up to 0.16 fg genomic DNA in a 25 μL PCR reaction system even effected with high concentrations of snail DNA, and no cross reaction was observed from the genomic DNA of Paramphistomum cervi, Clonorchis sinensis, Orientobilharzia turkestanicum, Metorchis orientalis, Dicrocoelium chinensis. To evaluate the transmission risk of this disease, 409 snail samples collected from different areas of Gansu province were used to detect and analyze the transmission risk of F. hepatica in this area. Of 409 snail samples, the overall prevalence is 43.76%. The prevalence was 92.75% in Gannan Tibetan Autonomous Prefecture, while no snail was positive for F. hepatica in Linxia Hui Autonomous Prefecture. The nest-PCR was firstly used to detect the infection of F. hepatica in snail. It is a novel, useful and convenient method with high sensitivity and specificity. This study is the first report about the epidemiological surveillance of snail infection by F. hepatica in northwestern China, which will help to evaluate the transmission risk of F. hepatica in northwestern China.
Molecular Detection and Genotyping of Intestinal Microsporidia from Stray Dogs in Iran.
Iranian journal of parasitology
BACKGROUND:Microsporidia as one of the most important pathogens in veterinary and agricultural settings, have emerged in immunocompromised patients in Iran. To date, different genotypes have been identified in humans and animals, supporting the possibility of zoonotic zoonosis transmission potential. The aim of this study was to evaluate the distribution of genotypes among overpopulated stray dogs in vicinity of Tehran, the capital city of Iran. METHODS:Totally, 75 stool and 75 urine samples were obtained from 75 stray dogs during the time period from Mar 2015 to Oct 2015. DNA extraction was performed on all the samples and specific fragment of small subunit ribosomal RNA gene of and spp. was amplified. Furthermore, specific primers targeting the internal transcribed spacer region of were applied to determine the genotype of the microorganism. RESULTS:Microsporidia was detected in 5.3% of stool samples, while none of the urine samples was positive for microsporidia species. Overall, 440 bp fragment of was amplified in all the samples and there was no amplification for spp. The results of sequencing of 410 bp fragment of internal transcribed spacer region showed that all the were genotype D. CONCLUSION: was the most prevalent microsporidian species in the stray dogs and all the positive isolates were characterized as genotype D.
A new pyrosequencing assay for rapid detection and genotyping of Shiga toxin, intimin and O157-specific rfbE genes of Escherichia coli.
Goji Noriko,Mathews Amit,Huszczynski George,Laing Chad R,Gannon Victor P J,Graham Morag R,Amoako Kingsley K
Journal of microbiological methods
Shiga toxin (stx)-producing Escherichia coli (STEC) contamination in food and water is one of the most recognized concerns and a major financial burden in human hygiene control worldwide. Rapid and highly reliable methods of detecting and identifying STEC causing gastroenteric illnesses are crucial to prevent foodborne outbreaks. A number of tests have been developed and commercialized to detect STEC using molecular microbiology techniques. Most of these are designed to identify virulence factors such as Shiga toxin and intimin as well as E. coli O and H antigen serotype specific genes. In order to screen pathogenic STEC without relying on O:H serotyping, we developed a rapid detection and genotyping assay for STEC virulence genes using a PCR-pyrosequencing application. We adapted the PyroMark Q24 Pyrosequencing platform for subtyping 4 major virulence genes, Shiga toxin 1 and 2 (stx1 and stx2), intimin (eae) and O157-antigen gene cluster target rfbE, using Single Nucleotide Polymorphism (SNP) analysis. A total of 224 E. coli strains including isolates from Canadian environment, food and clinical cases were examined. Based on the multiple alignment analysis of 30-80 base nucleotide pyrogram reads, three alleles of the Shiga toxin 1a gene (stx1a) (stx1a-I, stx1a-II, stx1a-III) were identified. Results of the stx1, stx2, eae and rfbE genotyping revealed that each group of O:H serotype shares distinctive characteristics that could be associated with the virulence of each genotype. O157:H7/NM carries stx1a-II (94%), stx2a (82%), λ/γ1-eae (100%) and rfbE type-H7/NM (100%). Whereas isolates of the "Top-6" serotypes (O26, O45, O103, O111, O121, O145) had a high incidence of stx1a-I (90%) and stx2a (100%). stx1a-III (60%) was only observed in non Top-7 (Top-6 plus O157) STEC and Shigella spp. The entire assay, from extracting DNA from colonies on a plate to the generation of sequence information, can be completed in 5h. The method of profiling these 4 STEC pathogenic genotypes as demonstrated in this paper is rapid, easily performed, informative and cost-effective, and thus has a potential to be deployed in the food industry for the routine screening of potentially pathogenic STEC isolates.
Relationship between Salmonella infection, shedding and serology in fattening pigs in low-moderate prevalence areas.
San Román B,Garrido V,Sánchez S,Martínez-Ballesteros I,Garaizar J,Mainar-Jaime R C,Migura-Garcia L,Grilló M J
Zoonoses and public health
Salmonella is a major foodborne pathogen causing important zoonosis worldwide. Pigs asymptomatically infected in mesenteric lymph nodes (MLN) can be intermittent shedders of the pathogen through faeces, being considered a major source of human infections. European baseline studies of fattening pig salmonellosis are based on Salmonella detection in MLN. This work studies the relationship between Salmonella infection in MLN and intestinal content (IC) shedding at slaughter and the relationship between the presence of the pathogen and the serologic status at farm level. Mean Salmonella prevalence in the selected pigs (vertically integrated production system of Navarra, Spain) was 7.2% in MLN, 8.4% in IC and 9.6% in serum samples. In this low-moderate prevalence context, poor concordance was found between MLN infection and shedding at slaughter and between bacteriology and serology. In fact, most of shedders were found uninfected in MLN (83%) or carrying different Salmonella strains in MLN and in IC (90%). The most prevalent Salmonellae were Typhimurium resistant to ACSSuT ± Nx or ASSuT antibiotic families, more frequently found invading the MLN (70%) than in IC (33.9%). Multivariable analysis revealed that risk factors associated with the presence of Salmonella in MLN or in IC were different, mainly related either to good hygiene practices or to water and feed control, respectively. Overall, in this prevalence context, detection of Salmonella in MLN is an unreliable predictor of faecal shedding at abattoir, indicating that subclinical infections in fattening pigs MLN could have limited relevance in the IC shedding.
A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds.
Haas Katrin,Overesch Gudrun,Kuhnert Peter
Journal of food protection
Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the "gold standard." Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.
Serological tools for detection of infection in animals and humans.
Yang Yong,Cai Ya Nan,Tong Ming Wei,Sun Na,Xuan Yin Hua,Kang Yan Jun,Vallée Isabelle,Boireau Pascal,Cheng Shi Peng,Liu Ming Yuan
One health (Amsterdam, Netherlands)
Trichinellosis is a serious foodborne zoonotic disease. It is an important threat to public health in both developing and developed countries. Human infections are strongly associated with consuming undercooked meat containing infective larvae. The development of serological tools has enabled seroepidemiological studies and contributed to our knowledge on the importance of this parasite. Serological tests can also help the diagnosis of parasite infections in humans and the surveillance of animals. Generally speaking, serological techniques include detection methods for specific antibodies and for circulating parasite antigens in the serum or tissue fluids. Here, we present a comprehensive review of various methods used in the detection of antibodies against and circulating parasite antigens in animals and humans.
Fascioliasis: a worldwide parasitic disease of importance in travel medicine.
