Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115 bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish.

A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA.

The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.     

Monitoring the spore dynamics of Aphanomyces astaci in the ambient water of latent carrier crayfish

The specialized crayfish parasite Aphanomyces astaci causes the devastating crayfish plague in European crayfish. Even though A. astaci sporulation has been thoroughly studied under pure culture conditions, little is known about the sporulation dynamic from its live host. Our purpose was to investigate the A. astaci spore dynamic in its native parasite-host relationship by monitoring the sporulation from carrier crayfish into the ambient water using agent specific qPCR. American signal crayfish (Pacifastacus leniusculus) with known positive carrier status were housed individually and communally in two experimental set-ups using multiple replicates and different temperatures. Water samples were collected weekly, and spore numbers were quantified. We demonstrate here that live latent carrier crayfish continuously released a moderate number of A. astaci spores (~2700 spores per crayfish/week) in the absence of death and moulting events. In contrast, a pronounced sporulation increase was seen already one week prior to death in moribund crayfish, suggesting a crayfish plague-like condition developing in weakened or stressed individuals. Significantly more spores were produced at 18°C compared to 4°C, while a negative correlation was detected between spore numbers and temperatures rising from 17 to 23°C. This study is the first attempt to quantify the spore release from carrier crayfish on the basis of qPCR applied on water samples, and demonstrate that the approach successfully unravel A. astaci sporulation patterns. The results emphasize that carrier crayfish pose a constant infection risk to highly susceptible crayfish species regardless of crayfish life cycle state.     

Physiological and genetic characterisation of some new Aphanomyces strains isolated from freshwater crayfish

Five Aphanomyces strains were isolated during suspected outbreaks of crayfish disease in Spain and Italy. Genetic and physiological evidence show that the strains isolated from the freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus, do not fit into any previously identified group of Aphanomyces astaci and are not capable of killing crayfish following standardised experimental infection. RAPD-PCR and ITS sequencing analysis show a high degree of similarity between the new isolates, while they are clearly different from the A. astaci reference strains. They do, however, possess some properties, which are commonly associated with parasitic species such as repeated zoospore emergence and the lack of sexual reproduction. The five isolates share some physiological properties i.e. a high growth rate, and germination in response to nutrients and, in contrast to A. astaci, they do not express chitinase constitutively during growth or sporulation. Until their taxonomic status is fully elucidated we suggest that the new isolates be given the tentative species name Aphanomyces repetans.     

Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum

Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was amplified and sequenced, and the resultant sequences were aligned with published sequences for Pythium aphanidermatum, P acanthicum, and P myriotylum. A pair of P insidiosum-specific primers (PI-1 and PI-2) were designed from variable regions within the ITSI region. A nested PCR assay was developed in which the 1st round amplified the ITSI region by use of universal fungal primers. Second-round amplification utilized the internal P insidiosum-specific primers PI-1 and PI-2. Specificity of the assay was tested with DNA extracted from cultures of the following: 10 clinical isolates of P insidiosum and 1 isolate each of P graminicola, P irregulare, P arrhenomanes, P myriotylum, P deliense, Basidiobolus ranarum, Conidiobolus coronatus, Aspergillus terreus, Lagenidium giganteum, and a canine-pathogenic Lagenidium species. Nested PCR produced a single 105-base pair amplicon for each of the P insidiosum isolates, but did not produce amplicons for any of the other isolates. Results of this study suggest that PCR is a useful tool for the identification of P insidiosum.     

Development of PCR assay for differentiation of some important wild animal meat of Sri Lanka

A polymerase chain reaction (PCR) assay was developed to differentiate meat of Ceylon spotted deer (Axis axis ceylonensis), Ceylon hog deer (A. porcius oryzus), Ceylon sambhur (Cervus unicolor unicolor) and barking deer (Muntiacus muntijak malabaricus) from meat of cattle, goat, buffalo, pig, dog and sheep. A set of primers was designed according to the sequence of the mitochondrial cytochrome b gene of C. elaphus canadensis and by PCR amplification about 450 bp band was observed for all four animal species and these primers were not cross reacted with DNA of other animal species tested in the study under the tested reaction conditions. A band of 649 bp size was observed for all animal species when DNA was amplified with the universal primers and that indicated the presence of mitochondrial DNA in the samples. Further, the results indicated that this technique was sensitive enough to differentiate rotten meat, at least 5 days after the killing of an animal. Under these PCR conditions, the DNA of bacteria, which is involved in decomposition of meat, was not amplified with both universal and specific primers. However, the method was not sensitive enough in differentiating cooked meat of these species. Slaughtering of these four wild animal species is banned, but the animals are being killed illegally. Lack of meat identification methods has been identified as one of the major constraints to implement legal procedures and conserve biodiversity in the country.     

