Species-specific PCR Amplification Research Articles

Histopathology, Isolation and Molecular Detection of Pasturella multocida from Sheep in Gadag Region of Karnataka, India

Bacterial pneumonia is the most common respiratory problem in small ruminants. The heart blood and lung tissue samples from two dead sheep suffering from pneumonia at the age of six month were subjected for histopathology, bacterial isolation & identification and antibiogram. Acute fibrinous bronchopneumonia was observed at necropsy and histopatholgy. The isolates were identified as P. multocida based on cultural and biochemical properties, and additionally confirmed by species-specific PCR amplification of KMT1 (Multocida toxin hydrolase gene) gene with the amplicon of 460 bp. The antibiogram showed susceptibility to chloramphenicol, β-lactams, quinolones, fluoroquinolones and aminoglycosides. In this study, the occurrence of ovine pasteurellosis caused by P. multocida with acute fibrinous bronchopneumonia was recorded.

Potential intermediate hosts of Angiostrongylus cantonensis in the European Mediterranean region (Mallorca, Spain)

Angiostrongylus cantonensis is the main etiological agent of eosinophilic meningitis, a disease that often leads to severe neurological manifestations in mammals and birds. In recent years, the prevalence of this zoonotic nematode has dramatically increased as it expands into new territories beyond its native range in Southeast Asia and the Pacific Basin.Its arrival to Europe, the last continent to be invaded, has raised many questions concerning the parasite's life cycle, particularly in the Mediterranean region, where it is now endemic. This study aims to collect evidence about intermediate hosts (snail and slug species) involved in the transmission of the A. cantonensis in Mallorca. We have conducted a systematic surveillance of gastropods within 2 km radius areas, surrounding the specific locations where infected hedgehogs were found. We employed a sequence-based detection approach that included a species-specific PCR amplification followed by DNA sequencing of the internal transcribed spacer I (ITS-1). This conservative approach was essential to avoid cross reactions with the numerous metastrongylid species known to be circulating in Mallorca. Overall, we collected and identified 398 gastropods belonging to 17 species, of which 11% tested positive for A. cantonensis. These gastropods were collected from touristic settlements to agricultural lands. Five gastropod species: Theba pisana, Cornu aspersum, Deroceras reticulatum, Milax gagates and Otala lactea, had been previously incriminated as A. cantonensis intermediate hosts, while 6 species: Papillifera papillaris, Cochlicella acuta, Eobania vermiculata, Ganula lanuginosa, Milax nigricans and Rumina decollata, are newly recorded hosts. The findings of this study have important epidemiological implications, and further measures are discussed to prevent neuroangiostrongylosis cases.

Time and cost-efficient identification of Trichophyton indotineae.

During the past 5 years, an outbreak of recalcitrant dermatophytosis due to a novel Trichophyton species generally resistant to terbinafine, T.indotineae, has spread out from South Asia to many countries around the World. These isolates cannot be reliably differentiated from other Trichophyton spp. on the basis of morphological traits and the current laboratory diagnostics relies on sequencing of ribosomal DNA ITS region. In this study, we aimed to introduce two inexpensive and rapid PCR-based assays for differentiation between T.indotineae and other dermatophytes. The first introduced assay is based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, involving the amplification of TOP2 sequences and the digestion of PCR products by Cfr13I restriction enzyme. The second assay is proposed as conventional endpoint species-specific PCR amplification of the C120-287 intergenic locus. To validate the assays, a total of 191 Trichophyton spp. and 2 Microsporum canis strains with known ITS region sequences were used. From the T.mentagrophytes / T.interdigitale species complex (TMTISC), strains with 18 different ITS genotypes were tested. The sample of TMTISC isolates included 41 T.indotineae strains. TOP2 PCR-RFLP and T.indotineae-specific PCR were positive with testing on DNA of all 41 T.indotineae isolates and two strains of T.mentagrophytes belonging to ITS Types XIII and XVI, but negative with other species and other TMTISC ITS genotypes (n=152). Therefore, the specificity of both new assays was 99%. The two developed diagnostic assays provide accurate and cost-effective means of identifying cultured T.indotineae isolates.

