Species-specific PCR Reactions Research Articles

Detection of Mycoplasma spp. in free-living seabirds.

This study aimed to investigate the presence of Mycoplasma spp. and identify the species of mycoplasma isolates obtained from seabirds found on Brazilian coastal beaches. Tracheal and cloacal swab samples were collected from 50 seabirds rescued by three conservation and marine animal rehabilitation centers located in Brazil. The tracheal and cloacal samples were subjected to mycoplasma culture and the isolates were identified through PCR. A "Mollicutes-specific" 16S rRNA PCR reaction was employed for triage. Four species-specific PCR reactions were used to detect Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or M. gallinarum. The Mollicutes positive and species negative samples were submitted do 16S rRNA sequencing. Eighteen (36%) of 50 seabirds tested positive for mycoplasma by culture. In the PCR for the genus, 28 (56%) of 50 seabirds were positive for Mycoplasma spp., with 13 (26%) detected in the trachea, one (2%) in the cloaca, and 14 (28%) in both sites. In the species-specific PCR, M. gallisepticum was detected in 17.8%, and M. meleagridis in 17.8%. Both species were detected in 14.3%. Of the isolates not characterized at species level, we obtained ten sequences and they were divided into three clusters. The first cluster was closely related to M. meleagridis, the second to M. synoviae, and the third grouped M. tully, M. gallisepticum, and M. imitans. Four and five of nine species of seabirds studied had mycoplasma detected by culture or PCR, respectively. Mycoplasmas were found in the majority of the animals studied, with the highest prevalence proportionally found in Sula leucogaster, and the lowest in Fregata magnificens. The phylogenetic analysis identified Mycoplasma spp. adapted to aquatic birds.

Circulation of Babesia Species and Their Exposure to Humans through Ixodes ricinus

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

Identification and quantification of fumonisin-producing Fusarium species in grain and soil samples from Egypt and the Philippines

Fumonisins are considered among the important mycotoxins associated with human esophageal cancer and livestock diseases. These mycotoxins are mainly produced by Fusarium verticillioides in tropical and subtropical regions such as the Philippines and Egypt and humid temperate regions of the world. The classical taxonomy of fumonisin-producing fungi is challenging, and species-specific PCR reactions are commonly used to clearly identify species within these complexes. The aim of this study was to isolate, identify and quantify fumonisin-producing species in maize, wheat and soil samples from Egypt and the Philippines, and to test Eppendorf-Agar as a long term preservation method. We isolated 44 single spore isolates (39 from Egypt and five from the Philippines) from the collected samples (25 isolates from maize, five from wheat and 14 from soil). In addition, we quantified the content of fumonisin-producing fungi DNA from 15 maize samples and six wheat samples from Egypt, and from six maize samples from the Philippines. morphological and microscopic identification indicated that 21 isolates from Egypt and five from the Philippines were F. verticillioides , one isolate was F. proliferatum and two isolates were F. nygamai . Molecular identification indicated that all these isolates belonged to F. verticillioides. Most were from maize, four were from soil and only one was from wheat. Other Fusarium species isolated included F. oxysporum and F. solani . No F. graminearum isolates were found. The quantitative PCR (qPCR) results obtained using the Taqfum-2f, Vpgen-3R primer pair and the FUMp probe for quantification of fumonisin-producing Fusarium species showed that fumonisin-producing Fusarium isolates were present in four maize samples from the Philippines and eight maize samples from Egypt. The Fusarium DNA levels from fumonisin-producing isolates were in the range of 13 × 10 -3 to 61 × 10 -1 ng ng -1 total DNA in positive samples, except in one maize sample from the Philippines with high concentration of >0.5 ng ng -1 total DNA. This indicates that >50 % of all DNA was Fusarium DNA. No fumonisin-producing Fusarium DNA was detected in the wheat samples and in the remaining maize samples. These results showed that PCR-techniques based on qPCR can be used to identify fumonisin-producing Fusarium species and quantify risks of mycotoxin contaminated grains.

Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs

BackgroundDirofilaria immitis and Dirofilaria repens are mosquito-borne zoonotic filarioids typically infecting dogs, causing a potentially fatal cardiopulmonary disease and dermatological conditions, respectively. The females are larviparous, releasing the larvae (microfilariae) into the bloodstream, which further develop in mosquito vectors. However, microfilaremia greatly fluctuates during a 24-h period. As the sampling time can greatly influence the accuracy of diagnosis, the aim of the present study was to assess the circadian periodicity of D. immitis and D. repens in naturally co-infected dogs in an endemic area of Romania and to investigate possible differences of periodicity between these two species.MethodsOverall, four dogs harbouring natural co-infection with D. immitis and D. repens were selected and sampled every two hours for two consecutive days: two dogs in July 2014 and two in July 2015. At each sampling time, a 0.7 ml blood sample was taken. Modified Knott’s test was performed on 0.5 ml, and the remaining 0.2 ml were used for DNA extraction and molecular amplification, both in single and duplex PCR reactions. Microfilariae of both species were morphologically identified and counted in each collected sample, microfilaremia was calculated, and fluctuation was charted.ResultsThe dynamics of microfilaremia showed similar patterns for both Dirofilaria species. In all four dogs, D. immitis was present at all sampling times, with several peak values of microfilaremia, of which one was common for all dogs (1 am), while minimum counts occurred between 5 and 9 am. Similarly, for D. repens, one of the peak values was recorded in all dogs at 1 am, while minimum counts (including zero) occurred at 9 and 11 am. Single species-specific PCR reactions were positive for both D. immitis and D. repens in all collected samples, while duplex PCR failed to amplify D. repens DNA in many cases.ConclusionsBoth Dirofilaria immitis and D. repens microfilariae are subperiodic, following a similar variation pattern, with peak values of microfilaremia registered during the night in Romania. Duplex PCR fails to identify the infection with D. repens in co-infected dogs when the ratio of microfilaremia is in favour of D. immitis.

Detection of Mycoplasma felis from the kenneled cats with pneumonia

Abstract Two cats were obtained from a cat kennel. Over the previous 7 days, the cats had shown cough, anorex-ia, depression and nasal discharge. In this study, the consensus PCR was able to detect successfully Mycoplasma species in nasal swab samples of the cats. To identify feline mycoplasma species from the lung tissue of the cats with pneumonia, Mycoplasma species-specific PCR reactions were conducted. As the results, we could identify M. felis by the positive amplified DNAs. On the other hand, we could not detect any positive reactions with the PCR reaction for M. arginini , M. canis , M. edwardii , M. cynos , M. gateae , M. maculosum , M. molared , M. opalescens , M. spumans and Mycoplasma HRC-689. In conclusion, we detected M. felis from the kenneled cats with pneumonia. We suggested that this con-sensus PCR would be useful and effective for monitoring Mycoplasma species in various kinds of ani-mals including cats. The application of preceding consensus PCR before the species-specific PCRs may be the most recommended strategy for the identification of

Occurrence of barley pathogenicPyrenophoraspecies and their mating types in Hungary

Net blotch and leaf stripe caused by Pyrenophora teres and P. graminea, respectively, are two major foliar diseases of barley. These two species are able to infect wheat, too. The species composition of these pathogens was examined, for the first time, in four different regions of Hungary in 2006–2010. Altogether 204 isolates were obtained from 99 winter barley, 55 spring barley and 50 wheat leaf samples collected in commercial fields and experimental stations, and species assignment was carried out using species-specific PCR reactions. Most isolates belonged to P. teres f. teres (68%), 26% to P. teres f. maculata and only 6% of the isolates were assigned to P. graminea. Interestingly, all but one of the P. graminea isolates came from the western part of Hungary, while both forms of P. teres occurred in each region. The distribution of mating type genes was also examined in 144 isolates. The overall ratio of MAT1 and MAT2 genes in P. graminea, P. teres f. maculata and P. teres f. teres was 5:3, and close to 2:1 and 1:1, respectively. Both MAT1 and MAT2 isolates of each fungal species/form were distributed in almost all regions over several years, indicating a high potential for sexual outcrossing within local populations of these pathogens. Our survey may be helpful to determine priorities in disease resistance breeding programs. Further studies are in progress to examine the population structure of the most abundant pathogen P. teres f. teres.

Evaluation of possibilities in identification and susceptibility testing for Candida glabrata clinical isolates with the Integral System Yeast Plus (ISYP)

The aim of this study was to evaluate possibilities of correct identification and susceptibility testing of C. glabrata clinical isolates with Integral System Yeast Plus (ISYP). For species identification, as the reference method, API Candida test and species-specific PCR reactions were used. The potential of antifungal susceptibility testing by the ISYP test was compared with the Sensititre Yeast One. Whilst the reference methods confirmed that the received population (n = 65 isolates) represented only C. glabrata, identification with the ISYP system showed correct data only in the case of 18 strains tested (27.7%). Species identification of the other 47 strains with the ISYP test was not possible at all. Significant differences were also observed for drug susceptibility testing carried out by the ISYP and the Sensititre Yeast One. The highest level of disagreement in classifying strains as resistant or susceptible estimated, as 73.9% and 40.0%, was observed for itraconazole and amphotericin B, respectively. Satisfactory results were only obtained for 5-fluorocytosine with 93.8% agreement between both methods. In our opinion the idea of the ISYP system is certainly good. The combination of identification ability and drug susceptibility testing in one test is very important, especially from a clinical point of view. However, the current version of the ISYP has many disadvantages. We would like to encourage the manufacturer to make an effort and develop a new, more accurate version of the test.

A comparative evaluation of PCR- based methods for species- specific determination of African animal trypanosomes in Ugandan cattle

BackgroundIn recent years, PCR has been become widely applied for the detection of trypanosomes overcoming many of the constraints of parasitological and serological techniques, being highly sensitive and specific for trypanosome detection. Individual species-specific multi-copy trypanosome DNA sequences can be targeted to identify parasites. Highly conserved ribosomal RNA (rRNA) genes are also useful for comparisons between closely related species. The internal transcribed spacer regions (ITS) in particular are relatively small, show variability among related species and are flanked by highly conserved segments to which PCR primers can be designed. Individual variations in inter-species length makes the ITS region a useful marker for identification of multiple trypanosome species within a sample.MethodsSix hundred blood samples from cattle collected in Uganda on FTA cards were screened using individual species-specific primers for Trypanosoma congolense, Trypanosoma brucei and Trypanosoma vivax and compared to a modified (using eluate extracted using chelex) ITS-PCR reaction.ResultsThe comparative analysis showed that the species-specific primer sets showed poor agreement with the ITS primer set. Using species-specific PCR for Trypanozoon, a prevalence of 10.5% was observed as compared to 0.2% using ITS PCR (Kappa = 0.03). For Trypanosoma congolense, the species-specific PCR reaction indicated a prevalence of 0% compared to 2.2% using ITS PCR (Kappa = 0). For T. vivax, species-specific PCR detected prevalence of 5.7% compared to 2.8% for ITS PCR (Kappa = 0.29).ConclusionsWhen selecting PCR based tools to apply to epidemiological surveys for generation of prevalence data for animal trypanosomiasis, it is recommended that species-specific primers are used, being the most sensitive diagnostic tool for screening samples to identify members of Trypanozoon (T. b. brucei s.l). While ITS primers are useful for studying the prevalence of trypanosomes causing nagana (in this study the species-specific primers did not detect the presence of T. congolense) there were discrepancies between both the species-specific primers and ITS for the detection of T. vivax.

Comparison of phenotypic and molecular tests to identify lactic acid bacteria.

Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable.

