Species-specific Multiplex PCR Assay Research Articles

Widespread establishment of adventive populations of Leptopilina japonica (Hymenoptera, Figitidae) in North America and development of a multiplex PCR assay to identify key parasitoids of Drosophila suzukii (Diptera, Drosophilidae)

In recent years, there has been an increase in the adventive establishment and spread of parasitoid wasps outside of their native range. However, lack of taxonomic tools can hinder the efficient screening of field-collected samples to document the establishment and range expansion of parasitoids on continent-wide geographic scales. Here we report that Leptopilina japonica (Hymenoptera, Figitidae), a parasitoid of the globally invasive fruit pest Drosophila suzukii (Diptera, Drosophilidae), is now widespread in much of North America despite not having been intentionally introduced. Surveys in 2022 using a variety of methods detected L. japonica in 10 of 11 surveyed USA States and one Canadian Province where it was not previously known to occur. In most surveys, L. japonica was the most common species of D. suzukii parasitoid found. The surveys also resulted in the detection of Ganaspis cf. brasiliensis (Hymenoptera, Figitidae), the recently-released biological control agent of D. suzukii, in six USA States where it had not previously been found. These new detections are likely a result of intentional biological control introductions rather than spread of adventive populations. A species-specific multiplex PCR assay was developed as a rapid, accurate and cost-effective method to distinguish L. japonica, G. cf. brasiliensis, the closely-related cosmopolitan parasitoid Leptopilina heterotoma (Hymenoptera, Figitidae) and other native parasitoid species. This dataset and the associated molecular tools will facilitate future studies of the spread and ecological impacts of these introduced parasitoids on multiple continents.

Detection of chicken and turkey in different beef matrix by species-specific multiplex PCR assay

Meat is food compatible with healthy nutrition. It is a useful nutrient at all stages of life and helps to meet the required nutritional intake. Consumers rely on food labelling to decide whether the purchased meat product is safe and reliable. Therefore, it is important to guarantee consumer consumption without fraud. Different methods have been used for this subject. Elisa test has the method adopted by Moroccan reference laboratories, following the spread of scandals concerning the fraud of meat products in Casablanca in 2013. At the same time, other molecular methods have been in development, principally polymerase chain reaction. This work aims to optimize a multiplex PCR test based on a sensitive, specific, and reliable technique to identify the contamination of beef products by chicken and turkey meat and prevent their adulteration. Mitochondrial primers that specifically amplify the genetic regions of cytochrome b and 16S rRNA are used. These primers sequences did not cross-react with DNA isolated from horses, sheep, goats, pigs and dogs. Moreover, this method can detect up to 0,05 ng of DNA per reaction. Verification of the forty samples of commercial meat by this method has shown that almost half of the samples contain unreported meat. Therefore, regulatory authorities can use this easily applicable test in a laboratory with excellent analytical precision in inspections, law enforcement and regulatory compliance.

Molecular survey of Babesia parasites in Kenya: first detailed report on occurrence of Babesia bovis in cattle

BackgroundAmong protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers.MethodsA species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis.ResultsA total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97–100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties.ConclusionThese findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established.Graphical Abtract

Rapid molecular identification of Trichogramma (Hymenoptera: Trichogrammatidae) parasitizing the eggs of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae) in Serbia

BackgroundThe European corn borer (ECB), Ostrinia nubilalis (Hübner, 1796) (Lepidoptera: Crambidae), is the major pest of maize (Zea mays Linnaeus, 1753) in Serbia. One potential method for managing this pest is the augmentative release of naturally occuring egg parasitoids of the genus Trichogramma. The first step in this process is accurately identifying the naturally occuring species and estimating their natural distribution and abundance. Molecular identification, based on differences in DNA sequences, has commonly been employed for the identification of Trichogramma species. A simple, quick, and accurate molecular assay is urgently required for the identification of two common Trichogramma species, associated with ECB in Serbia: T. brassicae Bezdenko, 1968 and Trichogramma evanescens Westwood, 1833. Such an assay will facilitate an expansive survey of resident populations of Trichogramma associated with ECB across agricultural growing regions of Vojvodina province.ResultsA species-specific multiplex PCR assay for the 2 species was developed and validated that assay using a sample of 79 parasitoid wasps reared from ECB egg masses collected from sample sites across Vojvodina province. Trichogramma brassicae was confirmed as the dominant egg parasitoid of ECB in this region, accounting for 77 of the 79 wasps (97.47%). The remaining 2 were confirmed as T. evanescens. Trichogramma brassicae was detected at all 12 sample sites, while T. evanescens was detected at only 2 plots, Mokrin and Nakovo.ConclusionsThe species-specific multiplex PCR assay presented herein can provide the basis of a quick, cheap, and reliable means for identifying the species of Trichogramma that parasitize ECB egg masses in Serbia. Two currently documented species, T. brassicae and T. evanescens, are readily diagnosed by the size of the PCR product they produce in the assay. Any additional species are expected to not produce a band of a diagnostic size. Such species would subsequently be identified by sequencing, which may also allow them to be promptly incorporated into a revised assay.