Pathology caused by three species of Eimeria that infect the turkey with a description of a scoring system for intestinal lesions

ABSTRACT Three-week-old turkey poults were infected with pure lines of three species of Eimeria (E. adenoeides, E. gallopavonis, and E. meleagrimitis) recently isolated from commercial turkey farms. The lines had been propagated from a single oocyst and identified by species-specific PCR amplification of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Five to six days after infection their intestines were removed and examined for the presence of intestinal lesions. A description and review of the pathology caused by these parasites is provided, and a scoring system developed by which the severity of the lesions can be evaluated. The system is similar to that described by Johnson, J. and Reid, W. M. [1970. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens. Experimental Parasitology, 28, 30–36] for chickens in which a score of zero to four is assigned to lesions of increasing severity. The intestinal lesions observed here, and their assigned scores, are supported by representative illustrations. It is hoped that they may prove a useful tool for evaluating the pathology caused by E. adenoeides, E. gallopavonis, and E. meleagrimitis in the turkey. RESEARCH HIGHLIGHTS A scoring system has been developed for intestinal lesions caused by three species of Eimeria that infect the turkey. The lesions attributable to these species are illustrated.

Molecular characterization and phylogenetic analysis of Pantoea stewartii subspecies stewartii causing bronzing disease of jackfruit in Malaysia based on cps and hrp gene sequences

Bronzing disease of jackfruit was first detected in plantation areas in Selangor and Pahang, Malaysia. The symptoms included reddish discoloration of the affected fruit pulp and rags, which could reduce the fruit quality and discourage purchasers. In this study, species-specific PCR amplification with primers encoded for cps and hrp virulence genes showed all bacterial strains isolated from infected jackfruits with bronzing symptoms, displaying a 1.1 kb and 0.9 kb amplicon. Phylogenetic analyses of the cps and hrp gene sequences clustered all strains to Pantoea stewartii subspecies stewartii (P. stewartii subsp. stewartii) reference strains (GenBank accession Nos. AF077292, AF282857, KY965964 and KY965965), with 62 to 80% similarity. Results revealed the presence of these virulence genes in P. stewartii subsp. stewartii strains is required for pathogenicity of the bacterium in jackfruit.

Duplex PCR Assay for Identification of Tissue of Cattle and Buffalo Origin Targeting Mitochondrial Cytochrome b Gene

The aim of this study was to develop PCR assay for simultaneous detection of tissues from cattle and buffalo in a single PCR reaction. Species-specific primers for cattle and buffalo species were designed through homology comparisons and alignment of partial sequences of Cyt.b gene from common food animals. The conditions for species-specific PCR amplification were optimized in terms of quantity and concentration of various components for PCR mix and annealing temperature for each set of designed species-specific primer pairs. The assay revealed the amplification of genomic DNA from cattle to amplicon size of 655 bp, whereas DNA template from buffalo to amplicon length of 249 bp. Simultaneous amplification of DNA templates from cattle and buffalo was observed in duplex PCR assay. Thus, the developed assay could identify the tissues of cattle and buffalo origin, detecting even both the species together and being advantageous over species-specific PCR.

Screening of natural yeast isolates under the effects of stresses associated with second-generation biofuel production

Robust microorganisms are required for sustainable second-generation biofuel production. We evaluated the growth and fermentation performance of six natural isolates that were derived from grape wine and medicinal herbs using a wide range of carbon sources, rice and wheat straw hydrolysates as well as stress conditions associated with second-generation ethanol production. Sequence analysis of the 5.8S internal transcribed spacer (ITS) and species-specific PCR amplification of the HO gene region assigned the natural isolates to Saccharomyces cerevisiae. Restriction fragment length polymorphism (RFLP) analysis of the mitochondrial DNA revealed that natural yeast isolates are genetically closer to the laboratory strain BY4741 than to the CEN.PK strains. Dextrose fermentation by a natural isolate, MTCC4780, under semi-anaerobic conditions produced maximum ethanol yields of 0.44 g/g and 0.39 g/g, respectively, with and without the stresses encountered during lignocellulosic ethanol fermentation. However, MTCC4780 produced ethanol yields of 0.48 g/g, 0.42 g/g and 0.45 g/g, respectively, with glucose, rice and wheat straw enzymatic hydrolysate fermentation in a bioreactor. The isolates MTCC4781 and MTCC4796 showed higher growth and fermentation performance than did MTCC4780 in the presence of elevated temperature and pre-treatment inhibitors. Taken together, the MTCC4780, MTCC4781 and MTCC4796 strains have the potential to serve as a platform for lignocellulosic ethanol production under stresses associated with second-generation biofuel production.