Ashrafi Keyhan,Bargues M Dolores,O'Neill Sandra,Mas-Coma Santiago
Travel medicine and infectious disease
Fascioliasis is a foodborne zoonotic disease caused by the two parasite species Fasciola hepatica and Fasciola gigantica. This trematodiasis has never been claimed special relevance for travellers and migrants. However, the situation has drastically changed in the last two decades, in a way that fascioliasis should today be included in the list of diseases to be enhanced in Travel Medicine. Different kind of travellers have been involved in human infection reports: business travellers, tourists, migrants, expatriated workers, military personnel, religious missionaries, and refugees. Europe is the continent where more imported cases have been reported in many countries. More cases would have been probably reported in Europe if fascioliasis would be a reportable disease. In the Americas, most of the reports concern cases diagnosed in USA. Relative few patients have been diagnosed in studies on travellers performed in Asia. In Africa, most cases were reported in Maghreb countries. Blood eosinophilia and the ingestion of watercress or any other suggestive freshwater plant in anamnesis are extremely useful in guiding towards a fascioliasis diagnosis in a developed country, although may not be so in human endemic areas of developing countries. Several suggestive clinical presentation aspects may be useful, although the clinical polymorphism may be misleading in many cases. Non-invasive techniques are helpful for the diagnosis, although images may lead to confusion. Laparoscopic visualization should assist and facilitate procurement of an accurately guided biopsy. Endoscopic retrograde cholangiopancreatography (ERCP) is the first choice in patients in the chronic phase. ERCP and sphincterotomy are used to extract parasites from the biliary tree. Fluke egg finding continues to be the gold standard and enables for burden quantification and establishing of the drug dose. Many serological and stool antigen detection tests have been developed. Immunological techniques present the advantages of being applicable during all periods of the disease, but fundamentally during the invasive or acute period, as well as to other situations in which coprological techniques may present problems. Triclabendazole is the drug of choice at present, although the spread of resistance to this drug is challenging. Prevention mainly concerns measures to avoid individual infection by considering the different human infection sources.
High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.
Jiang Hui,Jiang Donglei,Shao Jingdong,Sun Xiulan,Wang Jiasheng
Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.
Fishing in the Soup - Pathogen Detection in Food Safety Using Metabarcoding and Metagenomic Sequencing.
Grützke Josephine,Malorny Burkhard,Hammerl Jens Andre,Busch Anne,Tausch Simon H,Tomaso Herbert,Deneke Carlus
Frontiers in microbiology
In food safety the detection of food contaminations with pathogenic microorganisms is a race against time and often outpaced by error-prone epidemiological approaches. For evidence-based outbreak investigations fast and reliable techniques and procedures are required to identify the source of infection. Metagenomics has the potential to become a powerful tool in the field of modern food safety, since it allows the detection, identification and characterization of a broad range of pathogens in a single experiment without pre-cultivation within a couple of days. Nevertheless, sample handling, sequencing and data analysis are challenging and can introduce errors and biases into the analysis. In order to evaluate the potential of metagenomics in food safety, we generated a mock community containing DNA of foodborne bacteria. Herewith, we compare the aptitude of the two prevalent approaches - 16S rDNA amplicon sequencing and whole genome shotgun sequencing - for the detection of foodborne bacteria using different parameters during sample preparation, sequencing and data analysis. 16S rDNA sequencing did not only result in high deviations from the expected sample composition on genus and species level, but more importantly lacked the detection of several pathogenic species. While shotgun sequencing is more suitable for species detection, abundance estimation, genome assembly and species characterization, the performance can vary depending on the library preparation kit, which was confirmed for a naturally contaminated game meat sample. The application of the Nextera XT DNA Library Preparation Kit for shotgun sequencing did not only result in lower reference genome recovery and coverage, but also in distortions of the mock community composition. For data analysis, we propose a publicly available workflow for pathogen detection and characterization and demonstrate its benefits on the usability of metagenomic sequencing in food safety by analyzing an authentic metagenomic sample.
Loop-Mediated Isothermal Amplification-Lateral-Flow Dipstick (LAMP-LFD) to detect Toxoplasma gondii oocyst in ready-to-eat salad.
Lalle Marco,Possenti Alessia,Dubey Jitender P,Pozio Edoardo
The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a foodborne zoonosis with a global distribution and estimated to cause up to 20% of the total foodborne disease burden in Europe. Association between T. gondii infection and the consumption of unwashed raw fruits and vegetables contaminated with oocysts has been reported and the increasing habit to eat pre-washed ready-to-eat salads poses a new potential risk for consumers. It is therefore important to trace the occurrence of potential contamination with this parasite to guarantee the safety of ready-to-eat vegetables. Detection of T. gondii in vegetables by molecular techniques has been achieved but low sensitivity (PCR) or expensive equipments (qPCR) limit routine applicability. Here, we describe the development and validation of a sensitive and robust method relying on a LAMP assay, targeting the 529 bp locus, to detect T. gondii oocysts down to 25 oocysts/50 g in ready-to-eat baby lettuce. The LAMP has been also adapted for a faster visualization of the result by a lateral flow dipstick chromatographic detection method.
Electrochemical and Optical Biosensors for the Detection of and : An Update Look.
Vizzini Priya,Braidot Matteo,Vidic Jasmina,Manzano Marisa
Foodborne safety has aroused tremendous research interest in recent years because of a global public health problem. The rapid and precise detection of foodborne pathogens can reduce significantly infection diseases and save lives by the early initiation of an effective treatment. This review highlights current advances in the development of biosensors for detection of spp. and that are the most common causes of zoonosis. The consumption of pathogen contaminated food is responsible for humans hospitalization and death. The attention focused on the recognition elements such as antibodies (Ab), DNA probes and aptamers able to recognize cells, amplicons, and specific genes from different samples like bacteria, food, environment and clinical samples. Moreover, the review focused on two main signal-transducing mechanisms, i.e., electrochemical, measuring an amperometric, potentiometric and impedimetric signal; and optical, measuring a light signal by OLED (Organic Light Emitting Diode), SPR (Surface Plasmon Resonance), and Optical fiber. We expect that high-performance of devices being developed through basic research will find extensive applications in environmental monitoring, biomedical diagnostics, and food safety.
Molecular Detection, Typing, and Quantification of Campylobacter spp. in Foods of Animal Origin.
Frasao Beatriz da Silva,Marin Victor Augustus,Conte-Junior Carlos Adam
Comprehensive reviews in food science and food safety
The most frequently reported zoonosis and the main bacterial foodborne disease infection in humans is caused by Campylobacter spp., and C. jejuni and C. coli are the most common types. These bacteria can be found in the intestinal tracts of cattle, dogs, cats, sheep, poultry and pigs. The isolation of this microorganism is laborious because it requires specific media and a low oxygen concentration for growth. Additionally, differentiation between species through conventional bacteriology is difficult, as there are few different biochemical characteristics among the various species. Molecular microbiological techniques have become more important and are now broadly applied to help overcome difficulties in the identification, differentiation, and quantification of this pathogen. To date, there have been advances in the development and use of molecular techniques for the identification of microorganisms in foodstuffs. Tools such as pulsed-field gel electrophoresis and multilocus sequence typing are the most commonly used for typing. For the identification and confirmation of species, polymerase chain reaction (PCR) is crucial. Quantification by real-time PCR has wide applicability. To identify strains and antimicrobial resistance genes, sequencing technologies have been applied. This review builds on the discussion about the main and most widely used molecular methods for Campylobacter, as well as methods showing better potential for the classification, identification, and quantification of this important pathogen.
Rapid and specific detection of Salmonella infections using chemically modified nucleic acid probes.