Identification and evaluation of antimicrobial resistance of enterococci isolated from raw ewes' and cows' milk collected in western Sicily: a preliminary investigation.

The present work was carried out to investigate the Antimicrobial Resistance (AMR) of enterococci isolated from raw ewes’ and cows’ milk. The samples were collected from eighteen semi-extensive dairy sheep and cow farms throughout western Sicily. Plate counts, carried out on Rapid Enterococcus Agar commonly used to detect food enterococci, revealed a maximal enterococcal concentration of approximately 4.58 Log Colony Forming Unit (CFU)/mL. Colonies were isolated and differentiated based on genetic analysis by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Thirty-eight different strains were identified. Analysis by a species-specific multiplex PCR assay grouped the strains into three Enterococcus species such as Enterococcus durans, Enterococcus faecalis and Enterococcus faecium. The 38 strains were also investigated for their antimicrobial resistance by a phenotypic approach. All 38 Enterococcus displayed resistance to at least one or more of the antimicrobials tested confirmed that the dairy enterococci could be a vector for the dissemination of antimicrobial resistance. This work showed that enterococci with AMR traits are commonly present in semiextensive dairy sheep and cow farms of western Sicily pointed out the relevance of informing dairy makers and veterinary regarding the antimicrobial use in order to mitigate problems of public health and veterinary medicine.

Molecular detection of adulteration in commercial buffalo meat products by multiplex PCR assay

Abstract Authentication of commercial buffalo meat products has become a market concern. This study intended to develop and validate a highly species-specific multiplex PCR assay for authentication of buffalo meat products. Four pairs of species-specific primers were used to target mitochondrial cytochrome oxidase I (COI) gene. The assay generated the expected PCR products of 313, 255, 294 and 177 bp for buffalo meat, cattle meat, pork meat and duck meat, respectively. The multiplex PCR assay was sensitive enough to detect 1 pg pure DNA and 0.1% (w/w) adulterated meat under mixed matrices. Market survey revealed about 35.3% of buffalo meat products are adulterated with cattle meat, pork meat or duck meat in China. The adulteration was found in all food product types including minced meat, frozen rolls, boiled meat, meat ball, vacuum-packed meat and jerky. These findings showed that multiplex PCR assay are potentially reliable techniques for detection of adulteration in raw and processed buffalo meat products.

Assessment of the probiotic potential of lactic acid bacteria isolated from kefir grains: evaluation of adhesion and antiproliferative properties in in vitro experimental systems

The main objective of this study was to isolate lactic acid bacteria from kefir grains and investigate their probiotic potential. In this study, 48 bacterial strains were isolated from kefir grains, whereas 39 strains were categorized to the genus Lactobacillus. Evaluation of the probiotic potential of the isolated stains was performed, including resistance to low pH, tolerance to pepsin, pancreatin and bile salts, and antibiotic resistance. In addition, evaluation of adhesion and antiproliferative properties in in vitro experimental systems was also conducted. Strains SP2 and SP5 that displayed the best performance in the conducted in vitro tests were selected for further studies. Firstly, genotypic identification of the two strains was performed by partial 16S rRNA gene sequencing, BLAST analysis, and species-specific multiplex PCR assay. The two strains were confirmed to be Pediococcus pentosaceus SP2 and Lactobacillus paracasei SP5. Then, the adhesion properties of the two strains were examined in vitro. Both strains displayed substantial adherence capacity to HT-29 human colon cancer cells. Moreover, a significant decrease of HT-29 cell growth after treatment with viable P. pentosaceus SP2 or L. paracasei SP5 was recorded. In addition, downregulation of anti-apoptotic genes and over-expression of cell cycle–related genes was recorded by real-time PCR analysis. Treatment with conditioned media of the two strains also caused significant reduction of cancer cell proliferation in a time- and concentration-dependent manner. P. pentosaceus SP2 and L. paracasei SP5 displayed the best probiotic properties that exerted substantial adherence on human colon cancer cells as well as significant anti-proliferative properties.

