Full-length Nucleotide Sequence Research Articles

Identification and Characterization of Wheat Yellow Striate Virus, a Novel Leafhopper-Transmitted Nucleorhabdovirus Infecting Wheat.

A new wheat viral disease was found in China. Bullet-shaped viral particles within the nucleus of the infected wheat leave cells, which possessed 180–210 nm length and 35–40 nm width, were observed under transmission electron microscopy. A putative wheat-infecting rhabdovirus vectored by the leafhopper Psammotettix alienus was identified and tentatively named wheat yellow striate virus (WYSV). The full-length nucleotide sequence of WYSV was determined using transcriptome sequencing and RACE analysis of both wheat samples and leafhoppers P. alienus. The negative-sense RNA genome of WYSV contains 14,486 nucleotides (nt) and seven open reading frames (ORFs) encode deduced proteins in the order N-P-P3-M-P6-G-L on the antisense strand. In addition, WYSV genome has a 76-nt 3′ leader RNA and a 258-nt 5′ trailer, and the ORFs are separated by conserved intergenic sequences. The entire genome sequence shares 58.1 and 57.7% nucleotide sequence identity with two strains of rice yellow stunt virus (RYSV-A and RYSV-B) genomes, respectively. The highest amino acid sequence identity was 63.8% between the L proteins of the WYSV and RYSV-B, but the lowest was 29.5% between the P6 proteins of these viruses. Phylogenetic analysis firmly established WYSV as a new member of the genus Nucleorhabdovirus. Collectively, this study provided evidence that WYSV is likely the first nucleorhabdovirus described infecting wheat via leafhopper P. alienus transmission.

Bacterial Genes of 2,4-Dichlorophenoxyacetic Acid Degradation Encoding α-Ketoglutarate-Dependent Dioxygenase Activity

The tfdA gene encodes α-ketoglutarate-dependent dioxygenase, which catalyzes the first step of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation pathway. The entire range of 2,4-D-degrading bacteria is divided into three groups based on their phylogeny, physiological and biochemical features, and isolation source. Each of these groups has its own version of the tfdA gene. The first group is the most studied and consists of fast-growing copiotrophic β- and γ-Proteobacteria isolated from anthropogenic habitats. The bacteria of this group possess the canonical sequence of this gene. Within this group, tfdA forms at least three classes (I, II, and III) of highly homologous gene families. The tfdA gene of Cupriavidus necator JMP134 was recognized as a Class I type. Class II consists of tfdA sequences belonging only to the genus Burkholderia of β-Proteobacteria, whereas Class III includes tfdA sequences belonging to the genera Delftia, Cupriavidus, Variovorax, Achromobacter, Comamonas, Rhodoferax, Halomonas, and Pseudomonas of β- and γ-Proteobacteria. The similarity of full-length nucleotide sequences between Class I and other classes was about 77–78%, and that between Class II and Class III was 93%. The second and third groups of 2,4-D-degrading bacteria are closely related to the genera Sphingomonas and Bradyrhizobium, which belong to α-Proteobacteria. tfdA-Like genes were identified only in four Sphingomonas spp. This fact and their phylogenetically distinct position make it possible to suggest that these genes evolved independently from each other through vertical gene transfer. The tfdAα gene was identified in the third group of bacteria of the genus Bradyrhizobium, which are both able and unable to degrade 2,4-D. Nevertheless, 2,4-D-degrading bacteria of the genera Bradyrhizobium and Sphingomonas have cad genes, which initiate the first step of the chlorophenoxyacetic acid degradation pathway. Based on the data on tfdAα localization in bacteria isolated from pristine ecosystems, a theory has been proposed that the tfdAα gene is ancestral for tfdA. Horizontal transfer and further adaptation to anthropogenic habitats probably led to the emergence of tfdA. However, tfdA and tfdAα may have diverged from a common ancestor, because they show a high similarity (51–57%) and separate distribution in the β-Proteobacteria, γ-Proteobacteria, and α-Proteobacteria, respectively.

A Rice CPYC-Type Glutaredoxin OsGRX20 in Protection against Bacterial Blight, Methyl Viologen and Salt Stresses.

Glutaredoxins (GRXs) belong to the antioxidants involved in the cellular stress responses. In spite of the identification 48 GRX genes in rice genomes, the biological functions of most of them remain unknown. Especially, the biological roles of members of GRX family in disease resistance are still lacking. Our proteomic analysis found that OsGRX20 increased by 2.7-fold after infection by bacterial blight. In this study, we isolated and characterized the full-length nucleotide sequences of the rice OsGRX20 gene, which encodes a GRX family protein with CPFC active site of CPYC-type class. OsGRX20 protein was localized in nucleus and cytosol, and its transcripts were expressed predominantly in leaves. Several stress- and hormone-related motifs putatively acting as regulatory elements were found in the OsGRX20 promoter. Real-time quantitative PCR analysis indicated that OsGRX20 was expressed at a significantly higher level in leaves of a resistant or tolerant rice genotype, Yongjing 50A, than in a sensitive genotype, Xiushui 11, exposed to bacterial blight, methyl viologen, heat, and cold. Its expression could be induced by salt, PEG-6000, 2,4-D, salicylic acid, jasmonic acid, and abscisic acid treatments in Yongjing 50A. Overexpression of OsGRX20 in rice Xiushui 11 significantly enhanced its resistance to bacterial blight attack, and tolerance to methyl viologen and salt stresses. In contrast, interference of OsGRX20 in Yongjing 50A led to increased susceptibility to bacterial blight, methyl viologen and salt stresses. OsGRX20 restrained accumulation of superoxide radicals in aerial tissue during methyl viologen treatment. Consistently, alterations in OsGRX20 expression affect the ascorbate/dehydroascorbate ratio and the abundance of transcripts encoding four reactive oxygen species scavenging enzymes after methyl viologen-induced stress. Our results demonstrate that OsGRX20 functioned as a positive regulator in rice tolerance to multiple stresses, which may be of significant use in the genetic improvement of rice resistance.

Pituitary adenylate cyclase-activating polypeptide (PACAP) regulates the expression of PACAP in cultured tilapia astrocytes.

Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic hormone that is involved in numerous physiologic functions. The present study examines the presence and the functional significance of PACAP and its receptor in the brain and astrocytes of tilapia (Oreochromis mossambicus). This is the first demonstration of the full-length nucleotide sequence of tPACAP gene in tilapia pituitary, brain, and cultured astrocytes. Two cDNA variants of the growth hormone-releasing hormone (GHRH)-PACAP gene were identified in tilapia pituitary, brain, and cultured astrocytes as a result of exon skipping with a long form (271 bp) encoding both tPACAP(38) and tGHRH and a short form (166 bp) encoding only tPACAP(38). The short form was found to be more abundant in astrocytes. Addition of ovine PACAP(38) (1 nM) to cultured astrocytes significantly stimulated the expression of tPACAP(38) at 4 hrs, but the effect dropped after 8 hrs of treatment. By contrast, the expression of PACAP type I receptor (PAC(1)-R) mRNA in the astrocytes was not responsive to PACAP(38) treatment. The tPACAP(38) expression also was activated by the cAMP analog, dibutyryl-cAMP, in a dose-dependent manner. Adding high salinity (170 mM NaCl, 500 mOsm/kg osmolarity) to cultured medium substantially increased astroglial tPACAP(38) expression over 4 hrs to a level that was maintained for 16 hrs. This observation was not found when mannitol (270 mM) was supplemented as an osmolarity-enhancing agent (500 mOsm/ kg). Taken together, tPACAP expression in tilapia astrocytes was well regulated by exogenous PACAP, cAMP, and salinity and might be involved in the adaptation to high salinity when the fish is in a seawater environment.

Complete genome sequence and phylogenetic analysis of Senecavirus A isolated in Northeast China in 2016.

Senecavirus A (SVA) is associated with vesicular disease in swine and the acute death of neonatal piglets. Here, senecavirus A was isolated from a pig displaying vesicular disease in Northeast China. The virus was designated as SVA/HLJ/CHA/2016 and its full-length nucleotide sequence was determined and analyzed in comparison with other known SVA strains. The complete genome sequence of SVA/HLJ/CHA/2016 shares high nucleotide identities, of 93.8 to 99%, with previously reported SVA full-length genomes. Phylogenetic analysis of both the SVA full-length genomes and the VP1 genes revealed that the SVA/HLJ/CHA/2016 strain is closely related to the 2015 US strains, instead of other China isolates. Our finding provides evidence that SVA infection of pigs has occurred in Northeast China, and the importance of SVA surveillance in China should be emphasized.

Molecular characterization of complete genomic segment-2 of picobirnavirus strains detected in a cat and a dog

We report here molecular characterization of complete genomic segment-2 of picobirnavirus (PBV) strains PBV/Cat/KNA/K40/2014 and PBV/Dog/KNA/RVC7/2015 detected in a cat (Felis catus) and a dog (Canis lupus familiaris), respectively, on the Caribbean island of St. Kitts. To obtain the full-length nucleotide (nt) sequence of gene segment-2 of the canine and feline PBV strains, the 5′- and 3′- portions of gene segment-2 containing an overlapping region were amplified using a non-specific primer-based amplification method with modifications. The complete gene segment-2 of feline PBV strain K40 and canine PBV strain RVC7 was 1784nt and 1689nt long, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 534 amino acid (aa) and 531 aa, respectively. The complete gene segment-2 of strains K40 and RVC7 exhibited a high degree of genetic diversity between themselves, and with those of PBVs from other host species. On the other hand, both the canine and feline PBV strains retained the 5′- and 3′- end nucleotide sequences and the three domains of putative RdRp that are conserved in PBVs. To our knowledge, this is the first report on molecular characterization of complete gene segment-2 of PBV strains detected in cats and dogs, allowing us to study the features of putative RdRps of PBVs in these host species, and providing important insights into the genetic makeup and evolution of feline and canine PBV strains. PBVs were detected for the first time in cats and dogs from the Caribbean region.

Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes.

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter, Scortum barcoo (McCulloch & Waite).

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70-90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus-specific PCR. The full-length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV-like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.

Detection of picobirnaviruses in vervet monkeys (Chlorocebus sabaeus): Molecular characterization of complete genomic segment-2

During 2014–2015, 270 fecal samples were collected from non-diarrheic, captive and wild African green monkeys (AGMs) on the island of St. Kitts, Caribbean region. By RNA-PAGE, picobirnaviruses (PBVs) were detected in sixteen captive AGMs. By RT-PCR and sequencing of partial gene segment-2, PBVs in 15 of these 16 samples were assigned to genogroup-I. The full-length nucleotide (nt) sequence of gene segment-2 of one of the genogroup-I PBV strains, strain PBV/African green monkey/KNA/016593/2015, was obtained using a non-specific primer-based amplification method with modifications. Gene segment-2 of strain 016593 was 1707bp long, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 538aa. Furthermore, the nearly complete gene segment-2 sequences of three other AGM PBV strains were determined using primers designed from gene segment-2 sequence of 016593. The gene segment-2 of the 4 AGM PBV strains were almost identical to each other, and exhibited a high degree of genetic diversity (maximum nt and deduced aa sequence identities of 66.4% and 65.3%, respectively) with those of PBVs from other host species. The 5′- and 3′- (except for one mismatch) end nt sequences and the three domains of RdRps were retained in the AGM PBV strains. To our knowledge, this is the first report on detection, and molecular characterization of complete gene segment-2 of PBVs in vervet monkeys. PBVs were detected for the first time from the Caribbean region.

Identification of Klebsiella capsule synthesis loci from whole genome data.

Klebsiella pneumoniae is a growing cause of healthcare-associated infections for which multi-drug resistance is a concern. Its polysaccharide capsule is a major virulence determinant and epidemiological marker. However, little is known about capsule epidemiology since serological typing is not widely accessible and many isolates are serologically non-typeable. Molecular typing techniques provide useful insights, but existing methods fail to take full advantage of the information in whole genome sequences. We investigated the diversity of the capsule synthesis loci (K-loci) among 2503 K. pneumoniae genomes. We incorporated analyses of full-length K-locus nucleotide sequences and also clustered protein-encoding sequences to identify, annotate and compare K-locus structures. We propose a standardized nomenclature for K-loci and present a curated reference database. A total of 134 distinct K-loci were identified, including 31 novel types. Comparative analyses indicated 508 unique protein-encoding gene clusters that appear to reassort via homologous recombination. Extensive intra- and inter-locus nucleotide diversity was detected among the wzi and wzc genes, indicating that current molecular typing schemes based on these genes are inadequate. As a solution, we introduce Kaptive, a novel software tool that automates the process of identifying K-loci based on full locus information extracted from whole genome sequences (https://github.com/katholt/Kaptive). This work highlights the extensive diversity of Klebsiella K-loci and the proteins that they encode. The nomenclature, reference database and novel typing method presented here will become essential resources for genomic surveillance and epidemiological investigations of this pathogen.