Machado Isabel,Garrido Victoria,Hernandez Luiza I,Botero Juliana,Bastida Nora,San-Roman Beatriz,Grilló María-Jesús,Hernandez Frank J
Analytica chimica acta
Salmonella is a leading source of bacterial foodborne illness in humans, causing gastroenteritis outbreaks with bacteraemia occurrences that can lead to clinical complications and death. Eggs, poultry and pig products are considered as the main carriers of the pathogenic Salmonella for humans. To prevent this relevant zoonosis, key changes in food safety regulations were undertaken to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis were reported worldwide in the last decade. Thus, new strategies for Salmonella detection are a priority for both, food safety and public health authorities. Such detection systems should provide significant reduction in diagnostic time (hours) compared to the currently available methods (days). Herein, we report on the discovery and characterization of nucleic acid probes for the sensitive and specific detection of live Salmonella within less than 8 h of incubation. We are the first to postulate the nuclease activity derived from Salmonella as biomarker of infection and its utility to develop innovative detection strategies. Our results have shown the screening and identification of two oligonucleotide sequences (substrates) as the most promising probes for detecting Salmonella - Sal-3 and Sal-5. The detection limits for both probes were determined with the reference Salmonella Typhimurium (STM 1) and Salmonella Enteritidis (SE 1) cultures. Sal-3 has reported LOD values around 10 CFU mL for STM 1 and 10 CFU mL for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 10 CFU mL for STM 1 and 10 CFU mL for SE 1. Both selected probes have shown the capability to recognize 49 out of 51 different Salmonella serotypes tested in vitro and the most frequent serotypes in porcine mesenteric lymph nodes as a standard sample used in fattening-pig salmonellosis baseline studies. Notably, our results showed 100% correlation between nuclease detection and the PCR-InvA or ISO-6579 standard method, underlining the great potential of this innovative nucleic acids technology to be implemented as a rapid method for food safety testing.
Preliminary study on an innovative, simple mast cell-based electrochemical method for detecting foodborne pathogenic bacterial quorum signaling molecules (N-acyl-homoserine-lactones).
Jiang Donglei,Feng Dongdong,Jiang Hui,Yuan Limin,Yongqi Yin,Xu Xin,Fang Weiming
Biosensors & bioelectronics
This paper reports the a novel and simple mast cell-based electrochemical method for detecting of bacterial quorum signaling molecules, N-acylhomoserine lactones (AHLs), which can be utilized to preliminarily evaluate the toxicity of food-borne pathogenic bacteria. Rat basophilic leukemia (RBL-2H3) mast cells encapsulated in alginate/graphene oxide hydrogel were immobilized on a gold electrode, while mast cells as recognition elements were cultured in a 3D cell culture system. Electrochemical impedance spectroscopy (EIS) was utilized to record the cell impedance signal as-influenced by Pseudomonas aeruginosa quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC-HSL). The results indicated that cellular activities such as cell viability, apoptosis, intracellular calcium, and degranulation were markedly influenced by the AHLs. Importantly, the exposure of 3OC-HSL to mast cells induced a marked decrease in the electrochemical impedance signal in a dose-dependent manner. The detection limit for 3OC-HSL was 0.034μM with a linear range of 0.1-1μM. These results were confirmed via conventional cell assay and transmission electron microscope (TEM) analysis. Altogether, the proposed method appears to be an innovative and effective approach to the quantitative measurement of Gram-negative bacterial quorum signaling molecules; to this effect, it also may serve as a primary evaluation of the cytotoxicity of food-borne pathogens.
Old Friends in New Places: Exploring the Role of Extraintestinal E. coli in Intestinal Disease and Foodborne Illness.
Markland S M,LeStrange K J,Sharma M,Kniel K E
Zoonoses and public health
The emergence of new antibiotic-resistant Escherichia coli pathotypes associated with human disease has led to an investigation in terms of the origins of these pathogens. According to the Centers for Disease Control and Prevention, unspecified agents are responsible for 38.4 million of the 48 million (80%) cases of foodborne illnesses each year in the United States. It is hypothesized that environmental E. coli not typically associated with the ability to cause disease in humans could potentially be responsible for some of these cases. In order for an environmental E. coli isolate to have the ability to cause foodborne illness, it must be able to utilize the same attachment and virulence mechanisms utilized by other human pathogenic E. coli. Recent research has shown that many avian pathogenic E. coli (APEC) isolated from poultry harbour attachment and virulence genes also currently found in human pathogenic E. coli isolates. Research also suggests that, in addition to the ability to cause gastrointestinal illnesses, APEC may also be an etiological agent of foodborne urinary tract infections (FUTIs). The purpose of this article was to evaluate the evidence pertaining to the ability of APEC to cause disease in humans, their potential for zoonotic transfer along with discussion on the types of illnesses that may be associated with these pathogens.
Campylobacteriosis, Salmonellosis, Yersiniosis, and Listeriosis as Zoonotic Foodborne Diseases: A Review.
Chlebicz Agnieszka,Śliżewska Katarzyna
International journal of environmental research and public health
Zoonoses are diseases transmitted from animals to humans, posing a great threat to the health and life of people all over the world. According to WHO estimations, 600 million cases of diseases caused by contaminated food were noted in 2010, including almost 350 million caused by pathogenic bacteria. , , as well as and may dwell in livestock (poultry, cattle, and swine) but are also found in wild animals, pets, fish, and rodents. Animals, often being asymptomatic carriers of pathogens, excrete them with faeces, thus delivering them to the environment. Therefore, pathogens may invade new individuals, as well as reside on vegetables and fruits. Pathogenic bacteria also penetrate food production areas and may remain there in the form of a biofilm covering the surfaces of machines and equipment. A common occurrence of microbes in food products, as well as their improper or careless processing, leads to common poisonings. Symptoms of foodborne infections may be mild, sometimes flu-like, but they also may be accompanied by severe complications, some even fatal. The aim of the paper is to summarize and provide information on campylobacteriosis, salmonellosis, yersiniosis, and listeriosis and the aetiological factors of those diseases, along with the general characteristics of pathogens, virulence factors, and reservoirs.
Public Health Risk Associated with Botulism as Foodborne Zoonoses.
Rasetti-Escargueil Christine,Lemichez Emmanuel,Popoff Michel R
Botulism is a rare but severe neurological disease in man and animals that is caused by botulinum neurotoxins (BoNTs) produced by and atypical strains from other and non- species. BoNTs are divided into more than seven toxinotypes based on neutralization with specific corresponding antisera, and each toxinotype is subdivided into subtypes according to amino acid sequence variations. Animal species show variable sensitivity to the different BoNT toxinotypes. Thereby, naturally acquired animal botulism is mainly due to BoNT/C, D and the mosaic variants CD and DC, BoNT/CD being more prevalent in birds and BoNT/DC in cattle, whereas human botulism is more frequently in the types A, B and E, and to a lower extent, F. Botulism is not a contagious disease, since there is no direct transmission from diseased animals or man to a healthy subject. Botulism occurs via the environment, notably from food contaminated with spores and preserved in conditions favorable for growth and toxin production. The high prevalence of botulism types C, D and variants DC and CD in farmed and wild birds, and to a lower extent in cattle, raises the risk of transmission to human beings. However, human botulism is much rarer than animal botulism, and botulism types C and D are exceptional in humans. Only 15 cases or suspected cases of botulism type C and one outbreak of botulism type D have been reported in humans to date. In contrast, animal healthy carriers of group II, such as type E in fish of the northern hemisphere, and B4 in pigs, represent a more prevalent risk of botulism transmission to human subjects. Less common botulism types in animals but at risk of transmission to humans, can sporadically be observed, such as botulism type E in farmed chickens in France (1998-2002), botulism type B in cattle in The Netherlands (1977-1979), botulism types A and B in horses, or botulism type A in dairy cows (Egypt, 1976). In most cases, human and animal botulisms have distinct origins, and cross transmissions between animals and human beings are rather rare, accidental events. But, due to the severity of this disease, human and animal botulism requires a careful surveillance.
Main challenges in the control of zoonoses and related foodborne diseases in the South Mediterranean and Middle East region.
Seimenis Aristarchos,Battelli Giorgio
In the South Mediterranean and Middle East region, interactions between humans, animals, and the surrounding environment are frequently close. This fact is mainly manifested in traditional farming settings (by sedentary, semi-sedentary, and nomadic communities) as well as where livestock intensification has been introduced. A combination of complex factors in these settings (e.g. challenges in country infrastructures and cross-sectoral collaboration/ coordination, traditional habits, poor social information, etc.) contribute to the emergence and, occasionally, to the endemic pattern of zoonoses. The phenomenal growth of international travel and trade, population displacement, and unhygienic settlements has increased the speed and ease with which pathogens and vectors can cross continents and cause outbreaks and epidemics. Programmes for the prevention and control of zoonoses have been implemented in several countries in this region; however, the expected results have not always been realised. The conflicts and civil unrest affecting certain countries in this region during the last decade, together with the mass displacement of people seeking refuge, have resulted in serious epidemiological and social impacts. Zoonoses and related food-borne diseases are, indeed, a worldwide challenge, whose prevention and control mainly depend on the actions of national authorities. Once peace has been established in this region, authorities need to address the burden of these diseases through resource mobilisation, the implementation of international agencies technical guidance, and inter-country collaboration.
An overview of food safety and bacterial foodborne zoonoses in food production animals in the Caribbean region.
Guerra Maria Manuela Mendes,de Almeida Andre M,Willingham Arve Lee
Tropical animal health and production
Foodborne diseases (FBDs) in the Caribbean have a high economic burden. Public health and tourism concerns rise along with the increasing number of cases and outbreaks registered over the last 20 years. Salmonella spp., Shigella spp., and Campylobacter spp. are the main bacteria associated with these incidents. In spite of undertaking limited surveillance on FBD in the region, records related to bacterial foodborne zoonoses in food-producing animals and their associated epidemiologic significance are poorly documented, giving rise to concerns about the importance of the livestock, food animal product sectors, and consumption patterns. In this review, we report the available published literature over the last 20 years on selected bacterial foodborne zoonoses in the Caribbean region and also address other food safety-related aspects (e.g., FBD food attribution, importance, surveillance), mainly aiming at recognizing data gaps and identifying possible research approaches in the animal health sector.
Understanding the oral transmission of Trypanosoma cruzi as a veterinary and medical foodborne zoonosis.
Velásquez-Ortiz Natalia,Ramírez Juan David
Research in veterinary science
Chagas disease is a neglected tropical disease transmitted by the protozoan Trypanosoma cruzi that lately has been highlighted because several outbreaks attributed to oral transmission of the parasite have occurred. These outbreaks are characterized by high mortality rates and massive infections that cannot be related to other types of transmission such as the vectorial route. Oral transmission of Chagas disease has been reported in Brazil, Colombia, Venezuela, Bolivia, Ecuador, Argentina and French Guiana, most of them are massive oral outbreaks caused by the ingestion of beverages and food contaminated with triatomine feces or parasites' reservoirs secretions and considered since 2012 as a foodborne disease. In this review, we present the current status and all available data regarding oral transmission of Chagas disease, highlighting its relevance as a veterinary and medical foodborne zoonosis.
A brief review of foodborne zoonoses in China.
Shao D,Shi Z,Wei J,Ma Z
Epidemiology and infection
Foodborne zoonoses have a major impact on public health in China. Its booming economy and rapid socioeconomic changes have affected food production, food supplies and food consumption habits, resulting in an increase in the number of outbreaks of foodborne zoonoses. Both emerging and re-emerging foodborne zoonoses have attracted increasing national and international attention in recent years. This paper briefly reviews the main foodborne zoonoses that have had a major impact on public health over the last 20 years in China. The major causative microorganisms, including foodborne bacteria, parasites and viruses, are discussed. The prevention and control of foodborne zoonoses are difficult challenges in China. The information provided here may aid the development of effective prevention and control strategies for foodborne zoonoses.