Development of molecular assays for the identification of the 11 Eimeria species of the domestic rabbit (Oryctolagus cuniculus)

Coccidiosis are the major parasitic diseases in poultry and other domestic animals including the domestic rabbit (Oryctolagus cuniculus). Eleven distinct Eimeria species have been identified in this host, but no PCR-based method has been developed so far for unequivocal species differentiation. In this work, we describe the development of molecular diagnostic assays that allow for the detection and discrimination of the 11 Eimeria species that infect rabbits. We determined the nucleotide sequences of the ITS1 ribosomal DNAs and designed species-specific primers for each species. We performed specificity tests of the assays using heterologous sets of primers and DNA samples, and no cross-specific bands were observed. We obtained a detection limit varying from 500fg to 1pg, which corresponds approximately to 0.8-1.7 sporulated oocysts, respectively. The test reported here showed good reproducibility and presented a consistent sensitivity with three different brands of amplification enzymes. These novel diagnostic assays will permit population surveys to be performed with high sensitivity and specificity, thus contributing to a better understanding of the epidemiology of this important group of coccidian parasites.     

Genetic diversity,phylogenetic position and morphometric analysis of Astacus colchicus (Decapoda,Astacidae): a new insight into Eastern European crayfish fauna

The phylogeny of European crayfish fauna, especially with respect to Eastern European species, is still far from being completely resolved. To fill this gap, we analyzed most of the European crayfish species focusing on the phylogenetic position of the endemic crayfish Astacus colchicus, inhabiting Georgia. Three mitochondrial and one nuclear marker were used to study evolutionary relationships among European crayfish species, resulting in the unique phylogenetic position of A. colchicus indicating independent species status to A. astacus. Phylogenetic analyses revealed a deep molecular divergence of A. colchicus in comparison to A. astacus (6.5–10.9% in mtDNA and 1.1% in nDNA) as well as to Pontastacus leptodactylus and P. pachypus (5.5–10.0% in mtDNA and 1.4–2.4% in nDNA). Absent ventral process on second male pleopod and abdominal somites II and III with pleura rounded lacking prominent spines clearly indicate taxonomic assignment to the genus Astacus; however, the species is distributed almost in the middle of Ponto-Caspian area typical by occurrence of the genus Pontastacus. Several morphological indices linked to head length, carapace, and total body length and width were found to demonstrate apparent differences between A. colchicus and A. astacus. Although this study provides a novel insight into European crayfish phylogeography, we also point out the gaps in comprehensive study of the P. leptodactylus species complex, which could reveal details about the potential species status of particular species and subspecies within this genus.     

Detection of Rhodococcus equi by polymerase chain reaction using species-specific nonproprietary primers.

Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10-base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.     

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.     

饲料中马、驴源性成分的分子生物学检测技术

根据马、驴线粒体DNA中的保守区段设计了一对引物，通过聚合酶链式反应（polymerase chain reaction，PCR）可以专一地检测扩增出马、驴源性成分的DNA片段，再经限制性内切酶Sau 3A和Alu Ⅰ的酶切鉴定可以区分马源性成分和驴源性成分．PCR扩增产物的测序结果验证了酶切鉴定结果的正确性。引物灵敏度测试培果表明该方法的检测低限均达0．3％．该对引物可以成为检测马、驴源性成分高效准确的检测工具。     

Prevalence and identification of fungal DNA in the small intestine of healthy dogs and dogs with chronic enteropathies

Limited information is available about the prevalence and phylogenetic classification of fungal organisms in the gastrointestinal tract of dogs. Also, the impact of fungal organisms on gastrointestinal health and disease is not well understood. The aim of this study was to evaluate the prevalence of fungal DNA in the small intestine of healthy dogs and dogs with chronic enteropathies. Small intestinal content was analyzed from 64 healthy and 71 diseased dogs from five different geographic locations in Europe and the USA. Fungal DNA was amplified with panfungal primers targeting the internal transcriber spacer (ITS) region. PCR amplicons were subjected to phylogenetic analysis. Fungal DNA was detected in 60.9% of healthy dogs and in 76.1% of dogs with chronic enteropathies. This prevalence was not significantly different between the two groups (p=0.065). Fungal DNA was significantly more prevalent in mucosal brush samples (82.8%) than in luminal samples (42.9%; p=0.002). Sequencing results revealed a total of 51 different phylotypes. All sequences belonged to two phyla and were classified as either Ascomycota (32 phylotypes) or Basidiomycota (19 phylotypes). Three major classes were identified: Saccharomycetes, Dothideomycetes, and Hymenomycetes. The most commonly observed sequences were classified as Pichia spp., Cryptococcus spp., Candida spp., and Trichosporon spp. Species believed to be clinically more important were more commonly observed in diseased dogs. These results indicate a high prevalence and diversity of fungal DNA in the small intestine of both healthy dogs and dogs with chronic enteropathies. The canine gastrointestinal tract of diseased dogs may harbor opportunistic fungal pathogens.     

