Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Background: Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. Aim: It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. Methods: A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. Results: The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. Conclusion: This method provides a sensitive and specific approach to detect issues related to well-being and safety.

Characterization of Actinobacillus pleuropneumoniae biovar 2 isolates reportedly reacted with the serovar 4 antiserum, and development of a multiplex PCR for O-antigen typing.

We have analyzed the capsule (CPS) and the lipopolysaccharide O-Antigen (O-Ag) biosynthesis loci of twelve Spanish field isolates of Actinobacillus pleuropneumoniae biovar 2, eleven of them previously typed serologically as serovar 4 and one non-typable (NT) (Maldonado et al., 2009, 2011). These isolates have the common core genes of the type I CPS locus, sharing >98% identity with those of serovar 2. However, the former possesses the O-Ag locus as serovar 4, and the latter possesses the O-Ag locus as serovar 7. The main difference found between the CPS loci of the 11 isolates and that of serovar 2 reference strain S1536 are two deletions, one of an 8 bp sequence upstream of the coding sequence and one of 111 bp sequence at the 5' end of the cps2G gene. The deletion mutations mentioned lead to a defect in the production of CPS in these isolates, which contributed to their previous mis-identification. In order to complement the serotyping of A. pleuropneumoniae in diagnostics and epidemiology, we have developed a multiplex PCR for the comprehensive O-Ag typing of all A. pleuropneumoniae isolates.

Comparison of Giardia duodenalis point-of-care antigen faecal tests to reference laboratory assays in non-symptomatic dogs.

Giardia duodenalis is one of the most prevalent enteric parasites of dogs. Point-of-care antigen tests (POC) are rapid and do not require additional equipment, or a specialised diagnostic laboratory. The aim of this study was to compare diagnostic tests available in veterinary practices and in a diagnostic laboratory for the detection of G. duodenalis on a cohort of group-housed dogs from New South Wales, Australia. Two different POC tests were used for the detection of G. duodenalis. Laboratory tests used were the multiplexed-tandem PCR panel (MT-PCR) that includes detection of G. duodenalis DNA, and two reference tests (an in-house TaqMan real-time PCR and a direct immunofluorescence assay, DFA). Canine faecal samples (n = 40) were tested simultaneously for the detection of G. duodenalis. Using either DFA or TaqMan real-time PCR as reference tests, 77.5% (31/40) and 82.5% (33/40) of dogs tested positive, respectively. Agreement (Kappa) between the DFA and TaqMan real-time PCR was 0.84 (95% CI 0.64 to 1.00). There was substantial G. duodenalis test outcome agreement between the two POC tests, Kappa = 0.75. Combining the two POC tests yielded 77% sensitivity and 100% specificity with DFA as reference, and for TaqMan real-time PCR it was 73% sensitivity and 100% specificity. The MT-PCR was in excellent agreement with each reference test, DFA or TaqMan real-time PCR. Due to the high specificity of both POC tests, they can be confidently used as rule-in diagnostics. Confirmatory testing that detects different biological parameters such as DNA, e.g. PCR (inc. MT-PCR), should be implemented before concluding that a dog is negative for the presence of G. duodenalis.

Is Brucella excreted in cattle faeces? - Evidence from Punjab, India.

Brucellosis is a neglected zoonosis that affects animals and people in much of the underdeveloped world. The disease is endemic in cattle in Punjab, India and controlling it is a public health challenge. Dairy farmers and farm labour commonly handle cattle faeces with bare hands and personal protective equipments are not used. No studies have been conducted about the shedding of Brucella species in faeces of sero positive cattle in the state. This study aimed to isolate and identify the Brucella species from faeces of sero positive cattle in Punjab, India. Faecal samples were collected from 350 Brucella sero positive cattle in Ludhiana district of Punjab, India. Isolation was performed using a pre-enriched Brucella selective broth medium as well as Brucella selective medium agar plates containing horse serum and Brucella selective supplements. Isolates were identified using Gram staining technique and rapid slide agglutination test, and then confirmed by using bcsp31 and 16s rRNA genus specific PCR. Isolates were further identified up to species level by using Bruce-Ladder multiplex PCR. Fourteen Brucella species were isolated, all of which showed coccobacilli on gram staining, positive rapid slide agglutination test and amplification of bcsp31 and 16s rRNA genes. Of the 14 isolates, 11 were identified as Brucella abortus and 3 were identified as Brucella melitensis. The study demonstrates that animal faeces could pose a potential risk for animal and human health and faeces of seropositive cattle must be handled with care.

Molecular identification and characterisation of Mannheimia haemolytica.

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.

PCR-based methods in detection and identification of dermatophytes in dogs and cats with suspected dermatophytosis in 2021 in Poland.

Dermatophytes from Microsporum, Trichophyton and Epidermophyton genera are divided into geophilic, zoophilic and anthropophilic species which cause skin infection in humans and wide group of animals, mainly mammals. Main species causing dermatophytosis in dogs and cats are Microsporum and Trichophyton. Conventional mycological diagnostic technique includes Saburaud Dextrose Agar (SAD) and others medium cultures, 10% KOH mount and direct microscopy of hairs and scraping. Molecular diagnostic become more frequent in veterinary practice due to shortening of waiting time. In this study we based on two PCR methods. The nested PCR amplified CHS1 gene for dermatophytes detection, and multiplex PCR coding ITS1 and ITS2 fragments for species identification of detected derpatophytes. Most frequently detected species was Microsporum canis, mainly in young cats. Geophilic Microsporum gypseum and anthropophilic Trichophyton rubrum was found primarily in dogs. Molecular methods in dermatophytosis identification are rapid in contrast to routinely, long lasting culture.

Virulence gene profiles and antimicrobial susceptibility of Salmonella Brancaster from chicken.

BACKGROUND: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. OBJECTIVE: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. METHODS: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). RESULTS: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (ß-lactamase temoneira [blaTEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. CONCLUSION: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

Clinical evaluation of a dermatophyte RT-PCR assay and its impact on the turn-around-time: A prospective study.

Onychomycosis is an important public health problem whose prevalence continues to grow and impact public health at several levels. Nevertheless, today the main diagnostic methods used in routine practice have many drawbacks. The aim of this study was to evaluate, for the first time, the clinical performance of a new multiplex polymerase chain reaction (PCR) (Novaplex®) in the identification of the causative agent on nail samples, and its impact on the turnaround time, compared to our traditional laboratory methods. From June 2022 to December 2022, all nail samples sent to our laboratory for suspected onychomycosis were included in this prospective study. We collected for each sample the results obtained with the Novaplex® PCR method and with the traditional direct microscopy examination and culture. Each discordant result was checked using a third method, which is another PCR method (DermaGenius® kit) as a resolver. For culture-positive samples, a turnaround time was calculated and compared to the one obtained with the Novaplex® method. A total of 131 samples were included. Among them, 5 were positive (3.8%) on direct microscopy, 33 were positive (25.2%) after culture, and 98 were negative (74.8%). All positive (n = 33) and negative (n = 69) cultures were also positive/negative with the Novaplex® PCR. Twenty-nine samples were positive with the Novaplex® method but negative with culture (discordant results). The percentage agreement between the culture and the Novaplex® methods was 77.9% (102 out of 131). While tested with the resolver (DermaGenius® PCR), 28 out of 29 discordant results were similarly found positive. The percentage agreement between the two PCR methods (Novaplex® and DermaGenius®) was 96.6%. The Novaplex® PCR method evaluated proved to be very reliable and allowed the direct identification of 62 out of 131 positive samples (47.3%) with the following distribution: 79.0% of Trichophyton rubrum complex, 11.3% of Trichophyton mentagrophytes complex, 6.5% of both Trichophyton rubrum complex and Trichophyton mentagrophytes complex, and 3.2% of Candida albicans. The median time [± 95% CI] for positive culture (between incubation and validation of the final identification) was 15 [12-23] days, while the turnaround time for the Novaplex® method adapted to our clinical laboratory routine is ≤7 days. Laboratory confirmation of onychomycosis is crucial and should always be obtained before starting treatment. The evaluated PCR method offered a rapid, reliable, robust, and inexpensive method of identification of the causative agent compared to traditional methods.

The aim of this study was to evaluate the clinical performance of a multiplex PCR in the identification of the causative agent of onychomycosis on nail samples, and its impact on the turnaround time, compared to our traditional laboratory methods. This new method is rapid, reliable, robust, and inexpensive.

Application of a Multiplex PCR Assay for Molecular Identification of Pathogenic and Non-Pathogenic Leptospires based on lipL32 and 16S rRNA Genes.

Leptospirosis is a serious zoonotic infection and the most prevalence disease is in the tropical and subtropical region. The definitive diagnosis of Leptospirosis, caused by spirochetes of the genus Leptospira infection is already using culture methods, serological tests such as the microscopic agglutination test (MAT) and molecular detection methods (PCR) are possible. In this study, we used multiplex PCR method for detection of pathogenic and non - pathogenic Leptospira based on lipL32 and 16S rRNA genes. All serovars were obtained from the Leptospira Reference Laboratory of Microbiology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran. The PCR product for the lipL32 and 16S rRNA genes was 272 bp and 240 bp respectively. The sensitivity amplification for the multiplex assay was 10-6 pg / µl for 16S rRNA gene and 10-4 pg / µl for lipL32 gene. The sensitivity for multiplex PCR was 10-3 pg / µl. The results supported the idea that multiplex PCR can be used to detect Leptospira samples. This method was also able to differentiate between saprophytic and pathogenic leptospires and was able to do so much easily than conventional methodologies. Due to the slow growth of Leptospira and the importance of time in diagnosis, molecular methods such as PCR are suggested.

Simultaneous and rapid detection of avian respiratory diseases of small poultry using multiplex reverse transcription-Polymerase Chain Reaction assay.

Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput.

A novel time-saving multiplex PCR assay for detecting and discriminating the most common canine Babesia species in Europe.

In Europe, most cases of canine babesiosis are caused by Babesia canis, Babesia vogeli (large piroplasms) and Babesia vulpes (small piroplasm). Molecular diagnosis is recommended due to its high sensitivity. Species identification after sequencing allows applying a rapid and efficient treatment, leading to a better prognosis; however, it is expensive and time-consuming. Thus, the objective of the present study was to develop a time-saving multiplex polymerase chain reaction (PCR) for simultaneously detecting and discriminating between large and small forms without sequence analysis. A new multiplex PCR was designed and tested using blood samples from 79 dogs showing clinical signs compatible with babesiosis which were previously analysed using blood smears and molecular methods. Multiplex PCR successfully discriminated between both Babesia groups showing bands of 700 and 890 bp for B. canis/B. vogeli and B. vulpes, respectively. No significant differences in the results of both PCR were detected and a substantial agreement between protocols (κ = 0.64) was found. Our multiplex PCR represents a reliable tool for detecting infections by the major Babesia spp. in dogs from Europe. Since no sequence analysis is required for identifying the species involved, this PCR allows the rapid administration of an appropriate treatment, thus improving the survival rate of the infected animals. In addition, it will represent a helpful tool for unravelling the real prevalence and distribution of B. vulpes and its implication in clinical cases.

Isolation, identification, molecular typing, and drug resistance of Escherichia coli from infected cattle and sheep in Xinjiang, China.

BACKGROUND: Escherichia coli infections are common in Xinjiang, a major region of cattle and sheep breeding in China. Therefore, strategies are required to control E. coli. The aim of this study was to investigate the phylogenetic groups, virulence genes, and antibiotic resistance characteristics of E. coli isolates. METHODS: In this study, 116 tissue samples were collected from the organs of cattle and sheep that were suspected of having E. coli infections between 2015 and 2019. Bacteria in the samples were identified using a biochemical identification system and amplification of 16S rRNA, and the phylogenetic groupings of E. coli isolates were determined by multiplex polymerase chain reactions. In addition, PCR detection and analysis of virulence factors, antibiotic resistance genes, and drug-resistant phenotypes of E. coli isolates were performed. RESULTS: A total of 116 pathogenic E. coli strains belonging to seven phylogenetic groups were isolated, with the majority of isolates in groups A and B1. Among the virulence genes, curli-encoding crl had the highest detection rate of 97.4%, followed by hemolysin-encoding hlyE with the detection rate of 94.82%. Antimicrobial susceptibility test results indicated that the isolates had the highest rates of resistance against streptomycin (81.9%). CONCLUSION: These characteristics complicate the prevention and treatment of E. coli-related diseases in Xinjiang.

Multiplex PCR for rapid differential diagnosis of co-prevalent species of Theileria (Theileria annulata and Theileria orientalis) in cattle.

Theileriosis is a tick-borne disease that causes enormous losses in the dairy industry. There are several species of Theileria that can infect bovines. Generally, more than one species are prevalent in any geographical area; thus, chances of co-infections are high. Differentiation of these species may not be possible by microscopic examination or serological tests. Therefore, in this study, a multiplex PCR assay was standardized and evaluated for rapid and simultaneous differential detection of two species of Theileria viz., Theileria annulata and Theileria orientalis. Species-specific primers were designed to target the merozoite piroplasm surface antigen gene (TAMS1) of T. annulata and the major piroplasm surface protein gene of T. orientalis, yielding specific amplicon of 229 bp and 466 bp, respectively. The sensitivity of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively. The simplex and multiplex PCRs were specific and showed no cross-reactivity with other hemoprotozoa for either primer. For comparative evaluation, blood samples from 216 cattle were tested by simplex and multiplex PCR for both species. Using multiplex PCR, 131 animals were found infected for theileriosis, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infections. This is the first report of T. orientalis from Haryana, India. Representative sequences of T. annulata (ON248941) and T. orientalis (ON248942) were submitted in GenBank. The standardized multiplex PCR assay used in this study was specific, sensitive, for the screening of field samples.

Identification of staphylococcal cassette chromosome mec in Staphylococcus aureus and non-aureus staphylococci from dairy cattle in Belgium: Comparison of multiplex PCR and whole genome sequencing.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

Comparison of multiplex copro PCR with coproscopy followed by PCR on recovered eggs for the detection of Echinococcus granulosus and Taenia spp. infection in dogs.

Echinococcosis and taeniasis are important helminth diseases that carry considerable impact on human and animal health. Domestic dogs and other canids are definitive hosts for several parasites of this group, including Echinococcus granulosus, Taenia multiceps, T. ovis, T. hydatigena and E. multilocularis. Detection of infection in dog populations is imperative for estimating the risk to susceptible humans and animals, and for its mitigation through prevention measures in dogs, other animals and humans. To date, identification of taeniid eggs, antigens or DNA in fecal samples are the most practical diagnostic modalities available for canine definitive hosts. Although widely used for this purpose, there is limited information comparing copro PCR and combined coproscopy-PCR protocols for the detection of taeniids. In the current study, a widely used multiplex PCR was performed on a large number of dog fecal samples using DNA extracted directly from feces. The samples were also tested by fecal flotation and coproscopy, eggs were isolated from microscopically-positive samples and extracted DNA was tested using the same multiplex PCR. The total number of taeniid positive samples detected using both methods was 46/317 (14.5%), including 10/317 (3.2%) E. granulosus positive samples. Both copro PCR and coproscopy have identified an equal number of samples as taeniid positive (n = 32). However, for the purpose of identification to species level, the copro PCR was significantly more sensitive than coproscopy followed by PCR on isolated eggs (sensitivity 0.7 vs. 0.41, p = 0.012), with 32/317 (10.1%) and 19/317 (6%) positive samples identified, respectively. The difference in identification of E. granulosus was highly apparent, as the majority of the E. granulosus positive samples (8/10) were detected by the copro PCR only. Coproscopy and egg PCR have identified 5/317 (1.6%) positive samples not detected by the copro PCR, including only a single sample (0.3%) positive for E. granulosus. Adding these positive samples to those identified by the copro PCR did not significantly improve the overall sensitivity (p = 0.074). Therefore, using both copro PCR and coproscopy in parallel may not be advantageous for taeniid detection and identification, at least until the egg PCR is further optimized and performs better. These results should be weighed against the different advantages that coproscopy based approach may offer.

Development of multiplex PCR assay for simultaneous detection of African swine fever, porcine circo and porcine parvo viral infection from clinical samples.

A diagnostic method for simultaneously detecting and distinguishing African Swine Fever (ASF), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV) in clinical specimens is critical for differential diagnosis, monitoring, and control in the field. Three primer pairs were designed and used to create a multiplex PCR assay. In addition, 356 porcine post mortem tissue samples from various parts of India's North Eastern region were tested by the developed multiplex PCR assay to demonstrate its accuracy. Using the designed primers, each of the ASF, PCV2 and PPV target genes was amplified, but no other porcine virus genes were detected. The assay's limit of detection was 102 copies/µl of PCV2, PPV, or ASFV. The detection of PCV2, PPV, and ASF in postmortem tissue samples revealed that they are co-circulating in India's North-Eastern region. The percentage positivity (PP) for PCV2, PPV and ASF single infection were 7.02% (25/356), 3.93% (14/356), and 3.37% (12/356), respectively, while the PP for PCV2& PPV co-infection was 2.80% (10/356), ASF & PCV2 co infection was 1.4% (5/356) and the ASF, PPV& PCV2 co-infection was1.40% (5/356). The results also indicate that the ASF can infect pigs alongside PCV and PPV.

A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Multiplex gel-based PCR assay for the simultaneous detection of 5 genotypes of porcine astroviruses.

Porcine astrovirus (PAstV) has been associated experimentally with diarrhea in piglets, but much more knowledge is needed about this virus. PAstV has high genetic variability, and 5 genotypes have been identified, namely PAstV1-5. To obtain information on the epidemiology of PAstV, we established a multiplex PAstV PCR assay to detect and differentiate the 5 PAstV genotypes simultaneously. The assay utilized specific primers for each genotype, producing fragments of 307, 353, 205, 253, and 467 bp, representing PAstV1-5, respectively. Our multiplex PCR assay amplified all 5 DNA fragments from single or mixed viral genomes without cross-reactions with other PAstV genotypes or other viruses in pigs. The limit of detection of the multiplex PCR assay was 5 × 102 copies/µL for PAstV1 and PAstV4, and 5 × 103 copies/µL for PAstV2, PAstV3, and PAstV5. We examined 76 pig fecal specimens with our multiplex PCR assay. PAstV was detected in 36 of 76 (47.4%) samples; ≥2 PAstVs were found in 20 of 76 (26.3%) samples. The multiplex PCR assay results were essentially the same as the results using a monoplex PAstV PCR assay, with a coincidence rate of >96%. Our multiplex PCR method provides a simple, sensitive, and specific detection tool for PAstV detection and epidemiologic surveys.