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.

Revisiting Stagonosporopsis species associated with chrysanthemum and pyrethrum ray blight

Ray blight is a destructive disease of Asteraceae affecting chrysanthemum and pyrethrum industries worldwide. Three morphologically similar but phylogenetically distinct species of the family Didymellaceae; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti are associated with the disease. Despite their close evolutionary relationship and cross host pathogenicity, these species have marked differences in their biology and epidemiology. Stagonosporopsis chrysanthemi and S. inoxydabilis are both homothallic with a MAT locus that carries both mating type genes. Ascomata play a major role in survival and dispersal of S. chrysanthemi, contributing to the onset of ray blight epidemics on chrysanthemum. However, S. tanaceti is either asexual or heterothallic due to the presence of only MAT1-2 idiomorph in its genome. Morphological similarity of the species causing ray blight on various Asteraceae, multiple changes in their taxonomy and historical confusion with other Phoma-like species have resulted in lack of clarity in their taxonomy, host range and global distribution. Host specificity studies and reports on global distribution of S. chrysanthemi published before its separation from S. inoxydabilis should be treated with caution. A recently developed species-specific multiplex PCR assay provides a rapid and robust tool to study the global distribution of these important quarantine pathogens. When global populations from cultivated and/or wild Asteraceae are available, population genetic analyses may aid in understanding the origin, evolutionary history and global migration patterns of the Stagonosporopsis spp. associated with ray blight of Asteraceae.

Molecular barcoding of venomous snakes and species-specific multiplex PCR assay to identify snake groups for which antivenom is available in Thailand.

DNA barcodes of mitochondrial COI and Cytb genes were constructed from 54 specimens of 16 species for species identification. Intra- and interspecific sequence divergence of the COI gene (10 times) was greater than that of the Cytb gene (4 times), which suggests that the former gene may be a better marker than the latter for species delimitation in snakes. The COI barcode cut-off scores differed by more than 3% between most species, and the minimum interspecific divergence was greater than the maximum intraspecific divergence. Clustering analysis indicated that most species fell into monophyletic clades. These results suggest that these species could be reliably differentiated using COI DNA barcodes. Moreover, a novel species-specific multiplex PCR assay was developed to distinguish between Naja spp, Ophiophagus hannah, Trimeresurus spp, Hydrophiinae, Daboia siamensis, Bungarus fasciatus, and Calloselasma rhodostoma. Antivenom for these species is produced and kept by the Thai Red Cross for clinical use. Our novel PCR assay could easily be applied to venom and saliva samples and could be used effectively for the rapid and accurate identification of species during forensic work, conservation study, and medical research.

Risks of suffering tick-borne diseases in sheep translocated to a tick infested area: A laboratory approach for the investigation of an outbreak

This study was designed to investigate an outbreak of high mortality that occurred in naïve Assaf sheep introduced into a Latxa sheep flock in the Basque Country, a region where piroplasmosis is endemic. To identify the causes of this outbreak, a panel of different methods, including traditional pathological, biopathological and parasitological analyses combined with recently developed molecular methods, was used. These novel molecular methods included a multiplex real-time PCR assay to screen for the presence of the most important tick-borne pathogens (piroplasms and anaplasmas), followed by a second species-specific multiplex real-time PCR assay for the identification of Anaplasma-positive samples. The identification of piroplasm-positive samples was carried out by a multiplexed microsphere-based suspension array using a Luminex® xMAP technology-based procedure. Anaplasmas and/or piroplasms were detected in 7/10 lambs and 11/13 ewes, with Babesia ovis being detected in 12 of the 23 animals, Theileria ovis in 6 and Anaplasma ovis in 4, both as single and mixed infections. Most of the animals infected with B. ovis had a marked decrease in the values of the red blood cell parameters. Ticks collected from the animals were identified as Riphicephalus bursa, recognised vector of B. ovis. Other haemolytic pathologies (clostridial disease, copper poisoning and leptospirosis) were ruled out and, considering all clinical, laboratory and epidemiological data, babesiosis by B. ovis was diagnosed. A detailed description of the clinical outcome, with ca. 60% of mortality, laboratory results and epidemiological findings are provided. The implications of the introduction of naïve animals into a piroplasmosis endemic area are discussed.

Species-specific multiplex real-time PCR assay for identification of deer and common domestic animals

A multiplex real-time PCR method for discriminating deer and common domestic species, including cattle, goat, horse, donkey, pig, and chicken was developed. Species-specific primer pairs were designed and used to produce different size DNA fragments with diverse melting temperature (T m ) values. The specificity and sensitivity of these primer pairs were separately confirmed using simplex real-time PCR analysis. Multiplex real-time PCR analysis was performed using combined primers, yielding distinct melting curve profiles for each species. The sensitivity limit of the multiplex PCR method was evaluated. Trace DNA of other species in deer DNA could be identified. Common deer products, including blood, meat, and antler were tested using this multiplex PCR method and different species of deer and domestic animals were identified. The rapid multiplex real-time PCR assay described herein is a specific, sensitive, and reliable method for high-throughput authentication of deer and domestic animal products.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams

BackgroundInfectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections.ResultsThe multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples.ConclusionsThe species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Adaptation of a species-specific multiplex PCR assay for the identification of blood meal source in Culicoides (Ceratopogonidae: Diptera): applications on Palaearctic biting midge species, vectors of Orbiviruses

Culicoides are small biting midges involved worldwide in the transmission of bluetongue and African horse sickness viruses. Feeding behaviours of Palaearctic biting midge species and their spatio-temporal dynamics remain unclear at the specific level. Three multiplex species-specific PCR-based assays were developed and used to identify blood meal source of engorged females of Palaearctic midge species of veterinary interest. Species-specific primers of potential hosts from livestock, domestic animals and wildlife (cattle, goat, sheep, red deer, roe deer, chamois, dog, pig, cat, horse) were designed and multiplexed from the mitochondrial cytochrome b gene. The assays also make possible to identify whether multiple blood meals have been taken. The first results from several Culicoides populations sampled in France highlight the utility of this valuable diagnostic tool combined with species identification assays, and suggest that most of the Culicoides species may have an opportunistic feeding behaviour regarding the host distribution and density. Noteworthy is the peculiar trophic behaviour of Culicoides chiopterus showing clear trends to cattle. Information on host preference and feeding behaviours are crucial for a better understanding of vector–host interactions and disease epidemiology.

Development and Validation of a Multiplex PCR for Detection of Scedosporium spp. in Respiratory Tract Specimens from Patients with Cystic Fibrosis

The emergence of Scedosporium infections in diverse groups of individuals, which are often treatment refractory, warrants timely and accurate laboratory diagnosis. Species- or group-specific primers based on internal transcribed spacer (ITS) sequence polymorphisms were designed for Scedosporium aurantiacum, Scedosporium dehoogii, Scedosporium prolificans, Pseudallescheria boydii species complex (former clade 5)/Pseudallescheria apiosperma (formerly classified as S. apiospermum sensu lato) and Pseudallescheria minutispora. Primers for S. aurantiacum, S. prolificans, and P. boydii species complex/P. apiosperma were incorporated into a multiplex PCR assay for the detection and identification of the three major clinically important Scedosporium species and validated using sputum specimens collected from patients seen at a major Australian cystic fibrosis clinic. The multiplex PCR assay showed 100% specificity in identifying the three major clinically relevant Scedosporium species from pure culture. When evaluated using DNA extracts from sputa, sensitivity and specificity of the multiplex PCR assay were 62.1% and 97.2%, respectively. This highly species-specific multiplex PCR assay offers a rapid and simple method of detection of the most clinically important Scedosporium species in respiratory tract specimens.

Simultaneous identification of the Anopheles funestus group and Anopheles longipalpis type C by PCR-RFLP

BackgroundAnopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the An. funestus group, the genomic DNA of Anopheles longipalpis type C specimens can be amplified with the Anopheles vaneedeni and Anopheles parensis primers from this assay. The standard, species-specific An. funestus group PCR, results in the amplification of two fragments when An. longipalpis type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between An. vaneedeni and An. parensis. Anopheles longipalpis type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the An. funestus group, such as An. funestus, An. funestus-like and An. parensis, are included. The present study provides a multiplex assay that will identify An. longipalpis along with other common members of the African An. funestus group, including Anopheles leesoni.MethodsA total of 70 specimens from six species (An. funestus, An. funestus-like, An. parensis, Anopheles rivulorum, An. vaneedeni and An. leesoni) in the An. funestus group and An. longipalpis type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database.ResultsThe enzyme, EcoRI digested only An. longipalpis type C and An. funestus-like after the species-specific An. funestus group PCR assay. The An. longipalpis and An. funestus-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for An. longipalpis type C and two fragments, 375 bp and 15 bp for An. funestus-like.ConclusionsAn RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.

Brucella inopinata sp. nov., isolated from a breast implant infection

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed.

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

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

Evaluation of a Cytolethal Distending Toxin (cdt) Gene‐Based Species‐Specific Multiplex PCR Assay for the Identification of Campylobacter Strains Isolated from Poultry in Thailand

We have recently developed a cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR assay for identifying Campylobacter jejuni, C. coli and C. fetus. In the present study, the applicability of this assay was evaluated with 34 Campylobacter-like organisms isolated from poultry in Thailand for species identification and was compared with other assays including API Campy, 16S rRNA gene sequence, and hippuricase (hipO) gene detection. Of the 34 strains analyzed, 20, 10 and 1 were identified as C. jejuni, C. coli, and Arcobacter cryaerophilus, respectively, and 3 could not be identified by API Campy. However, 16S rRNA gene analysis, showed that all 34 strains are C. jejuni/coli. To discriminate between these 2 species, the hipO gene, which is specifically present in C. jejuni, was examined by PCR and was detected in 20 strains, which were identified as C. jejuni by API Campy but not in the remaining 14 strains. Collective results indicated that 20 strains were C. jejuni whereas the 14 strains were C. coli. When the cdt gene-based multiplex PCR was employed, however, 19, 20 and 19 strains were identified as C. jejuni while 13, 14 and 13 were identified as C. coli by the cdtA, cdtB and cdtC gene-based multiplex PCR, respectively. Pulsed-field gel electrophoresis revealed that C. jejuni and C. coli strains analyzed are genetically diverse. Taken together, these data suggest that the cdt gene-based multiplex PCR, particularly cdtB gene-based multiplex PCR, is a simple, rapid and reliable method for identifying the species of Campylobacter strains.

P556 Optimisation of afiow cytometry protocol for detection of Cryptosporidium parvum in hospital tap water and human stools

This study was designed to investigate an outbreak of high mortality that occurred in naïve Assaf sheep introduced into a Latxa sheep flock in the Basque Country, a region where piroplasmosis is endemic. To identify the causes of this outbreak, a panel of different methods, including traditional pathological, biopathological and parasitological analyses combined with recently developed molecular methods, was used. These novel molecular methods included a multiplex real-time PCR assay to screen for the presence of the most important tick-borne pathogens (piroplasms and anaplasmas), followed by a second species-specific multiplex real-time PCR assay for the identification of Anaplasma-positive samples. The identification of piroplasm-positive samples was carried out by a multiplexed microsphere-based suspension array using a Luminex® xMAP technology-based procedure. Anaplasmas and/or piroplasms were detected in 7/10 lambs and 11/13 ewes, with Babesia ovis being detected in 12 of the 23 animals, Theileria ovis in 6 and Anaplasma ovis in 4, both as single and mixed infections. Most of the animals infected with B. ovis had a marked decrease in the values of the red blood cell parameters. Ticks collected from the animals were identified as Riphicephalus bursa, recognised vector of B. ovis. Other haemolytic pathologies (clostridial disease, copper poisoning and leptospirosis) were ruled out and, considering all clinical, laboratory and epidemiological data, babesiosis by B. ovis was diagnosed. A detailed description of the clinical outcome, with ca. 60% of mortality, laboratory results and epidemiological findings are provided. The implications of the introduction of naïve animals into a piroplasmosis endemic area are discussed.