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows

The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found.

Cryptosporidium suis and Cryptosporidium scrofarum in Eurasian wild boars (Sus scrofa) in Central Europe

From 2011 to 2012, to identify Cryptosporidium spp. occurrence in Eurasian wild boars (Sus scrofa) 29 randomly selected localities (both forest areas and enclosures) across the Central European countries of Austria, the Czech Republic, Poland, and the Slovak Republic were investigated. Cryptosporidium oocysts were microscopicaly detected in 11 out of 460 faecal samples examined using aniline-carbol-methyl violet staining. Sixty-one Cryptosporidium infections, including the 11 infections that were detected by microscopy, were detected using genus- or species-specific nested PCR amplification of SSU rDNA. This represents a 5.5 fold greater sensitivity for PCR relative to microscopy. Combining genus- and species-specific PCR tools significantly changes the perspective on the occurrence of Cryptosporidium spp. in wild boars. While RFLP and direct sequencing of genus specific PCR-amplified products revealed 56 C. suis (20) and C. scrofarum (36) monoinfections and only 5 mixed infections of these species, species-specific molecular tools showed 44 monoinfections and 17 mixed infections with these species. PCR analysis of the gp60 gene did not reveal any other Cryptosporidium infections. Similar to domestic pigs, C. scrofarum was detected as a dominant species infecting adult Eurasian wild boars (Sus scrofa). Cryptosporidium infected wild boars did not show signs of clinical disease. This report is perhaps the most comprehensive survey of cryptosporidial infection in wild boars.

COLLETOTRICHUM: HOST SPECIFICITY AND PATHOGENICITY ON SELECTED TROPICAL AND SUBTROPICAL CROPS

Colletotrichum and its teleomorph Glomerella are considered major fungal plant pathogens worldwide. They cause significant economic damage to fruit crops in tropical, subtropical and temperate regions. Several Colletotrichum species or biotypes are known to cause disease in a single host such as C. acutatum and C. gloeosporioides on apple, avocado, mango, papaya, passiflora, strawberry and tamarillo. It is also common to find a single species infecting multiple hosts such as C. gloeosporioides on apple, avocado, mango, papaya, peach, strawberry, and other hosts. Cross-infection potential was shown for two species, C. gloeosporioides from limonium and C. acutatum from strawberry, when inoculating peach, pear, mango, nectarine and strawberry. Molecular analyses including species-specific PCR amplification and ITS sequencing was reliable for identification of Colletotrichum isolates infecting apple, avocado, mango, papaya, passiflora, peach, strawberry and tamarillo fruits. Sub-populations within C. acutatum, C. gloeosporioides and C. boninense were characterized using ITS sequence analysis, while the latter appears to be a diverse and emerging new species infecting multiple fruit crops.

Molecular characterization of Staphylococcus pseudintermedius strains isolated from clinical samples of animal origin

The aim of this study was to determine the species distribution among 44 randomly selected clinical isolates (30 mecA-positive and 14 mecA-negative) of animal origin previously identified as Staphylococcus intermedius by phenotypic tests and species-specific PCR amplification of the 16S rRNA gene. For this purpose, we used a multiplex PCR for the detection of the nuc gene and restriction fragment length polymorphism analysis of pta gene amplified by PCR. Both methods allow discrimination of Staphylococcus pseudintermedius from the other closely related members of the S. intermedius group and other coagulase-positive staphylococci isolated from animals. Genetic diversity of S. pseudintermedius strains was analyzed by staphylococcal protein A-encoding gene (spa) typing. Multiplex PCR method was used to identify staphylococcal cassette chromosome mec (SCCmec) type in mecA-positive strains. All isolates previously identified as S. intermedius were shown to belong to S. pseudintermedius. According to PCR-based SCCmec typing, SCCmecIII was the most prevalent type (n = 23), and solely seven isolates were designated as non-typeable. Furthermore, the assessment of spa-typing results revealed that the majority of all strains (n = 27) harbored spa type t02, and 17 strains were classified as non-typeable.

Identification of European commercial cockles (Cerastoderma edule and C. glaucum) by species-specific PCR amplification of the ribosomal DNA ITS region

We present a novel one-step PCR method for the identification of Cerastoderma edule and C. glaucum. Sequence differences found in the internal transcribed spacer region (ITS) of the ribosomal DNA of the two cockles allowed us to design two species-specific reverse primers. These two primers were used in a multiplex reaction, together with a forward universal primer to amplify specific fragments with different lengths in each cockle (190 and 470 bp in C. edule and C. glaucum, respectively). The successful and specific amplifications obtained for two natural populations in each species as well as in canned products lend support to the usefulness of these markers.

