North American Isolates Research Articles

Limited Genetic Diversity in North American Isolates of Phytophthora erythroseptica Pathogenic to Potato Based on RAPD Analysis.

Pink rot of potato (Solanum tuberosum), caused by Phytophthora erythroseptica, is found wherever potatoes are grown, and in the last decade, it has reemerged as an economically important disease in Canada and the United States. A selection of isolates of P. erythroseptica from major potato-growing regions in North America, namely Prince Edward Island and New Brunswick, Canada, and Maine and Idaho, U.S.A., was assessed for genetic diversity with randomly chosen decanucleotide primers which were used to amplify regions of DNA to reveal polymorphisms among templates (random amplified polymorphic DNA [RAPD]). The isolates varied in their geographic origin as well as in their sensitivity to mefenoxam, as determined by an in vitro assay. In three separate RAPD screens (I, II, and III) with 23 isolates of P. erythroseptica chosen from a larger collection, 1,410, 369, and 316 robust, scorable bands were amplified, respectively. However, among the bands amplified in screens I, II, and III, only 3, 1, and 3 bands, respectively, were polymorphic. When three primers yielding polymorphisms were used to screen 106 isolates from Prince Edward Island and New Brunswick, or a representative collection of 32 isolates from Prince Edward Island, New Brunswick, Maine, and Idaho, no major variation was discovered. RAPD markers were not correlated with geographic origin or mefenoxam sensitivity of the isolates. From an evolutionary standpoint, the absence of genetic diversity among the isolates of P. erythroseptica we examined may be attributable to the relatively recent introduction of a small founding population of the pathogen in North America.

Simultaneous Detection of North American and European Porcine Reproductive and Respiratory Syndrome Virus Using Real-Time Quantitative Reverse Transcriptase–PCR

Porcine reproductive and respiratory syndrome (PRRS) is 1 of the most economically important diseases of swine. Detection of the etiologic agent, PRRS virus (PRRSV), represents a diagnostic challenge due to the heterogeneity of field isolates as well as the propensity for swine to develop persistent infection in which virus is difficult to detect. Recently European (EU) lineage PRRSV isolates, which are genetically divergent from North American (NA) isolates, have been introduced into NA swine further complicating efforts to diagnose this disease. In this study, real-time (TaqMan) reverse transcriptase (RT)-PCR assays were developed for multiplex detection, differentiation, and quantification of NA and EU PRRSV field isolates. Oligonucleotide primers and dual-labeled probes were selected from conserved regions of open-reading frame 7 and the 3'-untranslated region. The real-time RT-PCR assays described for the NA or EU genotype of PRRSV detected viral RNA from 83/83 strains (74 NA; 9 EU) previously isolated by cell culture between 1992 and 2003. The analytical sensitivity of both assays was consistently found to be less than a single TCID50, which corresponded to 5-10 RNA molecules, and was not significantly reduced when the reactions were performed in a multiplex format. When performing multiplex reactions, sensitive detection was possible even when 1 viral RNA concentration was up to 5,000-fold higher than the second. The diagnostic sensitivity and specificity of the multiplex reaction was found to be at a minimum equivalent to that of both nested RT-PCR and virus isolation.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylogenetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis

Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylo-genetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.

Utility of Diagnostic Tests Used in Diagnosis of Infection in Dogs Experimentally Inoculated with a North American Isolate of Leishmania infantum infantum

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.

Infectious hematopoietic necrosis virus: monophyletic origin of European isolates from North American Genogroup M

Infectious hematopoietic necrosis virus (IHNV) was first detected in Europe in 1987 in France and Italy, and later, in 1992, in Germany. The source of the virus and the route of introduction are unknown. The present study investigates the molecular epidemiology of IHNV outbreaks in Germany since its first introduction. The complete nucleotide sequences of the glycoprotein (G) and non-virion (NV) genes from 9 IHNV isolates from Germany have been determined, and this has allowed the identification of characteristic differences between these isolates. Phylogenetic analysis of partial G gene sequences (mid-G, 303 nucleotides) from North American IHNV isolates (Kurath et al. 2003) has revealed 3 major genogroups, designated U, M and L. Using this gene region with 2 different North American IHNV data sets, it was possible to group the European IHNV strains within the M genogroup, but not in any previously defined subgroup. Analysis of the full length G gene sequences indicated that an independent evolution of IHN viruses had occurred in Europe. IHN viruses in Europe seem to be of a monophyletic origin, again most closely related to North American isolates in the M genogroup. Analysis of the NV gene sequences also showed the European isolates to be monophyletic, but resolution of the 3 genogroups was poor with this gene region. As a result of comparative sequence analyses, several different genotypes have been identified circulating in Europe.

Phylogenetic relationships among Rhizophydium isolates from North America and Australia

The order Chytridiales is the largest and most diverse of five orders in phylum Chytridiomycota. Rhizophydium is one of two genera in the Chytridiales with more than 220 described species. Because thallus characters used in classical descriptions of Rhizophydium species often intergrade into other species, as well as other genera, species distinctions frequently are unclear. Species often are delimited solely on substrate or host; many described species consequently may be synonymous. On the other hand, because the thallus is relatively simple morphologically similar forms actually may be genetically distinct. As a beginning for the revision of the genus Rhizophydium, this study used molecular and ultra-structural analyses to characterize cultures identified as Rhizophydium species. A broad geographic sampling of Rhizophydium-like organisms from North American and Australian soils was studied as a foundation for enhanced identification of soil chytrids. The first objective was to ascertain the genetic variability of Rhizophydium isolates with spherical to angular sporangia and multiple discharge pores, using nuclear large subunit rRNA gene sequence analysis. Sequences of 45 isolates of Chytridiales, including 29 isolates in the Rhizophydium clade were analyzed. Alignment based on LSU rRNA secondary structure revealed a similar reduced stem and loop structure in the C1_3 helix region that distinguished morphologically similar Rhizophydium clade members from other members of the Chytridiales. In our parsimony analysis, the Chytriomyces clade was sister of the Nowakowskiella, Lacustromyces and Rhizophydium clades. Six subclades within the Rhizophydium clade were resolved. Several closely related isolates appeared geographically widespread because North American and Australian isolates were found together in three of the six subclades. The second objective was to sample zoospore ultrastructure among isolates in the six subclades and an unresolved polytomy group within the Rhizophydium clade, thus evaluating the application of zoospore ultrastructure for lower level taxonomic decisions. All isolates were examined by transmission electron microscopy, and four types of zoospores were found. Thus, within the well-supported Rhizophydium clade, zoospore ultrastructure appeared divergent. Because similar zoospore types also were found in two distinct subclades, zoospore structure might be interpreted superficially as convergent. However, unresolved polytomys indicated molecular divergence among these taxa and the need for a more diverse taxa and gene sampling to resolve relationships. One of the zoospore types characterized represents the most simplified form of zoospore described so far in the Chytridiales. The range in molecular secondary structure composition and in zoospore morphology suggested that isolates we provisionally placed in Rhizophydium actually represent multiple genera.

Genetic variation and phylogeny ofSpongospora subterranea f.sp.subterranea based on ribosomal DNA sequence analysis

The nuclear rDNA regions of the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene from 52 field isolates ofSpongospora subterranea f.sp.subterranea obtained from the British Isles and North America were polymerase chain reaction-amplified, sequenced, and assessed for genetic variation. Two genetically distinct groups (I and II) were identified based on the ITS sequence diversity among the isolates, representing 34.6% and 65.4% of the isolates, respectively. British Isles isolates occurred in groups I and II, whereas North American isolates belonged only to group II. The British Isles groups ofS. subterranea were associated with particular potato cultivars. The full-length small-subunit rRNA gene ofS. subterranea was sequenced and analyzed by both neighbor-joining and parsimony methods to clarify the taxonomic position of this pathogen. The results of phylogenetic analysis showed thatS. subterranea grouped together with other species of plasmodiphorids, and this group clustered with the phylum Cercozoa, an assemblage of filose and reticulose amoebae and phylogenetically related zooflagellates. The recognition of the existence of different genetic groups withinS. subterranea will be important for the design of plant-breeding programs and in testing for plant resistance.

The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus

I have used indirect ELISA with overlapping synthetic peptides representing the GP5 ectodomain to study the generation and specificity of peptide-binding Abs in pigs that were infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) strain VR2332 and in North American field sera submitted for PRRSV infection diagnosis. Peptide-binding Abs appeared in sera of the VR2332-infected pigs within about 30 days post-farrowing (dpf), reaching maximum titers 100-200 dpf and then decreasing slowly to about half of maximum titer by about 400 dpf. The formation of peptide-binding Abs and of virus neutralizing Abs correlated and their initial appearance coincided with disappearance of virus from the circulation. The Abs were specific for VR2332-specific peptides. In contrast, anti-N-protein Abs as measured by HerdCheck ELISA appeared within 7 dpf, reached maximum levels at about 100 dpf and had decreased below detectable levels by about 200 dpf. Twenty-seven field serum samples with virus neutralizing activity all possessed high levels of peptide binding Abs, but the Abs bound about equally to VR2332 and strain Lelystad virus (LV)-specific peptides. The indirect ELISA results using various large peptides and competition ELISA using small peptides (8 or 9 amino acids long) confirmed that the epitope recognized by the Abs is located in the GP5 ectodomain sequence 37SHLQLIYNL of VR2332. Use of mutated peptides in the competition ELISA showed that 42I to T and 38HL to TY substitutions blocked Ab recognition, whereas deletion of 41L had no effect. In addition, 26 serum samples submitted by two farms for diagnostic tests were found to possess low levels of Abs that bound to GP5 ectodomain peptides, even though the sera were sero-negative in the HerdChek ELISA and lacked neutralizing activity. Competition ELISA showed that the Abs recognized one or more epitopes located downstream of the PRRSV neutralization epitope. An epitope(s) located in the same area was recognized by Abs generated in mice by immunization with a GP5 ectodomain peptide conjugated to BSA. These Abs also lacked neutralizing activity.

Cefdinir activity against contemporary North American isolates from community-acquired urinary tract infections

Cefdinir is an oral cephalosporin approved by the US Food and Drug Administration in 1997 for the treatment of community-acquired (CA) respiratory tract and uncomplicated skin and soft tissue infections. The objective of the present study was to evaluate the in vitro activity of cefdinir against recent clinical isolates collected from CA-urinary tract infections (UTIs), a possible expanded indication. A total of 456 isolates from CA-UTI were collected from medical centres in North America (NA; United States and Canada) in 2003 and susceptibility tested by NCCLS reference broth microdilution methods. Cefdinir and cefpodoxime were the most active compounds tested against Escherichia coli (98.7% susceptibility), followed by nitrofurantoin (97.0%) and ciprofloxacin (95.0%). Cefdinir was 8- to 16-fold more potent than cefuroxime axetil and cefprozil against E. coli, Klebsiella spp. and Staphylococcus saprophyticus. The activity of cefdinir was most similar to that of cefpodoxime against E. coli and Klebsiella spp., but cefpodoxime showed inferior activity against S. saprophyticus. The cefdinir spectrum was significant superior (+3.8 to 16.5%) to that of trimethoprim/sulphamethoxazole against all pathogens evaluated. The cefdinir spectrum and potency were comparable or superior to other orally administered β-lactams tested against recent (2003) clinical isolates from CA-UTI.

Restriction fragment length polymorphism of mitochondrial genome of the entomopathogenic fungus Beauveria bassiana reveals high intraspecific variation

Beauveria bassiana is an entomopathogenic fungus with a growing potential for pest control in different agro-ecosystems worldwide. Such potential brings the necessity of developing a strain specific typing system. In a previous study, we reported the identification of molecular variants in mitochondrial DNA (mtDNA) polymorphism in 15 North American isolates. Results indicated a highly conserved mitochondrial genome showing only two mitochondrial genotypes (mitotypes). In this study we used whole genomic DNA from 18 isolates of B. bassiana, two unidentified Beauveria spp., and one each of B. amorpha, B. cylindrospora and B. nivea from more diverse origins. By doing single- and double-restriction enzyme digestion of total genomic DNA with EcoRI, and HindIII and then probing with BbmtE2, the predominance of mitotypes A and B was observed again, along with three newly described mitotypes (C to E). Additionally, by using whole B. bassiana mtDNA digested with HpaII as probe, we further demonstrate up to nine different mitotypes within B. bassiana. With either of the two probes, distinguished between members of the genus Beauveria and from Paecilomyces farinosus and Metarhizium anisopliae. Phylogenetic analysis could not however distinguish B. amorpha and B. nivea isolates from B. bassiana, suggesting a close genetic relation between the three species of the genus. Altogether, these results show high variability in mitochondrial genome, which can be useful as a reliable tool for the biopesticide industry for both species and isolate specific identification.

Potency and spectrum reevaluation of cefdinir tested against pathogens causing skin and soft tissue infections: A sample of North American isolates

Cefdinir is a widely used orally administered cephalosporin for community-acquired respiratory tract infections and skin and soft tissue infections (SSTI). A total of 415 nonduplicate isolates of community-acquired SSTI (CA-SSTI) were collected from medical centers in North America and susceptibility tested against cefdinir and various compounds indicated for the treatment of CA-SSTI. The cefdinir MIC 50/90 in μg/mL/% susceptible for strains of the 7 principal CA-SSTI pathogens were: oxacillin-susceptible Staphylococcus aureus (0.5/0.5/100%), oxacillin-susceptible coagulase-negative staphylococci (0.06/0.12/100%), group A streptococci (≤0.03/≤0.03/100%), group B streptococci (≤0.03/0.06/100%), viridans group streptococci (0.25/2/88%), Klebsiella spp. (0.12/1/95%), and Escherichia coli (0.25/0.5/95%). Cefdinir was the most potent oral cephalosporin tested against staphylococci and the Enterobacteriaceae species, and 8-fold to 64-fold more potent than cephalexin against these pathogens. β-Hemolytic streptococci was highly susceptible to cefdinir (MIC 90, ≤ 0.03–0.06 μg/mL), while viridans group streptococci showed slightly elevated MIC results. Cephalexin MIC values for streptococcal strains (MIC 90, 1–32 μg/mL) were 32-fold to 64-fold higher than those of cefdinir or other oral cephalosporins evaluated. Only 0.5% of all 415 recent CA-SSTI pathogens were resistant to cefdinir (MIC, ≥ 4 mg/L). Cefdinir showed a spectrum and potency comparable or superior to other orally administered β-lactams (cephalexin).

North American populations of Entoleuca mammata are genetically more variable than populations in Europe

Entoleuca mammata (syn. Hypoxylon mammatum) is a damaging pathogen of Populus tremuloides and P. grandidentata in North America and P. tremula in Europe, where the fungus occurs only sporadically in alpine regions and Scandinavia. It has been hypothesized that E. mammata was introduced to Europe from North America. In this study, E. mammata isolates collected from Europe and North America were compared by a sequence analysis of two DNA markers derived from DNA fingerprints. The objective of the study was to elucidate the relationship between North American and European E. mammata populations by testing two hypotheses: (1) North American and European isolates are conspecific; and (2) the fungus was introduced between continents causing both a founder effect and a genetic bottleneck. North American populations were found to be more polymorphic, but no major phylogenetic differences between fungal isolates collected from different continents were found. This result combined with the historical observations of the disease in Europe implies that E. mammata was introduced to Europe several centuries ago.

Genetic diversity and molecular phylogeny of Anaplasma marginale isolates from Minas Gerais, Brazil

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world, and many isolates of A. marginale may occur in a given geographic area. Phylogenetic relationships have been reported for A. marginale isolates from the US using gene and protein sequences of MSP1a and msp4. These studies demonstrated that msp4 sequences, but not MSP1a DNA or protein sequences, provide phylogeographic information and also that MSP1a sequences are highly heterogeneous among A. marginale populations. However, little information is available on the genetic diversity of A. marginale isolates from other regions of the world. The present study was undertaken to examine genetic variation among 10 isolates of A. marginale obtained from infected cattle in the State of Minas Gerais, Brazil, where A. marginale is endemic. Neighbor-joining analysis of msp4 sequences of Brazilian and New World isolates of A. marginale from Argentina, Mexico and the US provided bootstrap support for a Latin American clade. The sequences of the MSP1a repeats of four Brazilian isolates of A. marginale were compared to sequences of Latin American and US isolates. The MSP1a repeated sequences of Latin American isolates of A. marginale had nine repeat forms, α–Φ, which have not been reported previously in North American isolates of A. marginale. Furthermore, the repeated forms τ, σ and μ were only present in the Brazilian isolates. The results demonstrated that the genetic heterogeneity observed among isolates of A. marginale is common in endemic areas, independent of the predominant tick vector and is consistent with previous studies in which msp4 provided phylogeographic information about A. marginale isolates, while MSP1a was found not to be a useful marker for phylogeographic characterization of A. marginale isolates.

Detecção e caracterização biológica e molecular de Rupestris stem-pitting associated virus e seu efeito na fotossíntese de videiras

Rupestris stem pitting associated virus (RSPaV) é o agente causal das "caneluras do tronco de Rupestris" da videira (Vitis spp.). Neste trabalho, um isolado de RSPaV, encontrado em videiras cv. Cabernet Franc no Rio Grande do Sul, foi estudado. O vírus foi detectado biologicamente, por enxertia em videira indicadora cv. Rupestris du Lot, em 26,2% das amostras avaliadas. A seqüência parcial do gene da replicase do RSPaV, isolado sul-brasileiro, com 831 nucleotídeos amplificados por RT-PCR e 276 aminoácidos deduzidos, apresentou maior identidade de nucleotídeos (98,1%) e aminoácidos deduzidos (99,6%), com dois isolados norte-americanos. O RSPaV estudado apresentou baixa homologia (37-41%) com outros vírus do gênero Foveavirus. A maioria das mudas de videira cv. C. Franc infetadas com RSPaV apresentou diminuição no potencial fotossintético (2,68 a 5,12 vezes) e aumento na taxa respiratória no escuro quando comparadas a mudas sadias, salientando os impactos que esse vírus pode proporcionar no potencial produtivo de videiras.

Detection and Confirmation of Potato mop-top virus in Potatoes Produced in the United States and Canada.

Potato mop-top virus (PMTV) was detected in potatoes grown in the United States and Canada during surveillance testing by a reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene in RNA3. Out of 3,221 lots of seed and ware potatoes that were tested, 4.3% were positive for PMTV. The reliability of the survey results was confirmed by reextraction of selected samples and additional RT-PCR tests using two primer sets targeting gene segments in RNA2 and RNA3. Amplicons generated from RNA2 and RNA3 were identified by analysis of fragment length polymorphisms after digestion with BamHI and HindIII, respectively. PMTV was further identified by enzyme-linked immunosorbent assay, bioassay on Nicotiana debneyi, and transmission electron microscopy. Sequencing of a portion of the coat protein gene revealed near 100% identity among isolates from the United States and Canada and >97% homology of the North American isolates with European isolates.

Characterization of emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States.

European-like field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) have recently emerged in North America. The full-length genomic sequence of an index isolate characterized in 1999, strain EuroPRRSV, served as the reference strain for further studies of the evolution and epidemiology of European-like isolates (type 1) in the United States. Strain EuroPRRSV shared 90.1 to 100% amino acid identity with the prototype European strain, Lelystad, within the structural and nonstructural open reading frames (ORFs) and 95.3% overall nucleotide identity. The 5' untranslated region and two nonstructural regions within ORF 1 were closely examined due to significant divergence from strain Lelystad. A 51-bp deletion in a region within ORF 1a, coding for nonstructural protein 2 (NSP2), was observed. Sequence analysis of the structural ORFs 2 to 7 of additional European-like isolates indicated that these isolates share 93% nucleotide identity with one another and 95 to 96% identity with the Lelystad strain but only 70% identity with the North American reference strain VR-2332. Phylogenetic analysis with published PRRSV ORF 3, 5, and 7 nucleotide sequences indicated that these newly emerging isolates form a clade with the Lelystad and United Kingdom PRRSV isolates. Detailed analysis of four of these isolates with a panel of 60 monoclonal antibodies directed against the structural proteins confirmed a recognition pattern that was more consistent with strain Lelystad than with other North American isolates.

