A phylogenetic analysis by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting of the Leishmania genome project Friedlin reference strain.

We have assessed the phylogenetic status of the Leishmania genome project Friedlin reference strain by MLEE and multiprimer RAPD including a set of 9 stocks representative of the main Leishmania species and of the whole genetic diversity of the Leishmania genus. To our knowledge, the detailed genetic characterization of the Friedlin strain has never been published before. As previously recorded (Tibayrenc et al. 1993), MLEE and RAPD data gave congruent phylogenetic results. The Friedlin reference strain was definitely attributed to Leishmania (Leishmania) major Yakimoff et Schokhor, 1914. Five specific RAPD patterns made it possible to distinguish between the Friedlin strain and the 2 other L. (L.) major stocks included in the study. Various specific MLEE and RAPD characters permitted to distinguish between the Leishmania species included in the study. All these characters are usable to detect accidental laboratory mix-ups involving the Friedlin reference strain. In confirmation with previous studies involving a more limited set of genetic markers, the general genetic diversity of the Leishmania genus proved to be considerable. It must be made clear that only one strain cannot be considered as representative of the whole genetic variability of the genus Leishmania. In the future, it is therefore advisable to complement the results obtained in the framework of the Leishmania genome project with data from other strains that should be selected on a criterion of important genetic differences with the Friedlin strain.     

Virulent and attenuated lines of Leishmania major: DNA karyotypes and differences in metalloproteinase GP63

The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host.     

Sand fly saliva: effects on host immune response and Leishmania transmission

The feeding success of sand flies (Diptera: Phlebotominae) is linked to the vast array of pharmacological substances in their saliva, which interferes with the host haemostasis and immune response. Modification of feeding site plays also an important role in Leishmania transmission. In naive hosts, co-inoculation of saliva and Leishmania parasites increases the chance of successful transmission. Disease exacerbation seems to be associated with enhanced production of type 2 cytokines and selective inhibition of some macrophage functions including the production of NO and H202. On the other hand, hosts repeatedly exposed to sand fly bites develop anti-saliva immune response that results in a protection against Leishmania infection. This led to a new interesting approach to anti-Leishmania vaccine--using salivary components to block parasite transmission. The review is therefore focused on the interactions that run between immunomodulatory molecules in sand fly saliva and host immune response, with the impact on Leishmania infection development. Recent studies revealed that saliva-based vaccine for leishmaniasis might be effective and feasible, however, several questions still require to be solved. The knowledge based on experimental mouse model cannot be fully extrapolated to dogs or humans and due to differences in salivary antigens between sand fly species the protective effect is species-specific. On the other hand, the specificity of salivary antigens enables the use of anti-saliva antibodies for monitoring the exposure of hosts to sand fly bites and might be used as a marker of risks for Leishmania transmission in endemic areas.     

Leishmania tropica (Kinetoplastida: Trypanosomatidae)--a perplexing parasite

Leishmania tropica is one of the causative agents of cutaneous leishmaniasis (CL), a disfiguring parasitic disease that recently was found to be viscerotropic. In urban areas it is transmitted from infected individuals by the bite of phlebotomine sand flies to na?ve persons (anthroponotic CL). In rural areas animals are thought to be the reservoir, but the full life cycle is still under investigation (zoonotic CL). For many years L. tropica was either confused or merely grouped with L. major while Phlebotomus sergenti was the only proven vector. In recent years new foci have erupted, but few have been investigated. This review describes some of the history, recent findings, epidemiology, potential vectors, and the search for possible reservoir hosts besides man.     

Biological, serological, and molecular variabilities of clover yellow vein virus

ABSTRACT A comparative study was made on the host reactions, serological properties, and nucleotide sequences of the coat protein (CP) gene of 10 clover yellow vein virus (C1YVV) isolates and one bean yellow mosaic virus (BYMV) isolate collected from different host plant species and locations in Japan. Two strains of C1YVV isolates, grouped on the basis of host reactions on Chenopodium amaranticolor, C. quinoa, Nicotianaclevelandii, N. benthamiana, Vicia faba, and Trifolium repens, corresponded to two serotypes determined by double-antibody sandwich- and triple-antibody sandwich-enzyme-linked immunosorbent assay using three polyclonal and nine monoclonal antibodies. These results were also confirmed by nucleotide sequence analysis of the CP gene. The CP gene of C1YVV isolates of strain 1, including the Australian isolate C1YVV-B, had 93 to 98% nucleotide identities and 97 to 99.6% amino acid identities. The CP of C1YVV isolates of strain 2, including the New Zealand isolate C1YVV-NZ, had 92 to 98% nucleotide identities and 95 to 98% amino acid identities. The nucleotide identities and the amino acid identities between the two C1YVV strains were 82 to 84%, and 90 to 94%, respectively. When compared with the CP sequences of 12 C1YVV isolates, the CP sequence of the BYMV isolate had 71 to 73% nucleotide identity and 73 to 77% amino acid identity. Amino acid sequence differences among C1YVV isolates from strains 1 and 2 were located mostly at the N-terminal regions of the CP. Our results indicated that the C1YVV isolates studied could be separated into two strains on the basis of host reactions, serology, and the nucleotide sequence of the CP gene.     

Genetic analysis of streptomycin-resistant (Sm(R)) strains of Erwinia amylovora suggests that dissemination of two genotypes is responsible for the current distribution of Sm(R) E. amylovora in Michigan

Streptomycin-resistant (Sm(R)) strains of the fire blight pathogen Erwinia amylovora were first isolated in southwest Michigan in 1991. Since that time, resistant strains have progressed northward to other apple-producing regions in the state. A total of 98.7% of Sm(R) strains isolated between 2003 and 2009 in Michigan harbored the strA-strB genes on transposon Tn5393. strA and strB encode phosphotransferase enzymes that modify streptomycin to a nonbactericidal form. Mutational resistance to streptomycin, caused by a point mutation-mediated target-site alteration of the ribosomal S12 protein, occurred in 1.3% of E. amylovora strains from Michigan. Tn5393 was originally introduced to E. amylovora on the plasmid pEa34; thus, the first Sm(R) strains isolated contained both pEa34 and the ubiquitous nonconjugative plasmid pEA29. More recently, we have observed Sm(R) strains in which Tn5393 is present on pEA29, suggesting that the transposon has moved via transposition from pEa34 to pEA29. Almost all of the strains containing Tn5393 on pEA29 had lost pEa34. Of 210 pEA29::Tn5393 plasmids examined, the transposon was inserted at either nucleotide position 1,515 or 17,527. Both of these positions were in noncoding regions of pEA29. Comparative sequencing of the housekeeping genes groEL and potentially variable sequences on pEA29 was done in an attempt to genetically distinguish Sm(R) strains from streptomycin-sensitive (Sm(S)) strains isolated in Michigan. Only 1 nucleotide difference within the total 2,660 bp sequenced from each strain was observed in 2 of 29 strains; multiple sequence differences were observed between the Michigan strains and E. amylovora control strains isolated in the western United States or from Rubus spp. Alterations in virulence observable using an immature pear fruit assay were detected in three of eight Sm(R) strains examined. Our current genetic data indicate that only two Sm(R) strain genotypes (strains containing pEA29::Tn5393 with Tn5393 inserted at either nucleotide position 1,515 or 17,527 on the plasmid) are responsible for the dissemination of Tn5393-encoded streptomycin resistance in Michigan, and that the Sm(R) and Sm(S) strains in Michigan compose a homogenous group.     

Serological Analysis and Coat Protein Sequence Determination of Potato Virus Y (PVY) Pepper Pathotypes and Differentiation from Other PVY Strains

The serological relationships of Potato Virus Y (PVY) isolates belonging to the pepper pathotypes 0, 1 and 1-2 were established by enzyme-linked immunosorbent assay (ELISA). PVY pepper pathotypes did not react with monoclonal antibodies which typically recognize non-pepper strains within the PVY group, leading to discrimination between these two groups of strains. No serological differences were found between the three PVY pepper pathotypes. The coat protein (CP) nucleotide and predicted amino acid sequences of the three different PVY pepper pathotypes were determined. The highest sequence similarity was found between pathotypes 0 and 1 (99.2%), while the lowest occurred between these two and pathotype 1–2 (98.1%). PVY strains from potato and tobacco appeared more distantly related. Phenetic analysis of the CP amino acid sequences showed that the PVY pepper pathotypes formed a tightly clustered group separate from other PVY strains.     

Assessing insecticide resistance and aversion to methomyl-treated toxic baits in Musca domestica L (Diptera: Muscidae) populations in southern California

Progeny of house flies (Musca domestica L) from ten California poultry operations, three dairies and one horse-riding facility were tested for methomyl- and muscalure-treated bait resistance using up to three different assays: a topical assay, a no-choice feeding assay and a choice feeding assay. LD50 resistance factors from the topical assay, compared with a locally-derived susceptible colony, ranged between 1 and 4. LC50 resistance factors from the no-choice feeding assay ranged mostly between 2 and 5, with one value of 18. Measurable LT50 resistance ratios for female flies in the choice feeding assay ranged from 43 to 159; two populations had <10% mortality at 48h and could not be measured. LT50 resistance ratios for male flies in the choice feeding assay ranged from 26 to 96, and one population was too resistant to measure. A behavioral assay tested the feeding preference of male and female flies provided a dish of sugar and a dish of methomyl- and muscalure-treated bait. Of eight strains tested, females from seven strains and males from six strains showed significant preference for sugar over bait. Behavioral factors appear to be important in the severe resistance of house flies to baits in California.     

Comparison of the minor coat protein gene sequences of aphid-transmissible and -nontransmissible isolates of <Emphasis Type="Italic">Citrus tristeza virus</Emphasis>

The nucleotide sequences for the minor coat protein (CPm) gene and its deduced amino acid sequences for two aphid-transmissible and two nontransmissible isolates of Citrus tristeza virus (CTV) from symptomless orchard trees of Miyagawa satsuma [Citrus unshiu (Macf.) Marc.] on trifoliate orange [Poncirus trifoliate (L.) Raf.] and declining Washington navel [C. sinensis (L.) Osb.] trees on sour orange (C. aurantium L.) rootstocks were analyzed and compared with those of highly transmissible CTV strains available in GenBank. The isolates produced severe symptoms on indicator plants and their aphid transmissibility was assayed through acquisition by A. gossypii of CTV and subsequent inoculation feeding on young Mexican lime seedlings. The CPm gene nucleotides and coded amino acid sequences were very similar among the nontransmissible isolates and among the transmissible. Five of 73 nucleotide substitutions that existed between CPm gene nucleotide sequence of nontransmissible and transmissible isolates caused changes in the deduced amino acid sequences of the nontransmissible isolates. Two nucleotide substitutions yielded new amino acids with similar properties. However, the three remaining mutations led to substitution of new amino acids with a different charge and polarity at positions 14, 238 and 239. The last two mutations occurred at the C-terminal region of the CPm, which is implicated in the formation of a salt bridge that helps to maintain the protein’s tertiary structure. Amino acid substitutions can affect aphid transmission efficiency by altering the conformation of the proteins or masking motifs involved in the interaction between CPm and aphid stylets.     

Use of pyrosequencing to quantify incidence of a specific Aspergillus flavus strain within complex fungal communities associated with commercial cotton crops

Atoxigenic strains of Aspergillus flavus have been used as aflatoxin management tools on over 50,000 hectares of commercial crops since 2000. To assess treatment efficacy, atoxigenic strain incidence is routinely monitored by vegetative compatibility analyses (VCA) that require culturing, generation of auxotrophs, and complementation with tester mutants. Two pyrosequencing assays (PA) that require no culturing were developed for monitoring incidences of atoxigenic strains on ginned cottonseed. The assays, which quantify frequencies of characteristic single nucleotide polymorphisms (SNPs) in the aflR and pksA genes, were validated against standard VCA on cottonseed collected from commercial gins in South Texas, Arizona, and Southern California where the atoxigenic strain AF36 is used to manage aflatoxin contamination. Cottonseed washings were subjected to both VCA and PA. PA was performed directly on DNA isolated from particulates pelleted from the wash water by centrifugation. Addition of CaCl(2) and diatomaceous earth prior to pelleting increased the amount of DNA isolated. Accuracy and reproducibility of the PA were contrasted with those for the VCA that has been used for over a decade. Correlation coefficients between VCA and PA indicated good correspondence between the results from the two assays (r = 0.91 for aflR assay and r = 0.80 for pksA assay). PAs were highly variable for samples with low incidences of A. flavus due to variability in the initial polymerase chain reaction step. This held for both DNA isolated from cottonseed washes and for mixtures of purified DNA. For samples yielding low quantities of A. flavus DNA, averaging of results from 4 to 5 replicates was required to achieve acceptable correlations with VCA. Pyrosequencing has the potential to become a powerful tool for monitoring atoxigenic strains within complex A. flavus communities without limitations imposed by traditional culturing methods.     

不同书虱品系的磷化氢半数击倒时间与抗性相关性研究

参照FAO推荐方法测定了磷化氢对嗜卷书虱(Liposcelis bostrychophila(Badonnel))2个品系、嗜虫书虱(L.entomophila(Enderlein))3个品系和小眼书虱(L.paeta(Pearman))3个品系等共8个不同来源书虱的毒力和抗性系数Rf,测定了磷化氢浓度为2mg/mL条件下供试各书虱品系的半数击倒时间KT50,分析了其LC50值与KT50值的相关性。结果为:3种书虱的8个品系对磷化氢的抗性差异明显,高抗性的嗜虫书虱的磷化氢抗性比敏感的嗜卷书虱高1000倍以上,各品系书虱的KT50值与Rf值呈正相关,相应LC50值之对数与KT50值的对数呈直线相关。结果表明,磷化氢对书虱成虫的半数击倒时间KT50值可以用来判断书虱抗性大小,并可用于快速判断抗性以指导现场熏蒸。     

The Complete Nucleotide Sequence of Potato virus X Strain OS: The First Complete Sequence of a Japanese Isolate

The complete nucleotide sequence of the genomic RNA of a Japanese isolate of Potato virus X (PVX), strain OS (PVX-OS), was determined. The PVX-OS genome is 6435 nucleotides in length, excluding the 3′ poly(A) sequence, and encodes for five open reading frames (ORFs), similar to other reported PVX strains. Comparisons of the nucleotide sequence of PVX-OS with those of European and South American PVX strains revealed that the PVX-OS genomic sequence showed 95-96% homology with European strains and 77-78% with South American strains. Phylogenetic analysis of PVX-OS and other PVX strains found that PVX-OS clusters with European strains. This is the first report on the complete nucleotide sequence of a PVX strain from Japan. Received 10 May 2001/ Accepted in revised form 26 October 2001     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.     

实时荧光RT-PCR-步法检测南方菜豆花叶病毒

南方菜豆花叶病毒(Southern bean mosaic virus,SBMV)是我国二类检疫性有害生物,以南方菜豆花叶病毒日本分离物(SBMV-J)总RNA为模板,采用RT-PCR方法扩增病毒外壳蛋白基因及其上游基因的cDNA片断并将其克隆到pMD18-T载体上。序列分析结果表明:SBMV-J cp基因由801个核苷酸组成,编码266个氨基酸,SBMV-J与其它分离物及株系cp基因的核苷酸序列同源性为83%～97%,氨基酸序列同源性为86%～97%。由于SBMV各分离物及株系cp基因的同源性较低,难于设计出较长的普通PCR引物。通过较短引物设计和TaqMan-MGB探针技术,建立了SBMV的实时荧光RT-PCR一步检测方法。该方法的检测低限是0．16 pg,最佳检测总RNA的量是0．16 ng。     

拮抗罗非鱼养殖水体中海豚链球菌功能性微生物的筛选

以罗非鱼链球菌病的病原——海豚链球菌(Streptococcus iniae)为目标菌株,研究了多株分离自罗非鱼养殖水体具有净化水质功能的微生物对其的拮抗作用。结果表明:通过初筛获得了11株对海豚链球菌具有拮抗功能的微生物菌株,其中7株拮抗效果较为显著,分别属于芽孢杆菌(B0903,B0910,B0916)、乳杆菌(L0914,L0917)、假丝酵母(C0909)和诺卡氏菌(N0906)。对这7株菌株从生长优势、共凝集率、净化水质能力等三方面进行进一步的筛选,发现7株菌株的生长系数均大于海豚链球菌,L0917、C0909、N0906的倍增时间显著低于海豚链球菌,且L0914、C0909、N0906能与海豚链球菌产生共凝集作用,共凝集率分别为3.03%、4.11%和1.85%。7株菌都具有一定的净化水质的能力,但C0909、L0914对氨氮的去除率显著低于菌株N0906。研究显示,诺卡氏菌N0906较适宜作为罗非鱼养殖中控制养殖生态环境和链球菌病害的微生态菌株。     

甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。     

Development of a real‐time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse‐grown lettuce,and its detection in irrigating water

Midrib rot is an emerging disease in greenhouse production of lettuce caused by Pseudomonas cichorii, and probably introduced through contaminated irrigation water. Concentrations of 100 CFU mL^?1 are enough to induce the typical midrib rot symptoms. A sensitive real‐time PCR assay was developed, based on a 90‐bp amplicon from the pathogenicity gene cluster hrcRST and a Taqman Minor Groove Binding probe. Specificity of the assay was tested with 39 P. cichorii strains, including the type strain, and 89 strains from 83 other Pseudomonas species. The relationship between detection signals and P. cichorii DNA concentrations was linear over 6‐logs. Detection threshold with excellent reproducibility was 500 fg of DNA or about 70 genome copies. Sample preparation and DNA isolation were optimized to allow detection in 1 L water samples. The assay was first evaluated with greenhouse irrigation water spiked with serial dilutions of P. cichorii. The calculated cell numbers obtained with real‐time PCR were 10‐fold lower than plate counts of actual spiked cells. However, the assay consistently detected 100 CFU per reaction, corresponding to the detection of 1 CFU mL^?1 of irrigation water, which is well below the concentration needed for midrib rot infection. Finally, the assay proved to be valuable for detecting infective P. cichorii concentrations in the irrigation water of a commercial lettuce production greenhouse.     

实时荧光RT-PCR一步法检测南方菜豆花叶病毒

南方菜豆花叶病毒（Southem bean mosaic virus，SBMV）是我国二类检疫性有害生物，以南方菜豆花叶病毒日本分离物（SBMV-J）总RNA为模板，采用RT-PCR方法扩增病毒外壳蛋白基因及其上游基因的cDNA片断并将其克隆到pMD18．T载体上。序列分析结果表明：SBMV-J cp基因由801个核苷酸组成，编码266个氨基酸，SBMV．J与其它分离物及株系印基因的核苷酸序列同源性为83％-97％，氨基酸序列同源性为86％-97％。由于SBMV各分离物及株系印基因的同源性较低，难于设计出较长的普通PCR引物。通过较短引物设计和TaqMan-MGB探针技术，建立了SBMV的实时荧光RT-PCR一步检测方法。该方法的检测低限是0．16pg，最佳检测总RNA的量是0．16ng。