Predominance of Ehrlichia chaffeensis in Rhipicephalus sanguineus ticks from kennel-confined dogs in Limbe, Cameroon

Rhipicephalus sanguineus ticks (n = 63) collected from five dogs (two adults and three puppies) housed in a kennel were screened for Ehrlichial agents (Ehrlichia canis, E. chaffeensis, and E. ewingii) using a species-specific multicolor real-time TaqMan PCR amplification of the disulphide bond formation protein (dsb) gene. Ehrlichia chaffeensis DNA was detected in 33 (56%) ticks, E. canis DNA was detected in four (6%) ticks, and one tick was coinfected. The E. chaffeensis and E. canis nucleotide sequences of the amplified dsb gene (374 bp) obtained from the Cameroonian R. sanguineus ticks were identical to the North American genotypes.

Isolation and characterization of two genetically distant groups of Acetobacter diazotrophicus from a new host plant Eleusine coracana L.

Acetobacter diazotrophicus strains were isolated from Eleusine coracana, a new host plant cultivated along the coast of Tamil Nadu in India. Using a species-specific oligonucleotide primer and PCR amplification, the presence of this bacterium was demonstrated directly in plant tissues, proving its endophytic nature, and it was absent in non-rhizosphere soils. The isolates were also characterized on the basis of typical morphology, electron microscopy and biochemical tests, including nitrogen-fixing efficiency, to assess their diversity. When RAPD analysis was performed on the isolates, they fell into two distinct genetically related groups when compared with the type strain PA1 5 (ATCC 49037). In view of the importance of E. coracana to this region, associated nitrogen-fixing Acetobacter strains may be agronomically important because they could supply part of the nitrogen that the crop requires.

Species-specific plasmid sequences for PCR identification of the three species of Borrelia burgdorferi sensu lato involved in Lyme disease.

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification.

Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.

A Variable 23S rDNA Region is a Useful Discriminating Target for Genus-Specific and Species-Specific PCR Amplification in Arcobacter Species

Summary A genus-specific PCR assay was created for the detection of all Arcobacter strains and three speciesspecific PCR assays were developed for the discrimination among A. cryaerophilus, A. butzleri and A. skirrowii . The PCR assays were based on a target sequence which comprises the most variable areas of 23S rDNA. The specificity of the developed PCR assays was extensively tested on an assortment of 54 Arcobacter and 71 phylogenetically related reference strains. All primer sets were further evaluated on a large number of field strains isolated from different sources in various geographical locations.

Rapid detection and identification of Candida albicans and Torulopsis (Candida) glabrata in clinical specimens by species-specific nested PCR amplification of a cytochrome P-450 lanosterol-alpha-demethylase (L1A1) gene fragment

PCR of a Candida albicans cytochrome P-450 lanosterol-alpha-demethylase (P450-L1A1) gene segment is a rapid and sensitive method of detection in clinical specimens. This enzyme is a target for azole antifungal action. In order to directly detect and identify the clinically most important species of Candida, we cloned and sequenced 1.3-kbp fragments of the cytochrome P450-L1A1 genes from Torulopsis (Candida) glabrata and from Candida krusei. These segments were compared with the published sequences from C. albicans and Candida tropicalis. Amplimers for gene sequences highly conserved throughout the fungal kingdom were first used; positive PCR results were obtained for C. albicans, T. glabrata, C. krusei, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, and Trichosporon beigelii DNA extracts. Primers were then selected for a highly variable region of the gene, allowing the species-specific detection from purified DNA of C. albicans, T. glabrata, C. krusei, and C. tropicalis. The assay sensitivity as tested for C. albicans in seeded clinical specimens such as blood, peritoneal fluid, or urine was 10 to 20 cells per 0.1 ml. Compared with results obtained by culture, the sensitivity, specificity, and efficiency of the species-specific nested PCR tested with 80 clinical specimens were 71, 95, and 83% for C. albicans and 100, 97, and 98% for T. glabrata, respectively.

Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells.

Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells.

Pigment-Cell-Specific Genes from Fibroblasts Are Transactivated after Chromosomal Transfer into Melanoma Cells

Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells.