Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Desarrollo de un ensayo de PCR múltiple para la diferenciación rápida de los virus de la viruela aviar y la viruela de paloma. El objetivo de este estudio fue desarrollar un ensayo de PCR múltiple capaz de diferenciar rápidamente dos especies principales de Avipoxvirus (APV) (viruela del pollo), el Fowlpox virus (FWPV) y el Pigeonpox virus (PGPV), (viruela de la gallina), que causan enfermedades en especies de aves. A pesar de la importancia de un ensayo de diferenciación rápida, no existe ningún ensayo que pueda diferenciar las especies de APV sin secuenciación. Para lograr esto, se seleccionaron fragmentos blanco de ADN específicos de especie del gene fpv122 de FWPV y el gene HM89_gp120 de Pigeonpox virus, que son únicos para cada genoma. Nueve muestras recolectadas de pollos, palomas y un pavo que no fueron vacunados con lesiones típicas de la viruela se identificaron genéticamente como FWPV y PGPV. Los iniciadores diseñados y los fragmentos de ADN blanco se validaron mediante análisis in silico mediante la herramienta de búsqueda de alineación local básica de nucleótidos (BLAST). El ensayo de PCR múltiple consistió en iniciadores específicos de especie y cebadores PanAPV previamente descritos (específicos de género) y fue capaz de diferenciar entre Fowlpox virus y Pigeonpox virus, de acuerdo con los resultados filogenéticos. Este estudio representa la primera diferenciación exitosa de los genomas de Fowlpox virus y Pigeonpox virus utilizando una prueba de PCR múltiple convencional. Este ensayo tiene el potencial de facilitar el diagnóstico rápido y el control de las infecciones por Avipoxvirus.

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 A TRIPLEX REAL-TIME PCR ASSAY TO DETECT ECHINOCOCCUS SPECIES IN CANID FECAL SAMPLES.

Echinococcosis is a zoonotic disease with great significance to public health, and appropriate detection and control strategies should be adopted to mitigate its impact. Most cases of echinococcosis are believed to be transmitted by the consumption of food and/or water contaminated with canid stool containing Echinococcus spp. eggs. Studies assessing Echinococcus multilocularis, Echinococcus granulosus sensu stricto, and Echinococcus shiquicus coinfection from contaminated water-derived, soil-derived, and food-borne samples are scarce, which may be due to the lack of optimized laboratory detection methods. The present study aimed to develop and evaluate a novel triplex TaqMan-minor groove binder probe for real-time polymerase chain reaction (rtPCR) to simultaneously detect the 3 Echinococcus spp. mentioned above from canid fecal samples in the Qinghai-Tibetan Plateau area (QTPA). The efficiency and linearity of each signal channel in the triplex rtPCR assay were within acceptable limits for the range of concentrations tested. Furthermore, the method was shown to have good repeatability (standard deviation ≤0.32 cycle threshold), and the limit of detection was estimated to be 10 copies plasmid/µl reaction. In summary, the evaluation of the present method shows that the newly developed triplex rtPCR assay is a highly specific, precise, consistent, and stable method that could be used in epidemiological investigations of echinococcosis.

Simultaneous detection and differentiation of three oncogenic viral diseases of chicken by use of multiplex PCR.

Avian oncogenic or tumor diseases are common in poultry industry causing significant economic loss. Marek's disease (MD), avian leukosis (AL) and Reticuloendotheliosis (RE) are the three major viral oncogenic infections that are difficult to differentiate with gross lesions. Multiplex PCR for simultaneous detection and differentiation of these three viruses was developed and validated. The primers targeting the genes of pp38, pol and LTR for MDV, ALV and REV were designed to yield 206, 429, and 128 bp, respectively. The sensitivity of the PCR primers was checked with serial dilution of positive template DNA for each virus and found to be in the range of 10-5 to 10-7 of 1 µg/µl of initial template DNA. Out of 114 suspected tumor samples screened, 8 samples were positive for MDV, 13 samples were positive for ALV and 31 samples positive for REV. Five samples were positive for both MD and ALV; 3 samples were positive for MD and REV and 25 samples were positive for ALV and REV. Eight samples were positive for all three viruses. Multiplex PCR demonstrated to be a useful technique for simultaneous, rapid detection and differentiation of major tumor causing and immunosuppressive viral diseases of chicken.

Reprint of: The European badger, Meles Meles, as a new host for Trichinella britovi in Romania.

The European badger, Meles meles (Carnivora, Mustelidae) is a widespread opportunistic omnivorous mammal. Its food spectrum comprises a wide variety of plants and small animals, occasionally including rodents, such as mice or rats. Considering that rodents are known to play a key role in the life cycle of Trichinella spp., the aims of this study were to investigate the occurrence of these parasites in badgers from Romania and to identify the species. Overall, 61 badgers originating from 14 counties were examined by trichinoscopy and artificial digestion. For species determination, the positive muscle samples, and the larvae recovered from the artificial digestion were used for DNA isolation, and further processed by multiplex PCR. A single badger, originating from Sibiu County, Central Romania, was found positive for Trichinella spp. Five cysts were identified using trichinoscopy: four in the diaphragm and one in the foreleg muscles. Artificial digestion revealed an infection rate of 70 larvae/100 g of muscle. The PCR indicated the occurrence of T. britovi, which is the most commonly detected species in wild carnivores in temperate areas. Although T. britovi has previously been reported in Romania, this represents the first report of its occurrence in the European badger in the country. However, the low prevalence indicates a minor reservoir role of this species.

Standardization of a rapid quadruplex PCR method for the simultaneous detection of bovine, buffalo, Salmonella spp, and Listeria monocytogenes DNA in milk / [Padronização de um método rápido de PCR quadriplex para a detecção simultânea de DNA de bovino, bubalino, Salmonella spp. e Listeria monocytogenes em leite]

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.

O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.

Research Note: Simultaneous detection of infectious laryngotracheitis virus, fowlpox virus, and reticuloendotheliosis virus in chicken specimens.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

Development of tetra-primer ARMS-PCR protocol to genotype the prolactin receptor SNP 39136666 and assessment of this SNP in Brazilian locally adapted cattle breeds / [Desenvolvimento de protocolo de tetra-primer ARMS- PCR para genotipagem do SNP 39136666 do receptor da prolactina e avaliação desse SNP em raças bovinas brasileiras localmente adaptadas]

As raças taurinas de origem ibérica Limonero e Carora (Bos primigenius taurus) possuem o fenótipo de pelo curto, liso e com baixa densidade folicular, o que confere a esses animais maior tolerância térmica e melhor produtividade em regiões quentes. Diferentes mutações associadas a esse fenótipo foram descritas no gene do receptor de prolactina PRLR, localizado no cromossomo bovino BTA20. Uma mutação recentemente encontrada é a substituição do nucleotídeo C por T, SNP 39136666 (p. R497*), no exon 11, que gera um códon de parada e, consequentemente, uma menor isoforma desse receptor. Neste trabalho, desenvolveu-se um protocolo rápido e de baixo custo para detecção desse SNP, utilizando-se a técnica de tetra-primer ARMS-PCR. Assim, foi possível detectar essa mutação nas raças brasileiras de origem ibérica localmente adaptadas: Caracu, Crioulo Lageano, Mocho Nacional e Pantaneiro. O alelo T foi mais frequente na raça Caracu (80%), enquanto o alelo C foi mais frequente na raça Crioulo Lageano (84%). Essa simples metodologia pode ser usada para genotipar esse SNP e ajudar na aplicação dessas informações moleculares em programas de melhoramento focados na tolerância térmica em bovinos taurinos e seus mestiços.(AU)

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein.

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID50/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China.

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV-A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT-PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV-A, and the polyprotein genes of PKV and PSaV. This multiplex RT-PCR could specifically detect TGEV, PEDV, PDCoV, PRV-A, PKV and PSaV without cross-reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 100 -101  ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT-PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV-A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV-A, PRA-V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV-A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT-PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China.

Detection of fraud by addition cow's milk in cheese buffalo and its connection with seasonality / Detecção de fraude por adição de leite de vaca em queijo de búfala e a relação com a sazonalidade

The seasons influence the production of buffalos’ milk. Because of this, the producers may produce a mixture of buffalo and bovine milk during cheese production in periods of low production. Therefore, the present work aimed to investigate fraud in buffalo cheese and the relationship between seasonality and different physicochemical properties of buffalo cheeses produced and marketed in eastern Amazonia. We obtained commercial samples of buffalo cheese during two Amazonian climatic periods from commercial points of Marajó-Pará, Brazil. After collection, there were lipid, protein, ash, and humidity analyses. Determination of carbohydrates and energy values was also performed for the nutritional characterization of samples, as well as for mPCR analysis to detect buffalo and/or bovine DNA. DNA extraction protocol of the samples was standardized and two pairs were used for the mPCR reaction, amplifying fragments of approximately 220 bp for Bubalus bubalis DNA and 346 bp fragments for Bos taurus DNA. Among the samples acquired in the rainy season, we observed that 33% were inadequately labeled, indicating fraud from cow’s milk incorporation and fraud from substitution of raw material. From the nine samples obtained in the dry season, all the samples showed cow’s milk incorporation fraud. The highest fraud rate coincided with the period of low milk production from buffalo and there was a difference in composition between fraudulent and non-fraudulent cheeses. Therefore, seasonality influences increase in cattle milk for the production of buffalo cheese, and this adulteration may decrease the nutritional content of the product.

As estações climáticas influenciam a produção de leite de búfala. Isso pode levar os produtores a misturarem os leites de búfala e bovino durante a produção de queijo em períodos de baixa produção. Portanto, o presente trabalho teve como objetivo verificar fraudes em queijo de búfala, a relação com a sazonalidade e as diferenças físico-químicas de queijos de origem bubalina, produzidos e comercializados no leste da Amazônia. Foram coletadas amostras comerciais de queijo de búfala em dois períodos climáticos da Amazônia em pontos comerciais do Marajó-Pará, Brasil. Após a coleta foram realizadas análises de lipídios, proteínas, cinzas e umidade. A determinação dos carboidratos e do valor energético também foi feita para a caracterização nutricional das amostras, bem como a análise de mPCR para a detecção de DNA de búfalo e/ou bovino. Para isso, padronizou-se um protocolo de extração de DNA das amostras e utilizou-se dois pares na reação mPCR, amplificar fragmentos de aproximadamente 220 pb para o DNA de Bubalus bubalis e fragmentos de 346 pb para o Bos taurus. Entre as amostras adquiridas na estação chuvosa, observou-se que 33% foram rotuladas inadequadamente, caracterizando fraude por incorporação de leite de vaca e fraude por substituição de matéria-prima. Das 9 amostras coletadas no período seco, todas as amostras apresentaram fraude na incorporação do leite de vaca. Este estudo revelou que a maior taxa de fraude coincide com o período de baixa produção de leite e que há uma diferença na composição entre queijos fraudulentos e não fraudulentos. Portanto, a sazonalidade influencia no acréscimo de leite de bovinos na produção de queijo de búfala e que esta adulteração pode diminuir o conteúdo nutricional do produto.

Development and use of a triplex real-time PCR assay for detection of three DNA viruses in psittacine birds.

Aves polyomavirus 1, psittacine beak and feather disease virus, and psittacid herpesvirus 1 are important pathogens of psittacine birds with the potential to cause substantial morbidity and mortality. Using publically available nucleotide sequences, we developed and validated a triplex real-time PCR (rtPCR) assay to rapidly detect these 3 viruses. The assay had high analytical sensitivity, detecting <6 copies of viral DNA per reaction, and 100% analytical specificity, showing no cross-reactivity with 59 other animal pathogens. Archived formalin-fixed, paraffin-embedded tissues from psittacine birds diagnosed at postmortem as infected with each of the viruses as well as virus-negative birds were used to validate the utility of the assay. Birds were selected for the positive cohort if they showed histologic evidence of infection (i.e., characteristic inclusion bodies in tissues); birds in the negative cohort had final diagnoses unrelated to the pathogens of interest. The triplex rtPCR assay confirmed 98% of histopathology-positive cases, and also identified subclinical infections that were not observed by histologic examination, including coinfections. Birds that tested positive only by rtPCR had significantly higher cycle threshold values compared to those with histologic evidence of infection. Positive, negative, and overall percentage agreements as well as the kappa statistic between the results of the assay and histopathology were high, demonstrating the usefulness of the assay as a tool to confirm disease diagnoses, and to improve detection of subclinical infections.

Development and application of multiplex PCR method for simultaneous detection of seven viruses in ducks.

BACKGROUND: Major viruses, including duck-origin avian influenza virus, duck-origin Newcastle disease virus, novel duck parvovirus, duck hepatitis A virus, duck Tembusu virus, fowl adenovirus, and duck enteritis virus, pose great harm to ducks and cause enormous economic losses to duck industry. This study aims to establish a multiplex polymerase chain reaction (m-PCR) method for simultaneous detection of these seven viruses. RESULTS: Specific primers were designed and synthesized according to the conserved region of seven viral gene sequences. Then, seven recombinant plasmids, as the positive controls, were reconstructed in this study. Within the study, D-optimal design was adopted to optimize PCR parameters. The optimum parameters for m-PCR were annealing temperature at 57 °C, Mg2+ concentration at 4 mM, Taq DNA polymerase concentration at 0.05 U/µL, and dNTP concentration at 0.32 mM. With these optimal parameters, the m-PCR method produced neither cross-reactions among these seven viruses nor nonspecific reactions with other common waterfowl pathogens. The detection limit of m-PCR for each virus was 1 × 104 viral DNA copies/µL. In addition, the m-PCR method could detect a combination of several random viruses in co-infection analysis. Finally, the m-PCR method was successfully applied to clinical samples, and the detection results were consistent with uniplex PCR. CONCLUSION: Given its rapidity, specificity, sensitivity, and convenience, the established m-PCR method is feasible for simultaneous detection of seven duck-infecting viruses and can be applied to clinical diagnosis of viral infection in ducks.

Multiplex TaqMan qPCR assay for specific identification of encapsulated Trichinella species prevalent in North America.

BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.

A multiplex xTAG assay for the simultaneous detection of five chicken immunosuppressive viruses.

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.

Pathological and molecular identification of porcine cysticercosis in Maharashtra, India.

Porcine cysticercosis, caused by metacestodes of Taenia solium is an important emerging zoonotic disease with public health and economic significance. Pigs acquire the disease through consumption of Taenia solium eggs excreted by human tapeworm carriers. The present study was conducted to investigate the prevalence of porcine cysticercosis in Nagpur and Mumbai region of Maharashtra, India by P/M examination of carcasses followed by histopathology of affected organs in infected animals and molecular identification of cysts for confirmation. Out of 1000 pigs examined during slaughter, three pigs were found to be heavily affected with T. solium cysts giving a prevalence of 0.3%. Histological section of brain in infected animals revealed marked vascular congestion of meninges, mild neuronal degeneration, perivascular cuffing and gliosis while the liver showed the infiltration of mononuclear cell, predominantly eosinophils throughout the parenchyma. Some degree of calcification was observed in the cysts lodged in liver while calcification was not evident in case of cysts lodged in brain, tongue, diaphragm and skeletal muscle. Molecular identification by PCR using two sets of oligonucleotide primers against LSU rRNA gene and Mt-Cox1 gene of T. solium confirms the cysts to be that of T. solium. The molecular diagnostics methods have been considered for validation in conjunction with P/M inspections, parasitological and histopathological examinations. The study confirms the presence of porcine cysticercosis in the two regions and demands proper sanitary measures to minimize the risk of infection from zoonoses and food safety point of view.

Multiple zoonotic helminth infections in domestic dogs in a rural area of Khuzestan Province in Iran.

BACKGROUND: Echinococcosis and toxocarosis caused by the genus of Echinococcus and Toxocara spp. are among important helminthic diseases worldwide. Limited data on the prevalence of these parasites persuaded us to determine the prevalence of E. granulosus, E. multilocularis, and T. canis infections in domestic dogs in rural areas of Ahvaz, southwestern Iran. Fecal samples from 167 domestic dogs were examined using both microscopy and PCR techniques. Multiplex PCR was performed for the presence of Echinococcus, and Taenia spp. and single PCR for detection of T. canis and Toxascaris leonina. RESULTS: The total occurrence of identified parasites was 65 (38.9%). The microscopic examinations showed that 40 (24%), 18 (10.8%), and four (2.4%) of dogs were infected with taeniid-like, ascarid, and both genera eggs, respectively. Echinococcus granulosus was identified in seven (4.2%), Taenia spp. in 29 (17.4%), and mixed infection with both in 11 (6.6%) samples. Sequencing of PCR-positive samples identified E. granulosus s.s. (G1), 18 T. hydatigena (10.8%), five T. multiceps (3%), three T. serialis (1.8%), one T. ovis (0.6%), one Spirometra erinaceieuropaei voucher (0.6%), and two Mesocestoides corti (1.2%). This is the first report of S. erinaceieuropaei voucher and M. corti in dogs in Iran. Nine (5.4%) and 16 (9.6%) dogs showed infection with T. canis and T. leonina, respectively. Two samples showed coinfection with both ascarids. CONCLUSIONS: Several studies have reported echinococcosis and toxocarosis in intermediate hosts from the southwest of Iran; however, this study is the first molecular research on E. granulosus and T. canis in domestic dogs in a rural area of southwestern Iran. Furthermore, issues of soil contamination with dogs' feces and recent dust storms in Khuzestan may have a role in the spreading of these zoonotic infections to other provinces close to it, and neighboring countries such as Iraq.

Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice.

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.

Development of LAMP assays for the molecular detection of taeniid infection in canine in Tibetan rural area.

For field-identification of taeniid cestodes in canine animals in Tibetan area, loop-mediated isothermal amplification (LAMP) assays for Echinococcus multilocularis, E. shiquicus, Taenia hydatigena, T. multiceps, T. pisiformis and T. crassiceps were developed and evaluated along with the reported assay for E. granulosus. The LAMP assays showed specific reaction with their corresponding target species DNA with the detection limit of 1 to 10 pg. Moreover, the assays for E. granulosus, E. multilocularis, T. hydatigena and T. multiceps could detect DNA extracted from 3 or more eggs of their corresponding target species. Then, the LAMP assays were applied on samples containing 3 to 35 taeniid eggs obtained from 61 field-collected canine feces in Qinghai, and the result was compared with a reported multiplex PCR and sequence analysis. The LAMP assays and the PCR detected single species DNA of E. granulosus, E. shiquicus, T. hydatigena and T. multiceps in 5, 2, 44 and 2 samples, respectively. In the rest 8 samples, DNA of both E. granulosus and T. hydatigena were detected by the PCR but the LAMP assays detected those DNAs in 2 samples and only T. hydatigena DNA in 6 samples. It was assumed that less than 3 E. granulosus eggs were mixed in the samples although the samples contained 21 to 27 eggs in total. In conclusion, the LAMP assays were less sensitive than the multiplex PCR, but would have adequate sensitivity for field use in Tibetan area.

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site.