Differences in Etiology Affect Mefenoxam Efficacy and the Control of Pink Rot and Leak Tuber Diseases of Potato.

Data supplementing a previously published survey of North American isolates of Phytophthora erythroseptica and Pythium ultimum demonstrated that the proportion of the populations sensitive to mefenoxam remains high, 79.6 and 96.9% with EC50 sensitivities ranging from <0.01 to 0.9 µg ml-1 and <0.01 to 0.8 µg ml-1, respectively. Mefenoxam should provide control of these pathogens in most potato production areas. Factors affecting the development of pink rot and leak in potato tubers and the efficacy of mefenoxam to control these diseases with different etiologies were examined. Results confirmed that P. erythroseptica is capable of directly infecting potato tubers causing pink rot, whereas Pythium ultimum requires a wound to infect and cause leak. Mefenoxam was applied to replicated field plots as a single in-furrow application at planting, as an in-furrow application at planting followed by an additional sidedress application 3 weeks after planting, as a single foliar application when tubers were 7 to 8 mm in diameter, and as two foliar applications when the tubers were 7 to 8 mm in diameter and 14 days later. The recommended label rate plus two additional lower application rates were used with each method. For tubers challenge-inoculated after harvest, mefenoxam was found to be more effective in controlling pink rot relative to leak over all application methods. The greatest level of pink rot control (89%) was attained with the in-furrow at planting and sidedress application. All rates tested provided similar levels of control with this application method, but this method provided only a modest level of leak control (35%), and leak was not controlled by foliar applications of mefenoxam at any rate tested. In contrast, the foliar applications of mefenoxam resulted in 10 to 50% control of pink rot. Since the isolates of both pathogens were highly sensitive to me-fenoxam, disease-specific control was attributed to differences in disease etiology. Therefore, the use of mefenoxam to control pink rot in the field and storage appears to be well founded.

Heterogeneity in Nsp2 of European-like porcine reproductive and respiratory syndrome viruses isolated in the United States

Recently, isolates of porcine reproductive and respiratory syndrome virus (PRRSV) that possess nucleotide sequences similar to European isolates have been reported in United States herds. The origin, diversity and prevalence of European-like North American PRRSV isolates in the U.S. remain unknown. Nucleotide sequence analysis of the 12 kb ORF1 of a North American isolate, SDPRRS 01-08 (01-08), showed 93.7% identity with Lelystad virus (LV), the prototypic European isolate, but only 58% identity with VR-2332, the prototypic North American isolate. Comparisons between LV and 01-08 at the peptide sequence level of the predicted non-structural proteins (Nsp) showed that Nsp9 (98.9% amino acid identity) was the most conserved and the least conserved was Nsp2 at 90.6% identity. For the purpose of comparison, GP5, the principal envelope structural protein, showed a 93.5% identity between 01-08 and LV. The most dramatic differences between the Nsp2 proteins of LV and 01-08 were a single 17 amino acid deletion between residues 734 and 750, as well as several amino acid differences. The same deletion was identified in the Nsp2 in five of seven other EuroPRRSV isolates submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. The remaining two isolates contained small deletions, but in other regions of Nsp2. Peptide sequence diversity in the form of hypervariability and deletions in Nsp2 demonstrate that European-like PRRSV isolates in the USA represent a heterogeneous group. Furthermore, areas in Nsp2 with deletions and amino acid hypervariability localize to regions that are predicted to be immunologically important.

The Israeli strain IS-98-ST1 of West Nile virus as viral model for West Nile encephalitis in the Old World

West Nile virus (WNV) recently became a major public health concern in North America, the Middle East, and Europe. In contrast with the investigations of the North-American isolates, the neurovirulence properties of Middle-Eastern strains of WNV have not been extensively characterized. Israeli WNV strain IS-98-ST1 that has been isolated from a white stork in 1998, was found to be highly neuroinvasive in adult C57BL/6 mice. Strain IS-98-ST1 infects primary neuronal cells from mouse cortex, causing neuronal death. These results demonstrate that Israeli strain IS-98-ST1 provides a suitable viral model for WNV-induced disease associated with recent WNV outbreaks in the Old World.