玉蜀黍属物种间遗传关系的RAPD分析

用136个RAPD引物对玉蜀黍属大刍草和玉米种质基因组DNA的多态性进行检测,共得到5 303条条带,其中多态性带4 500条。遗传相似系数分析结果表明,玉米与大刍草种质遗传相似性系数变幅为0.585～0.809,相同种遗传相似性系数为0.767～0.809,而种间或亚种间的相似性系数为0.585～0.745,表明利用RAPD技术能准确地揭示出玉米与大刍草种间的遗传关系。聚类结果表明,玉蜀黍属内所有大刍草和玉米可分类为繁茂亚属和玉蜀黍亚属,繁茂亚属包括四倍体多年生类玉米种、二倍体多年生类玉米种、繁茂类玉米种和尼加拉瓜类玉米种;玉蜀黍亚属包括小颖类玉米亚种、墨西哥类玉米亚种、委委特南戈类玉米亚种和玉米。运用RAPD技术证实在玉蜀黍属中尼加拉瓜类玉米种与繁茂类玉米种亲缘关系最近。     

PCR方法特异性鉴定狮源性制品

目的建立可对狮DNA进行特异性检测的PCR方法,应用在检验检疫过程中获得的狮疑似样品和保护执法工作中难以检查辨认的狮产品进行物种鉴定。方法针对狮属动物线粒体细胞色素b(Cytb)基因设计特异性引物,用该引物分别对从亚洲狮、非洲狮和虎、豹、熊等14种非狮源性哺乳动物的肌肉、血液、毛发和肉骨粉中提取的DNA进行PCR(聚合酶链反应)扩增。结果试验表明所设计的引物对狮DNA具有特异性,从而达到了对该物种进行特异性检测鉴定的目的。检测灵敏度可达到在骨粉中检出0.5%含量的狮源性成分。结论该PCR方法具有方便、经济、灵敏度高和重复好等特性,可用于狮源性制品的检测鉴定。     

PCR-based differentiation of three porcine Eimeria species and Isospora suis

Isospora suis and Eimeria are frequent coccidian parasites of pigs. The unsporulated oocysts of Eimeria species and of I. suis are difficult to differentiate. Therefore, a species-specific PCR was developed. PCR products were amplified from Eimeria polita, Eimeria porci, and Eimeria scabra using primers from the conserved 18S rRNA regions and were subsequently sequenced. Based on variable sequence regions, primers were constructed for the differentiation of the three Eimeria species and I. suis. Using a combination of PCRs detecting one or two species, all four coccidian species were detected (theoretical lower detection level: DNA content of 250 oocysts of each Eimeria species or 25 oocysts of Isospora in 1microl) and differentiated. The PCR-based differentiation of the above mentioned species provides a useful alternative to microscopy.     

Identification of trichomonadid protozoa from the bovine preputial cavity by polymerase chain reaction and restriction fragment length polymorphism typing.

Accurate identification of the bovine pathogen Tritrichomonas foetus is sometimes complicated by the presence of other trichomonadid protozoa in clinical samples. A highly specific and reproducible approach for differentiating 3 common types of bovine trichomonadid protozoa found in the bovine preputial cavity, T. foetus, Pentatrichomonas hominis, and a Tetratrichomonas species, was developed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Universal trichomonadid protozoa primers, TFR1 and TFR2, were used to amplify the 5.85 rRNA gene and internal transcribed spacer regions (ITSRs), and the products were digested with the restriction enzyme HpyCH4IV. Restriction fragment length polymorphism analysis was performed on 55 trichomonad isolates from bovine preputial washing and scraping samples. The RFLP results correlated 100% with 5.85 rRNA gene and ITSR sequence resultsand PCR results with primers specific for T. foetus. The results of this study demonstrate that PCR and RFLP analysis can be used in lieu of DNA sequencing to identify the specific trichomonadid protozoa isolated from the bovine preputial cavity.     

羊丝状支原体簇环介导等温扩增检测方法的建立

本试验利用环介导等温扩增技术(LAMP)建立了羊丝状支原体簇的快速检测方法,该方法以羊丝状支原体簇成员的保守性基因16S rRNA为靶序列,设计了4条特异性引物,在65 ℃等温条件下,60 min一步完成反应。在反应管中预先添加羟基萘酚蓝(HNB),阳性呈蓝色,阴性呈紫色。丝状支原体簇成员——丝状支原体山羊亚种(Mmc)、丝状支原体丝状亚种LC型(Mmm LC)、山羊支原体山羊肺炎亚种(Mccp)的LAMP检测为阳性;对其他病原菌没有交叉反应。以Mmc的核酸为模板进行灵敏度检测,LAMP的最低检出限为10 pg/μL。结果表明,本试验建立了一种特异、敏感、快速、简便的羊丝状支原体簇的LAMP方法。     

高寒草甸8种植物种子内生细菌和真菌群落的多样性

种子是微生物从母本植物到子代植物的传播介质,将有益微生物代代相传。种子传播的微生物存在于植物不同部位,为寄主植物提供了一系列的益处。以往种子微生物研究主要集中于农作物,鲜有关于草地植物的研究。本研究利用Illumina-Miseq DNA测序技术,对青藏高原高山草甸8种优势植物的种子微生物群进行多样性分析。结果表明:8种草地植物的种子中有多种细菌和真菌(115个细菌属和135个真菌属),且细菌和真菌群落均存在显著差异(P<0.05)。8种草地植物的优势种细菌门和真菌门分别为Proteobacteria和Ascomycota。在8种草地植物的种子中均发现了细菌属Pseudomonas、Pantoea和Ochrobactrum及真菌属Botrytis、Bullera和Didymella。另外,在8种草地植物的种子中有其独特的细菌和真菌属。本研究表明,植物的个体特征是塑造种子微生物群落的重要因素,同时还发现了一些有利于寄主植物生长且可用于未来种子微生物组调控的微生物。     

Amplification of feline sarcoid‐associated papillomavirus DNA sequences from bovine skin

Feline sarcoids are uncommon dermal neoplasms that are thought to be caused by papillomaviral (PV) infection. Feline sarcoid‐associated PV (FeSarPV) has been consistently detected in sarcoids from North American and New Zealand cats but has not been detected within any other feline sample. This suggests that feline sarcoids may develop due to cross‐species infection by a PV from an unidentified reservoir host. While there is some epidemiological evidence to suggest that cattle are the reservoir host of FeSarPV, this PV has never been identified within any bovine sample. In this study both consensus PCR primers and primers specific to FeSarPV were used to investigate the presence of PV DNA within five fibropapillomas and 18 samples of inflammatory skin disease from cattle. Consensus primers amplified bovine PV‐2 DNA from four fibropapillomas, but none of the dermatitis samples. However, specific primers amplified FeSarPV DNA from four fibropapillomas and five inflammatory skin lesions. To the best of our knowledge this is the first time that FeSarPV has been detected within any sample other than a feline sarcoid. The ability of FeSarPV to asymptomatically infect bovine skin suggests that cattle are the reservoir host of this PV and feline sarcoids could be the result of cross‐species infection of a dead‐end host by a bovine PV.     

Molecular biology can differentiate morphologically indistinguishable Myxosporean species: Myxobolus elegans and M. hungaricus

Two, morphologically indistinguishable myxosporean species, Myxobolus elegans Kashkovsky, 1966 and M. hungaricus Jaczó, 1940 were differentiated using molecular biological methods. Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify an approximately 1600 base pairs (bp) long fragment of the 18S ribosomal RNA gene. In restriction fragment length polymorphism (RFLP) study with HinfI, MspI and TaqI enzymes, the two parasite species were easily distinguishable. The genetic distinctness was also confirmed by the DNA sequence of their PCR products. Although M. elegans and M. hungaricus are morphologically very similar, based on the results of the PCR-RFLP and the DNA sequences, we concluded that they are valid species.     

Polymerase Chain Reaction–Restriction Fragment Length Polymorphism of Mitochondrial 12S rRNA Gene: A Simple Method for Identification of Poultry Meat Species

Chicken (Gallus gallus), duck (Anas platyrhynchos), turkey (Meleagris gallopavo), guinea fowl (Numida meleagris) and quail (Coturnix japonica) are the common poultry species consumed as meat throughout the world. In this work, a molecular technique has been developed for identification and differentiation of meat originating from these species. This tool helps in detection of misrepresentation of different poultry meats. The technique involves the extraction of DNA from the given sample, polymerase chain reaction (PCR) amplification of mitochondrial 12S rRNA gene using universal primers, restriction analysis with selected restriction enzymes, followed by identification of meat species based on restriction fragment length polymorphism (RFLP) pattern. In this study, we used HinfI, Mph1103I, MvaI, and Eco47I to identify and differentiate to poultry species referred to above. This species identification technique has also been applied successfully to processed meat products including those cooked at 120^∘C for 30 min. Simplicity of interpretation of results combined with versatility makes this a convenient and appropriate technique in the hands of meat analysts for identifying poultry meat species.