Complete genome sequences of two highly divergent Japanese isolates of Plantago asiatica mosaic virus.

Plantago asiatica mosaic virus (PlAMV) is a member of the genus Potexvirus and has an exceptionally wide host range.It causes severe damage to lilies. Here we report on the complete nucleotide sequences of two new Japanese PlAMV isolates, one from the eudicot weed Viola grypoceras (PlAMV-Vi), and the other from the eudicot shrub Nandina domestica Thunb. (PlAMV-NJ). Their genomes contain five open reading frames (ORFs), which is characteristic of potexviruses. Surprisingly, the isolates showed only 76.0-78.0% sequence identity with each other and with other PlAMV isolates, including isolates from Japanese lily and American nandina. Amino acid alignments of the replicase coding region encoded by ORF1 showed that the regions between the methyltransferase and helicase domains were less conserved than other regions, with several insertions and/or deletions. Phylogenetic analyses of the full-length nucleotide sequences revealed a moderate correlation between phylogenetic clustering and the original host plants of the PlAMV isolates. This study revealed the presence of two highly divergent PlAMV isolates in Japan.

Proteomics and antivenomics of Papuan black snake (Pseudechis papuanus) venom with analysis of its toxicological profile and the preclinical efficacy of Australian antivenoms

The Papuan black snake (Pseudechis papuanus Serpentes: Elapidae) is endemic to Papua New Guinea, Indonesian Papua and Australia's Torres Strait Islands. We have investigated the biological activity and proteomic composition of its venom. The P. papuanus venom proteome is dominated by a variety (n≥18) of PLA2s, which together account for ~90% of the venom proteins, and a set of low relative abundance proteins, including a short-neurotoxic 3FTx (3.1%), 3–4 PIII-SVMPs (2.8%), 3 cysteine-rich secretory proteins (CRISP; 2.3%) 1–3 l-amino acid oxidase (LAAO) molecules (1.6%). Probing of a P. papuanus cDNA library with specific primers resulted in the elucidation of the full-length nucleotide sequences of six new toxins, including vespryn and NGF not found in the venom proteome, and a calglandulin protein involved in toxin expression with the venom glands. Intravenous injection of P. papuanus venom in mice induced lethality, intravascular haemolysis, pulmonary congestion and oedema, and anticoagulation after intravenous injection, and these effects are mainly due to the action of PLA2s. This study also evaluated the in vivo preclinical efficacy of Australian black snake and polyvalent Seqirus antivenoms. These antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of P. papuanus venom in mice. On the other hand, all of the Seqirus antivenoms tested using an antivenomic approach exhibited strong immunorecognition of all the venom components. These preclinical results suggest that Australian Seqirus11Seqirus is a division of Australian company CSL Limited. antivenoms may provide paraspecific protection against P. papuanus venom in humans. Significance paragraphThe toxicological profile and proteomic composition of the venom of the Papuan black snake, Pseudechis papuanus, a large diurnal snake endemic to the southern coast of New Guinea and a handful of close offshore islands, were investigated. Intravenous injection of P. papuanus venom in mice induced intravascular hemolysis, pulmonary congestion and edema, anticoagulation, and death. These activities could be assigned to the set of PLA2 molecules, which dominate the P. papuanus venom proteome. This study also showed that Australian Seqirus black snake or polyvalent antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of the venom. These preclinical results support the continued recommendation of these Seqirus antivenoms in the clinical management of P. papuanus envenoming in Australia, Papua New Guinea or Indonesian Papua Province.

Full-length nucleotide sequence of ERMAP alleles encoding Scianna (SC) antigens.

Scianna (SC) blood group system comprises two antithetical antigens, Sc1 and Sc2, and five additional antigens. The antigens reside on a glycoprotein encoded by the erythroblast membrane-associated protein (ERMAP) gene. For the common ERMAP alleles, we determined the full-length nucleotide sequence that encodes the Scianna glycoprotein. Blood donor samples from five populations were analyzed including 20 African Americans, 10 Caucasians, 10 Thai, five Asians, and five Hispanics for a total of 100 chromosomes. An assay was devised to determine the genomic sequence of the ERMAP gene in one amplicon, spanning 21.4 kb and covering Exons 2 to 12 and the intervening sequence (IVS). All alleles (confirmed haplotypes) were resolved without ambiguity. Among 50 blood donors, we found 80 single-nucleotide polymorphisms (SNPs), including six novel SNPs, in 21,308 nucleotides covering the coding sequence of the ERMAP gene and including the introns. The noncoding sequences harbored 75 SNPs (68 in the introns and seven in the 3'-UTR). No SNP indicative of a nonfunctional allele was detected. The nucleotide sequences for 48 ERMAP alleles (confirmed haplotypes) were determined by allele-specific polymerase chain reaction and sequencing in 100 chromosomes. We documented 48 ERMAP alleles of 21,308 nucleotides each. The two nucleotide sequences available in GenBank for ERMAP alleles of similar length have not been found in our 100 chromosomes. Alleles determined without ambiguity can be used as templates to analyze next generation sequencing data, which will enhance the reliability in clinical diagnostics.

Molecular detection and genome characterization of porcine circovirus type 2 in rats captured on commercial swine farms.

Porcine circovirus type 2 (PCV2) is considered the major etiological pathogen of porcine circovirus-associated diseases (PCVADs) in pigs. Recently, PCV2 was also found in non-porcine animals such as cattle, rats, and mice. However, there was no record of PCV2 in rats in China. The goal of this study was to investigate whether PCV2 was present in rats (Rattus norvegicus, RN) on three swine farms, using molecular tools. PCR results showed that 30 of 95 (31.6%) rat samples were positive for PCV2. Moreover, further genotype analysis suggested that 10 of 30 (33.3%) were positive for PCV2a, 19 of 30 (63.3%) were positive for PCV2b, and only one sample (1/30, 3.33%) was co-infected by PCV2a and PCV2b. To determine the possible origin of PCV2, 60 serum samples were also collected from weaned pigs on those swine farms, and 23 out of 60 samples were positive for PCV2. In addition, two distinct RN-origin and two distinct porcine-origin PCV2 full-length nucleotide sequences were obtained from the farms. Sequence and phylogenetic analysis indicated that they had the highest nucleotide similarity and closest genetic relationships to each other. In this study, we report the infection and genome characterization of PCV2 in rats and compare RN-origin and porcine-origin PCV2 sequences obtained from the same pig farm, revealing possible cross-species transmission of PCV2.

Sequence and phylogenetic analysis of chicken reoviruses in China

Avian reovirus (ARV) has been responsible for many cases of chicken tenosynovitis in China in recent years, causing high morbidity among layer and broiler chickens. To study the degree of genetic divergence and evolution among ARVs, the full-length nucleotide sequences of the σC-encoding gene of eight ARV field isolates and the entire coding-region sequences of four isolates were determined and analyzed. The sequence analysis revealed that the eight σC-encoding genes shared 99.0–99.9% nucleotide sequence identity with each other and over 99% with the chicken reovirus reference strain S1133. However, the nucleotide sequences of the eight σC-encoding genes varied extensively from that of isolate AVS-B (GenBank accession no. FR694197), with only 55.5% identity. A sequence analysis of the whole ARV-coding region showed some nucleotide substitutions in the open reading frames encoding λA, λB, λC, μA, μB, μNS, σC, σA, σB, and σNS in the field strains. A phylogenetic analysis showed that all eight isolates clustered in group I with S1133, but that four field isolates shared less homology with strain S1133 than the others, indicating that they had been evolved in the field. We also studied the pathogenicity of two strains. No characteristic lesions were observed in vaccinated chickens, and no virus was detected in sampled tissues. However, an enzyme-linked immunosorbent assay revealed significant differences between the antibody responses of the inoculated groups and the negative controls. These results revealed that Chinese isolates shared the highest sequence homologies with S1133, grouped together in one cluster. Although the vaccination against ARV is used in farms, the pathogens still persist in Chinese poultry flocks.

Identification and functional activity of a staphylocoagulase type XI variant originating from staphylococcal food poisoning isolates.

Staphylocoagulase, an extracellular protein secreted by Staphylococcus aureus, has been used as an epidemiological marker. At least 12 serotypes and 24 genotypes subdivided on the basis of nucleotide sequence have been reported to date. In this study, we identified a novel staphylocoagulase nucleotide sequence, coa310, from staphylococcal food poisoning isolates that had the ability to coagulate plasma, but could not be typed using the conventional method. The protein encoded by coa310 contained the six fundamental conserved domains of staphylocoagulase. The full-length nucleotide sequence of coa310 shared the highest similarity (77·5%) with that of staphylocoagulase-type (SCT) XIa. The sequence of the D1 region, which would be responsible for the determination of SCT, shared the highest similarity (91·8%) with that of SCT XIa. These results suggest that coa310 is a novel variant of SCT XI. Moreover, we demonstrated that coa310 encodes a functioning coagulase, by confirming the coagulating activity of the recombinant protein expressed from coa310. This is the first study to directly demonstrate that Coa310, a putative SCT XI, has coagulating activity. These findings may be useful for the improvement of the staphylocoagulase-typing method, including serotyping and genotyping. This is the first study to identify a novel variant of staphylocoagulase type XI based on its nucleotide sequence and to demonstrate coagulating activity in the variant using a recombinant protein. Elucidation of the variety of staphylocoagulases will provide suggestions for further improvement of the staphylocoagulase-typing method and contribute to our understanding of the epidemiologic characterization of Staphylococcus aureus.

Goose Parvovirus and Circovirus Coinfections in Ornamental Ducks

Clinical observations and diagnostic procedures carried out to elucidate the cause of high mortality in 2-8-wk-old ornamental ducks (mandarin, wood, falcated, and silver teal ducks) are described. At necropsy, ducklings showed general pallor of skeletal and heart muscles, subcutaneous gelatinous transudates, pericarditis, ascites, and severe edema and hyperemia of lungs. Histopathologic examination revealed that the most important changes were located in the crop, bursa of Fabricius, and lungs with presence of amorphic basic intracytoplasmic inclusions. No bacteria or fungi could be detected from affected organs and ascitic fluid. Viral diagnosis included molecular detection for the presence of goose parvovirus (GPV), circovirus, avian influenza, herpesviruses, paramyxovirus, reovirus, and polyomavirus. Both GPV and circovirus could be detected by real-time PCR and nested broad-spectrum PCR, respectively. Phylogenetically, full-length nucleotide sequence of GPV showed a close similarity ranging from 95.6% to 97.9% with European and Asian pathogenic GPV. On the other hand, the detected circovirus showed nucleotide identity of 90% to 98% with goose circoviruses (GoCVs). This is the first report of GoCVs and GPV in ornamental ducks. The concurrence of GPV and GoCV infections is thought to contribute to the high mortality.

Megalocytivirus infection in cultured Nile tilapia Oreochromis niloticus.

Megalocytiviruses, such as infectious spleen and kidney necrosis virus (ISKNV), induce lethal systemic diseases in both ornamental and food fish species. In this study, we investigated an epizootic affecting Nile tilapia Oreochromis niloticus cultured in the US Midwest. Diseased fish displayed lethargy, gill pallor, and distension of the coelomic cavity due to ascites. Histopathological examination revealed a severe systemic abundance of intravascular megalocytes that were especially prominent in the gills, kidney, spleen, liver, and intestinal submucosa. Transmission electron microscopic examination revealed abundant intracytoplasmic polygonal virions consistent with iridovirus infection. Comparison of the full-length major capsid protein nucleotide sequences from a recent outbreak with a remarkably similar case that occurred at the same facility many years earlier revealed that both epizootics were caused by ISKNV. A comparison of this case with previous reports suggests that ISKNV may represent a greater threat to tilapia aquaculture than previously realized.

HLA-C*16:01:01:02, a recently isolated HLA class I allele from Southern Africa.

The full-length nucleotide sequence of HLA-C*16:01:01:02 differs from that of HLA-C*16:01:01:01 with a SNP at 2996C>T.

Direct evidence of recombination in the recA gene of Aeromonas bestiarum

Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures.