特异性引物PCR鉴定鸡球虫种类与卵囊纯度的研究

为了建立鸡球虫种类的快速分子生物学鉴定方法,检测实验室保存虫株受污染状况,分别对单卵囊分离繁殖及实验室长期传代保存的6株巨型艾美耳球虫（EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01）、4株柔嫩艾美耳球虫（ETDS01、ETGD01、ETAD和ETAM）、1株堆形艾美耳球虫（EA1201）、1株变位艾美耳球虫（EMIS01）、1株毒害艾美耳球虫（ENGD01）收集卵囊,纯化、提取总DNA,根据巨型艾美耳球虫、柔嫩艾美耳球虫、堆形艾美耳球虫的RAPD和SCAR分子标记、ITS-1区序列分别设计Tn-F与Tn-R、Mx-F与Mx-R、Ac-F与Ac-R、ET-1与ET-2、EM-1与EM-2、EA-1与EA-2等6对特异性引物,对13个虫株分别进行PCR扩增,1%琼脂糖电泳分析片段大小.结果显示：用引物Tn-F与Tn-R、ET-1与ET-2对ETDS01、ETGD01、ETAD、ETAM、EMTO01扩增出特异性条带,其余4种8个虫株未见条带;用引物Mx-F与Mx-R、EM-1与EM-2对EMSH01、EM4101、EMES01、EMBA01、EMTY01和EMTO01扩增出特异性条带,其余4种7个虫株未见条带;用引物Ac-F与Ac-R、EA-1与EA-2对EA1201扩增出特异性条带,其余4种12个虫株未见条带.结果说明特异性引物PCR方法鉴定的5种球虫与原常规生物学方法鉴定的结果一致,检测出保存的巨型艾美耳球虫EMTO01虫株受柔嫩艾美耳球虫污染,其余虫株未发现交叉污染.研究证明利用特异性引物建立的PCR方法可用于鸡球虫种类与卵囊纯度的鉴定.     

Humoral immune response to structural and nonstructural proteins of tick-borne encephalitis virus in the natural history of the disease

A hundred and ninety-one patients who had fallen ill with tick-borne encephalitis (TBE) after bites by virus-carrying ticks were examined to reveal the characteristics of antibody production of the major classes of immunoglobulins (IgM and IgG) against structural protein E of the TBE virus and nonstructural protein NS1 of the TBE viral replicative complex in the natural history of an infectious process in different forms and variants of the disease. In the two-wave course of TBE, the infectious process is characterized by a delayed antibody production, lower levels of specific antibodies against virion protein E in the acute phase of disease during the first wave of fever. The development of meningocerebral syndromes at the peak of the second fever wave is observed in patients with the delayed accumulation and low levels of IgG antibodies. Of great diagnostic and prognostic value is the detection of nonstructural protein NS1 antibodies on the first days of TBE, by taking into account the uncertainty of clinical criteria for the disease at its first (febrile) wave and the low titers (or their absence) of specific antibodies against virion protein E, detectable at that time in the sera of patient with the two-wave course of TBE.     

Immune Responses in Infections with Coccidia: Macrophage Activity

Peritoneal exudate cells from chickens immunized with two species of coccidia, Eimeria tenella or Eimeria maxima, were examined for their capacity to phagocytose stages of the parasite in vitro. True phagocytosis of the sporozoite stage is difficult to estimate because of its ability to invade cells, but may be evaluated by comparison with control suspensions. Peak activity (compared with cells from coccidia-free chickens) was found 3 to 5 weeks after the first inoulum of oocysts of E. tenella, and 1 week after the first inoculum of E. maxima- times which correspond to the onset of complete immunity to infection. Cells from coccidia-free chickens, in the presence of serum from birds immunized with E. tenella, phagocytosed sporozoites of E. tenella in a similar manner to cells from immunized birds. The immune serum had both cytophilic and opsonic adherence properties and the latter was species specific (for the two species tested).     

Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic .Eimeria species of the chicken.

We describe a polymerase chain reaction (PCR)-based assay for the detection, identification and differentiation of pathogenic species of .Eimeria in poultry. The internal transcribed spacer 1 (ITS1) regions of ribosomal DNA (rDNA) from .Eimeria acervulina, E. brunetti, E. necatrix and .E. tenella were sequenced and regions of unique sequences identified. Four pairs of oligonucleotide primers, each designed to amplify the ITS1 region of a single .Eimeria species, were synthesised for use in the PCR assay. In tests on purified genomic DNA from all seven species of .Eimeria that infect the chicken, each of the four primer pairs amplified the ITS1 region from only their respective target species. The robustness of the approach was further demonstrated by the amplification of specific DNA fragments from tissues of experimentally infected animals and from oocysts recovered from field samples. We conclude that the ITS1 regions of .Eimeria species contain sufficient inter-specific sequence variation to enable the selection of primers that can be applied in PCR analyses to detect and differentiate between species. In future work they may provide excellent markers for epidemiological studies.     

First record of Eimeria furonis infection in a ferret, Japan, with notes on the usefulness of partial small subunit ribosomal RNA gene sequencing analysis for discriminating among Eimeria species

Eimeria is a genus of apicomplexan parasites found in a variety of vertebrates including the weasel. At present, three species have been reported in members of the weasel family, but the presence of weasel Eimeria in Japan have been quite unclear. The identification of Eimeria species has been performed based on oocyst morphology, host species, and habitat in the host, but sometimes discriminating among morphologically similar species under light microscopy is impossible. The present study detected for the first time the weasel coccidium, E. furonis, in a ferret in Japan. Since molecular information for E. furonis has been quite unclear, this species was compared molecularly with other Eimeria species, and also the usefulness of sequencing analysis of the small subunit ribosomal ribonucleic acid gene (SSUrDNA) for discriminating among Eimeria species was examined. About the 350-bp sequence of SSUrDNA of all species including E. furonis compared differed from each other, and its differences found in Eimeria species were also reflected in the phylogram constructed using the partial nucleotide sequences. The sequencing analysis of partial SSUrDNA examined in the present study thus appears useful for discriminating among morphologically similar Eimeria species.     

Identification of seven Eimeria species in Swedish domestic fowl.

The aim of the study, conducted during the period 1992 to 1996, was to identify the Eimeria species present in Swedish chickens. All samples, including litter, faeces and guts from dead birds submitted for coccidial diagnosis, were obtained from farms where no live coccidiosis vaccines had ever been used. Identification of the different species was based on the criteria of oocyst morphology, location and characteristics of intestinal lesions, morphology of parasite endogenous stages, prepaient time and isoenzyme electrophoresis profiles of glucose phosphate isomerase. All seven Eimeria species of the domestic fowl were identified, namely E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella. Furthermore, Swedish monospecific isolates of E. maxima, E. necatrix and E. tenella were established.     

A survey of the inter- and intraspecific RAPD markers of <Emphasis Type="Italic">Eimeria</Emphasis> spp. of the domestic fowl and the development of reliable diagnostic tools

Coccidiosis of domestic fowl is a protozoan disease, caused by seven distinct species of the genus Eimeria, which is responsible for important economic losses in poultry production. In order to select RAPD primers for the discrimination of these seven Eimeria species, we carried out an initial screening using samples of E. acervulina, E. tenella and E. maxima.Out of 150 primers tested, 110 generated band profiles specific for each one of these species. A subset of 14 oligonucleotides were also tested for the simultaneous differentiation of the seven species, resulting in 11 discriminative primers. The intraspecific discrimination was assessed for five different species, using samples from different geographic regions including three continents. Numerous primers exhibited highly discriminative band profiles containing strain-specific markers, with a higher variability being observed among strains of E. acervulina than among E. tenella and E. maximastrains. However, no major differences were observed in the band patterns from strains collected in locations near to one another compared to strains originating from distantly located regions. Because RAPD is a technique performed under low stringency conditions, it suffers from poor reproducibility. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific RAPD fragments into SCAR markers. An initial conversion of 25 RAPD markers into SCARs, followed by validation of their specificity, resulted in 14 totally new Eimeria species-specific markers that can be used for the molecular diagnosis of the seven species that infect domestic fowl. This work represents a first step in the development of a set of species-specific SCARs that will be useful as tools for molecular diagnosis, genome mapping, and genetic diversity studies.     

Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.     

The observation of coccidia in swine

The coccidia species of domestic pig and wild boar were compared and defined morphologically and the sporogony of both was examined. No difference could be detected between the coccidia species of domestic pig and wild boar. Six Eimeria and one Isospora species were found: Eimeria debliecki, E. polita, E. porci, E. scabra, E. spinosa, E. suis and Isospora suis. E porci could be found in Austria for the first time. E. cerdonis Vetterling, 1965 is a synonym of E. polita Pellérdy, 1949.     

Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil Nadu, India

Coccidosis is one of the most commonly prevalent and economically important parasitic diseases of poultry worldwide. Chicken coccidia are protozoan parasites of the genus Eimeria. This study aimed at analysing the molecular prevalence of seven species of Eimeria infecting chickens in Tamil Nadu, India. Tissue samples (caecum, rectum and upper and mid intestines) collected from chickens exhibiting symptoms of coccidiosis were used for DNA extraction, followed by amplification of the internal transcribed spacer (ITS) region of Eimeria genome with genus-specific primers and speciation in nested polymerase chain reaction (PCR) with species-specific primers. Of 43 tissue samples examined, 25 were positive in ITS PCR and all the seven species could be identified. However, the prevalence of each species varied. In broilers, Eimeria necatrix was present in all infected chickens with Eimeria brunetti, Eimeria tenella, Eimeria maxima and Eimeria acervulina present in more than 50% of infected chickens, while Eimeria praecox and Eimeria mitis were only present in 11% to 16%. Although only 7 samples were positive among layers, the prevalence was largely similar, but with a higher prevalence of E. praecox and E. mitis and a lower prevalence of E. tenella. Multiple infections were most common, with 2–6 Eimeria species infecting the same chickens. In order to estimate the preponderance of each infecting species of Eimeria, a random cloning technique was adopted. The genus-specific ITS PCR product was cloned in a TA vector and ten clones were randomly picked and used as template for amplification of all the seven genera of Eimeria. If the specific species of Eimeria is preponderant, then the frequency of the clones showing that species-specific PCR amplification would be higher. Using this method, the most preponderant species present in the rectum, mid and upper intestines of layers was assessed to be E. acervulina, E. brunetti and E. necatrix. E. acervulina was present in 60–90%, E. necatrix in 10–30% and E. brunetti in 10–20% of the clones screened, indicating that these species could be the most preponderant Eimeria species. Intervention strategies should aim at these species. This new method of estimating preponderance of infecting Eimeria species could be used to assess the relative importance of each species at the farm or region level instead of relying only on prevalence estimates.     

Comparison of ion-pair and amide-based column reversed-phase liquid chromatography for the separation of thiamine-related compounds.

Two reversed-phase chromatographic methods for the separation of thiamine and related compounds are compared. The first procedure is based on the ion-pair technique using an octadecylsilica column, while the second uses a new amide-based stationary phase, which avoids the need to form ion-pairs, leading to narrower peaks and a simpler mobile phase. Analyses were performed by gradient elution and a photo-diode array was used for detection. Specificity was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity index with commercial standards. The procedures were applied to the determination of thiamine-related compounds in pharmaceutical preparations and urine. No preliminary sample treatment was required.     

Molecular phylogenies suggest the oocyst residuum can be used to distinguish two independent lineages of Eimeria spp in rodents

Using plastid 23S and nuclear 18S rDNA partial sequences for 16 Eimeria species from rodents, we compared their molecular phylogenetic inferences with morphological features and host specificity. The 16 ingroup taxa included Eimeria species which had different morphological features, but were from the same host genus or species, and species which had similar morphological features, but were from different host families or genera. Molecular phylogenies grouped the 16 rodent Eimeria species into two major lineages with high bootstrap support: lineage A included E. albigulae (from Neotoma), E. arizonensis (Peromyscus, Reithrodontomys), E. chaetodipi (Chaetodipus), E. chobotari (Dipodomys), E. dipodomlysis (Dipodomys), E. leucopi (Peromyscus), E. onychomysis (Onychomys), E. peromysci (Peromyscus) and E. reedi (Perognathus); and lineage B included E. falciformis (Mus), E. langebarteli (Peromyscus, Reithrodontomys), E. nieschulzi (Rattus), E. papillata (Mus), E. scholtysecki (Dipodomys), E. separata (Rattus) and E. sevilletensis (Onychomys). Examination of the morphological features of all 16 Eimeria species indicates that only the oocyst residuum shows a clear correlation to the phylogenetic relationships determined by the molecular data. Species in lineage A all contain one (or more) oocyst residuum in their sporulated oocysts, while species in lineage B lack an oocyst residuum in their sporulated oocysts. Considering that the host range of the Eimeria species used in this study includes nine genera in two families and that each eimeriid lineage contains species from both families, it seems likely that the two Eimeria lineages split before their host families diverged.     

A survey of Eimeria species in commercially-reared chickens in France during 1994

In a survey of chicken coccidia in France during 1994, samples of litter were collected from 41 farms. On 31 of these farms, eimerian oocysts were abundant enough to allow monitoring of their numbers in the litter. Peak total oocyst counts on these farms ranged from 16,200 to 1,254,000/g of litter, but no coccidiosis was observed. The chickens reared without anticoccidial agents in their food (poulets biologiques) produced higher and earlier peak oocyst counts in litter than the chickens given medicated food (poulets labels). The oocysts in litter samples from 22 farms (13 poulet biologique, five poulet label, two standard broiler, one breeder and one layer) of the original 41 were identified. Six of the seven eimerian species known to parasitize chickens were found, using combinations of five methods (oocyst morphology, intestinal lesions, enzyme electrophoresis, growth in embryonating eggs and prepatent time). Multispecific infections predominated (95% of 22 farms), up to six species occurring together. Of farms where oocysts were detected, the percentages with each species were: Eimeria acervulina (100%), E. mitis (82%), E. tenella (77%), E. maxima (73%), E. praecox (45%) and E. brunetti (27%). These appear to be the first definite records of E. mitis and E. praecox for France. Although E. necatrix was not found in this survey, it had recently been detected by other workers in France, so that all seven chicken Eimeria species were known to be contemporaneous.     

Cross-reactivity of anti-Eimeria tenella antibody fragments on merozoites and sporozoites of different chicken Eimeria species

Eimeria tenella-specific antibodies were examined for the cross-reactivity on the sporozoites and merozoites of E. tenella, Eimeria maxima, Eimeria acervulina and Eimeria brunetti in an indirect fluorescence antibody test. Two of nine antibodies showed cross-reactivity with sporozoites of E. maxima, E. acervulina and E. brunetti; however, the localization of specific fluorescence differed between species. No antibody binding was observed on merozoites. The suitability of these antibodies to alter the infectivity of Eimeria sporozoites and/or merozoites must be verified in cell culture models and in vivo experimental infections.     

和缓艾美耳球虫激发宿主免疫应答的初步研究

和缓艾美耳球虫Eimeriamitis是7种鸡球虫中致病力弱的虫种,对于其免疫原性的认识至今仍有争论。本研究以和缓艾美耳球虫涿州株为研究对象,以初次免疫及二次免疫后鸡的卵囊排出量为标准评估了其免疫原性。同时利用ELISA检测了免疫后鸡群血清中球虫特异性IgY抗体的水平,并通过ELISPOT技术分析了二免后鸡群外周血单核淋巴细胞（PBMC）中分泌IFN-γ的特异性淋巴细胞的数量。结果显示,二次免疫后,鸡的卵囊排出量显著低于初次免疫,免疫组的血清特异性IgY抗体水平较低,而分泌IFN-γ的特异性淋巴细胞的数量较高。这些结果表明和缓艾美耳球虫涿州株具备良好免疫原性,且其激发宿主产生的保护性免疫以细胞免疫应答为主,有作为疫苗株的潜力。     

Reporter gene expression in cell culture stages and oocysts of <Emphasis Type="Italic">Eimeria nieschulzi</Emphasis> (Coccidia,Apicomplexa)

The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria. We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo. The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection. Pyrimethamine resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene. Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages. Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts. Co-transfection with YFP/DHFR-TS allowed selection of resistant parasites in vivo. Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms.     

A laboratory method for the single-passage selection of drug-resistant mutants from populations of Eimeria species in chickens and its potential value in the choice of field use concentrations for new anticoccidial drugs.

Populations of Eimeria species in chickens frequently include mutants that are resistant to an anticoccidial drug without having been previously exposed to it. Such mutants are described here as 'inherently resistant'. A novel laboratory method of selecting them from their parent populations is described. Selection may be achieved in a single in vivo passage in chickens. Decoquinate-resistant mutants were selected from a previously unexposed E. tenella strain: they tolerated < 0.625% w/w decoquinate in the food of chicks, whilst the rest of the population was sensitive to > 0.002% w/w. This single-passage method has a unique advantage over others; development of resistance to different drugs may be validly compared by using a single criterion, the lowest selective concentration (LSC), all other experimental factors remaining constant. Furthermore, the method is economical with time, facilities and experimental animals, and may have a predictive value in the choice of use concentrations for new anticoccidial agents. A potential selection index (PSI), the proposed or actual field use concentration of a drug divided by its LSC, is defined. It is suggested that a field use concentration for a new drug should be somewhat lower than the LSC, and the overall PSI for all Eimeria species and strains tested should therefore be < 1. In the present study, the PSI for decoquinate and E. tenella was 8.00, which is consistent with the very rapid development of resistance to quinolones that occurred in the field.     

Prevalence of Eimeria bovis and Eimeria zuernii in German cattle herds and factors influencing oocyst excretion

The present study was designed to investigate the prevalence of the pathogenic coccidia species E. bovis and E. zuernii in shed-reared animals in German dairy and fattening facilities.Samples were obtained from 65 cattle farms distributed randomly across all the regions of Germany, regardless of the occurrence of clinical problems. The samples were obtained rectally. Faecal consistency and the total number of oocysts per gram of faeces (OPG) were determined for Eimeria spp., along with the separate OPG values for Eimeria (E.) bovis and E. zuernii. A questionnaire was completed for each farm to record information about herd size and management together with individual animal data. Eimeria oocysts, regardless of the kind of Eimeria spp., were detected in 62 of these farms, which gives a prevalence of 95.4 %. The farm prevalence of the pathogenic species was 76.9 % for E. bovis and 83.1 % for E. zuernii. The average oocyst excretion level was 2,950 OPG in terms of total Eimeria spp. oocyst excretion, 700 OPG for E. bovis and 1,500 OPG for E. zuernii.The number of oocysts excreted could not be correlated significantly with farm type or farm management but depended on the floor type which influences the infection pressure, on the age of the calves and the time after rehousing. In general, higher oocyst excretion rates were found in calves kept on litter compared to rearing on slatted floor. Younger calves and calves sampled early after housing shed higher amounts of oocysts than older calves and calves stabled a longer period before sampling, respectively. Furthermore, there was a positive correlation between OPG and the observation of diarrhoea, defined as observation of a loose to liquid faecal consistency. Excretion of E. zuernii oocysts was more closely linked to the occurrence of diarrhoea than E. bovis oocyst excretion. This study confirms that the pathogenic coccidia E. bovis and E. zuernii are ubiquitous in German cattle populations and a significant cause of diarrhoeal disease in calf rearing.     

Automatic detection of ultrasonic Doppler signal episodes resulting from fetal breathing movements

A method for automatic detection of fetal breathing movements has been proposed, based on the time-frequency structure of the corresponding continuous wave ultrasonic Doppler signals. The method uses spectral analysis of the envelope of the directional Doppler signal and cross-correlation analysis of both directional envelopes. Detection rule comprises the following criteria: presence of the peak in the envelope spectrum and of the adequate signal level in the frequency range corresponding to the fetal breathing rhythm, the peak value and the position limits of the peak of the cross-correlation coefficient of the both directional envelopes. The effect of the criteria setting on the rule performance and the tradeoff between the specificity and sensitivity was investigated. The rule is most sensitive to the threshold value of the cross-correlation coefficient of the envelopes. The limits of the position of this peak are crucial for the distinction between the breathing episodes and hiccups. The optimal settings of the criteria, resulting in average sensitivity and specificity exceeding, respectively, 0.70 and 0.80, are proposed.     

Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid chromatography.

A complete two-dimensional separation technique for the determination of acidic compounds in plasma was developed by using column-switching reversed-phase liquid chromatography. This technique was based on solute peak enrichment at the top of the second column during heart-cutting and an ion-pair chromatographic separation in the second column using tetrabutylammonium ion, where different separation modes in the first and second columns and solute peak enrichment at the top of the second column during heart-cutting were achieved coincidentally. Retention behaviors of two solutes, zidovudine-beta-D-glucuronide (AZT-beta-D-Gluc) and probenecid, in the first and second column and solute peak enrichment at the top of the second column were investigated for establishment of the system. Different retention behaviors of the solutes in the first and second column, which were evaluated by changes in capacity factor versus acetonitrile concentration in the mobile phases, and peak enrichment could be accomplished by using ion-pair chromatography in the second column. System suitability was confirmed by assessing the number of theoretical plates (N) of the second column for the solutes after heart-cutting. The N values in the second column after column switching were almost same as those in the case that the solutes were directly injected onto the second column. These results indicate that complete two-dimensional separation should be achieved by using this system. Furthermore, this technique was applied to method development for the determination of AZT-beta-D-Gluc and probenecid in rat plasma. The peaks of each analyte in the plasma extract obtained by deproteinization were well separated from those of endogenous substances, and easy determination of the analytes could be accomplished at the ng/ml level only by changing the acetonitrile concentration in the mobile phases.