Mass blood survey for malaria: pooling and real-time PCR combined with expert microscopy in north-west Thailand

BackgroundAsymptomatic carriage of Plasmodium falciparum and Plasmodium vivax is common in both low-and high-transmission settings and represents an important reservoir of infection that needs to be targeted if malaria elimination is to succeed.MethodsMass blood examinations (475 individuals) were conducted in two villages in Mae Hong Son, an area of endemic but low-transmission malaria in the north-west of Thailand. The microscopist at the local malaria clinic did not detect any infections. Pools of four samples were screened by real-time PCR; individual members of all of the positive pools were then re-examined by expert microscopy and by a second species-specific PCR reaction.ResultsEight subjects were found to be positive by both PCR and expert microscopy and one was found to be positive by PCR alone. The slides contained asexual stage parasites of P. vivax, P. falciparum and Plasmodium malariae, but no gametocytes. The local clinic was notified within two to eight days of the survey.ConclusionA combination of pooling, real-time PCR and expert microscopy provides a feasible approach to identifying and treating asymptomatic malaria infections in a timely manner.

Molecular identification ofMycoplasma cynosfrom laboratory beagle dogs with respiratory disease

In this study, we examined a colony of 20 beagle dogs in a laboratory animal facility. Mycoplasma was detected by consensus PCR assay in 1 dog with respiratory and constitutional symptoms. None of the other dogs were affected. The dog was euthanized and necropsied. In postmortem examinations, gray or plum-colored gross lesions were found on the lung, most commonly in the apical and cardiac lobes. Some lesions showed clear demarcation and consolidation. Microscopic examination showed peribronchiolar lymphoid hyperplasia and interstitial thickening, lesions pathognomonic for mycoplasma pneumonia. To identify canine Mycoplasma species, we used species-specific PCR reactions for M. arginini, M. canis, M. cynos, M. edwardii, M. felis, M. gateae, M. maculosum, M. molare, M. opalescens, M. spumans, Mycoplasma sp. HRC 689, and M. collis. As the result, we identified Mycoplasma cynos by amplification of DNA extracted from lung tissue of the laboratory beagle dog with respiratory disease.

Species-specific PCR discrimination of species of the calanoid copepod Pseudocalanus, P. acuspes and P. elongatus, in the Baltic and North Seas

Two species of the calanoid copepod Pseudocalanus, P. acuspes and P. elongatus, have been reported from the Baltic and North Seas. Morphometric analysis based on Frost (Can J Zool 67:525–551, 1989) did not identify the species in samples collected during the German GLOBEC program; the majority of measurements fell outside reported ranges for Baltic and North Sea specimens. A multiplexed species-specific PCR (SS-PCR) protocol was designed from a ~700 bp sequence of mitochondrial cytochrome oxidase I (mtCOI), with primers designed to amplify regions of different sizes for the two species to allow detection by gel electrophoresis. The accuracy and reliability of the multiplexed SS-PCR reaction was confirmed by sequencing the mtCOI region. Preliminary SS-PCR analysis of geographic distributions of the two species indicated that P. acuspes occurred exclusively at sampling sites in the Baltic Sea, whereas P. elongatus was found in the southern North Sea and at one site in the Baltic. The SS-PCR protocol can be used for routine identification of the two species for studies of population, community and ecosystem dynamics.

Benomyl sensitivity assays and species-specific PCR reactions highlight association of two Colletotrichum gloeosporioides types and C. acutatum with rumple disease on Primofiori lemons

Rumple is a serious peel collapse of Primofiori lemons in the southeast of Spain with an unresolved aetiology. Symptoms typically occur on fruits at ripening under wet conditions as dark sunken lesions producing premature fruit drop and damaged fruits unacceptable for fresh commercialization. A total of 16 Colletotrichum spp. isolates established from rumple-affected lemons collected during the autumn of 2007 from two different orchards were characterized by molecular and phenotypic assays and compared with reference isolates. Species-specific PCR reactions using β-tubulin 2 nucleotide sequences showed Colletotrichum gloeosporioides to predominate (81.5%) with limited occurrence of C. acutatum (18.75%). Among the C. gloeosporioides isolates, five (38.5%) showed benomyl resistance and eight (61.5%) were highly sensitive to the fungicide. The limited occurrence of C. acutatum could be related to factors such as the presence of both species on the same fruit, unfavourable meteorological conditions and low disease incidence. This work reveals an association of C. gloeosporioides and C. acutatum isolates with rumple disease of lemons and expands the range of C. acutatum on citrus.

Genotypic and technological characterization ofLactococcus lactisisolates involved in processing of artisanal Manchego cheese

Genotypic and technological characterization of wild lactococci isolated from artisanal Manchego cheese during the ripening process for selection of suitable starter cultures. A total of 114 isolates of lactococci were typed using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Sixteen distinct RAPD-PCR patterns, at a similarity level of 73%, were obtained. On the basis of species-specific PCR reaction, the isolates were assigned to the species Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris with L. lactis subsp. lactis being predominant at both dairies. Twenty-six isolates were technologically characterized to select those with the best properties. Most of them showed good technological properties although some could produce tyramine. The presence of coincident genotypes at both dairies has been demonstrated, which would suggest that they are well adapted to the Manchego cheese environment. Interesting differences were found in the technological characterization and the potential role of autochthonous lactococci strains as starter culture has been displayed. The great economic importance of Manchego cheese encouraged a deeper knowledge of its microbiota, to select strains with the best properties to use as starter cultures in industrial Manchego cheeses, preserving the autochthonous characteristics.

An ecological study of lactic acid bacteria from Almagro eggplant fermentation brines

Identification of the predominant lactic acid bacteria (LAB) involved in spontaneous fermentations of Almagro eggplants, and evaluation of the biodiversity by molecular typing. Almagro eggplant fermentations in three factories (A, B and C) enjoying Protected Designation of Origin (PDO) status were monitored by chemical and microbiological analysis of brines. LAB isolates from brines were identified by phenotypic analysis and by species-specific PCR reactions and typed by randomly amplified polymorphic DNA (RAPD)-PCR. All isolates from factories A and C belonged to the genus Lactobacillus (Lact.), whereas isolates from factory B belonged to Lactobacillus (50%), Leuconostoc (Ln.) (25%) and Lactococcus (Lc.) (25%); 1.9% of this microbiota was considered cosmopolitan. The genera Leuconostoc and Lactococcus and the species Lact. acidophilus and Lact. paracasei had never previously been reported in Almagro eggplant fermentations. Considerable differences in the composition of the lactic acid microbiota participating in the Almagro eggplant fermentations exist. Brine NaCl concentration has a notable influence both in number and in the species participating. The original aspect of this work consists of an ecological study of the LAB taking part in spontaneous Almagro eggplant fermentations from different factories. Participation of Leuconostoc and Lactococcus species and of Lact. acidophilus and Lact. paracasei, which had never before been described for this pickle, and the evidence that a lactic fermentation does not always take place, were the most relevant results.

Identification of anisakid nematodes from the Southern Baltic Sea using PCR-based methods

Four common species of Baltic anisakids Anisakis simplex, Hysterothylacium auctum,Contracaecum osculatum from fish (the last one also from the grey seal) and C. rudolphii from cormorants were examined using PCR-RFLP technique for working out the method allowing their identification and differentiation. A fragment of nuclear DNA containing intergenic regions (ITS-1 and ITS-2) of ribosomal DNA together with adjacent sequences of genes coding 18S and 28S rRNA (in total 1500 bp) was amplified and the products were digested using three endonucleases Hin fI,Hae III and Pvu II. Digestion with Hin fI endonuclease enabled species identification and differentiation between all nematode species studied. Digestion with Hae III endonuclease enabled identification of A. simplex and H. auctum while electrophoretic patterns obtained for both Contracaecum species were similar (differentiation between A. simplex, H. auctum and the genus Contracaecum). Pvu II endonuclease was not suitable to this purpose. On the basis of nucleotide sequences obtained, the pairs of species-specific primers were designed for each species studied. By the use of these pairs of primers the species-specific PCR reactions were carried out. The sizes of specific products were: 486 bp for A. simplex, 491 bp for C. osculatum, 505 bp for C. rudolphii and 663 bp for H. auctum. The methods presented herein can be useful in diagnostics of human anisakidosis, especially when parasites are disrupted.