Restriction Endonuclease Profiles Research Articles

Effective Isolation and Characterization of Mycobacteriophages with the Ability to Lyse Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Currently there is no cure for JD and test-based culling has proved ineffective at preventing the spread. To isolate new mycobacteriophages (mbps) that can potentially be used to control JD transmission and infection on dairy farms, we optimized an isolation protocol by fecal spiking and the testing of different isolation solution compositions. Using this protocol, we successfully enhanced the yield of mbps from spiked fecal samples, elevating it from less than 1% to 59%. With this method, we isolated 14 mbps from 475 environmental samples collected from MAP-positive dairy farms, after in-sample enrichment with MAP and the fast-growing M. smegmatis. The sample sources included soil, manure pits, lactation barns, feces, milk, and drain water. After fingerprinting these mbps by restriction enzyme profiling, we concluded that 12 were distinct and novel. Further characterization of their host range revealed that eight were capable of lysing multiple MAP strains. We also studied the cross-resistance, lysogeny, the effect of pH and their antimycobacterial properties in milk replacer. Each novel mbp showed limited cross-resistance and prophage immunity and showed no reduction in the titer in a range of pHs after 4 h. The novel phages were also able to reduce the mycobacterial counts to zero after 8 h in milk replacer. In conclusion, these novel mbps could be considered to be used in the control strategies of JD on farms.

Comprehensive analysis of 66 complete molluscum contagiosum virus (MOCV) genomes: characterization and functional annotation of 47 novel complete MOCV genomes, including the first genome of MOCV genotype 3, and a proposal for harmonized MOCV genotyping indexing.

Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.

Can low concentration flufenoxuron treatment increase the pathogenicity or production of nucleopolyhedrovirus occlusion bodies in Spodoptera exigua (Hübner) or Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae)?

Nucleopolyhedroviruses (Baculoviridae) are lethal pathogens that naturally infect lepidopteran larvae. The chitin synthesis inhibitor flufenoxuron has been reported to potentiate the insecticidal properties of nucleopolyhedrovirus occlusion bodies (OBs) by degrading the integrity of the peritrophic matrix in lepidopteran larvae. In the present study we examined the effect of flufenoxuron treatment on susceptibility of Spodoptera exigua (Hübner) and Spodoptera frugiperda (JE Smith) fifth instars to their homologous nucleopolyhedroviruses, SeMNPV and SfMNPV, respectively. Preliminary bioassays indicated that a 1 mg/l concentration of flufenoxuron resulted in 6.6 - 13.3% mortality of larvae, which could be used in combination with OB inoculum concentrations of 1.5 × 104 OBs/ml of SeMNPV OBs or 1.3 × 10⁶ OBs/ml of SfMNPV OBs. Treatment of larvae with flufenoxuron did not significantly affect virus-induced mortality of either S. exigua (16.7 - 17.2%) or S. frugiperda (28.9 - 34.4%) and had no significant effect on the mean yield of OBs from virus-killed insects of either species. The identity of both SeMNPV and SfMNPV in experimental insects was confirmed by examination of the restriction endonuclease profiles of each virus. These findings contrast with the results of previous studies in which high concentrations of flufenoxuron (100 mg/l) greatly potentiated virus-induced mortality in the silkworm Bombyx mori L., although insect mortality due to flufenoxuron treatment alone was not reported, for reasons that are unclear. We conclude that flufenoxuron (1 mg/l) failed to potentiate the insecticidal activity of SeMNPV or SfMNPV in their respective hosts.

Insecticidal Traits of Variants in a Genotypically Diverse Natural Isolate of Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV).

Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype.

Escherichia coli ST155 as a production-host of three different polyvalent phages and their characterisation with a prospect for wastewater disinfection

Bacteriophages are generally specific, and a cocktail of phages is needed to combat different bacterial targets. Their production usually requires pathogenic isolation hosts. We identified a novel strain, Escherichia coli ST155, that could serve as a production host for three different polyvalent phages (ϕPh_SE03, ϕPh_SD01, and ϕPh_EC01), thus superseding the use of individual isolation hosts. Upon propagation in E. coli ST155, the phages demonstrated differential intergeneric infectivity against Salmonella enterica, E. coli OP50, Shigella dysenteriae, E. coli MDR, and Acinetobacter baumannii. Phages were characterised based on morphology, latent period, burst size, the efficiency of plating, and restriction enzyme profile. Survival assay on Caenorhabditis elegans, the absence of Shiga toxin, and enterotoxigenic E. coli virulence genes indicated that E. coli ST155 could be non-pathogenic. Lack of antibiotic resistance and absence of functional prophages rendered the host suitable for environmental applications. As a proof-of-concept, phage ϕPh_SE03 was produced in ST155 by employing a unique Bacteriophage Amplification Reactor-Lytics Broadcasting System and was simultaneously disseminated into S. enterica augmented wastewater, which resulted in a 3-log reduction in 24 h. The study establishes the potential of E. coli ST155 as a phage production host thereby minimising the possibility of accidental release of pathogenic hosts into wastewater.

Diversity analysis of BmNPV Isolates Infecting Mulberry Silkworm (Bombyx mori) using Restriction Endonuclease Digestion Profiling

Understanding the genetic diversity of BmNPV isolates causing grasserie viral disease of mulberry silkworm Bombyx mori L. is essential for adoption of management strategies including biotechnological tools. The present study was aimed at the use of restriction fragment length polymorphism (RFLP) profiling for studying the molecular diversity analysis of six BmNPV isolates collected from Devanahalli, Kolar, Shidlaghatta, Hosakote, Tumakuru, and Ramanagara areas in Karnataka, India (BmNPV-Ko, BmNPV-Ho, BmNPV-SG, BmNPV-DH, BmNPV-TM, BmNPV-Ram respectively). DNA was extracted from each of these isolates and subjected to digestion with different restriction enzymes EcoR1, BamH1, Sma1, Nco1, and a combination of BamH1+Nco1 and electrophoresed in 0.8% w/v agarose gel to visualize restriction enzyme profile. The analysis revealed that all the six BmNPV isolates had similar Nco1 and Sma1 restriction patterns, although there was variation in low molecular weight fragments. The EcoR1 and BamH1 restriction patterns were nearly the same for all the isolates except for the presence of an approximately 4kb and an additional 1.5kb polymorphic band only in BmNPV-TM and BmNPV-Ram isolates. BamH1+Nco1 digestion of the DNA from each isolate yielded numerous fragments, which was different in BmNPV-Ram isolate. Molecular diversity analysis can helps in understanding the evolution and phylogeny of the virus, enhance the knowledge on its pathogenicity and can help to develop and adopt suitable measures to combat and diagnose the disease to reduce crop loss and increase income generating ability of the farmer.

Genetic and Serological Diversity of Chlamydia trachomatis Strains Detected from Japanese Infants

Background: Classical fifteen serovars and variants of Chlamydia trachomatis were originally classified by the micro-immunofluorescence test which principally distinguished epitopes on the major outer-membrane protein (MOMP). The sequences of MOMP gene including four variable domains (VDs) have been determined for all classic 15 serovars. The PCR assay was used to amplify a large part of the MOMP gene (ompA or omp1) including four VDs and then cataloged restriction fragment length polymorphism (RFLP) was used to identify the serovars from genotypes of C. trachomatis as reported previously. Materials and Methods: Nasopharyngeal specimens were collected from 15 Japanese infants with pneumonia, from 1 month to 1 year old, during the period May 1990 to September 2013. Conjunctival swabs were also collected from 12 Japanese neonates under 1 month old with inclusion conjunctivitis during the same period. The specimens were collected and placed into Abbott LCR proprietary medium and then tested by PCR for typing. The sequencing strategy used was the double-stranded DNA cycle sequencing method. Results: Applied to 15 reference strains, this PCR-RFLP procedure allowed us to determine 13 of the 15 serovars of C. trachomatis. Thirteen of 15 isolates of nasopharyngeal origin and 11 of 12 isolates of conjunctival origin could be un-equivocally assigned to one of the 15 reference serovars with a restriction endonuclease profile identical to that of the prototype. The typing of 15 nasopharyngeal isolates gave the following results: 11 as E, 2 as H, and 2 as unclassified serovars. The typing of 12 conjunctival isolates gave the following results: 5 as D, 4 as E, 1 as F, 1 as H and 1 as unclassified serovar. Among the unclassified serovars, one was B-like and the rest was D-like. Discussion: The sequences of MOMP gene for all classic 15 serovars allowed the construction of restriction endonuclease cleavage-site maps that confirm the fragment-size patterns observed by electrophoresis. Sequencing the entire MOMP gene and cataloging the sequences of VDs of all serovars has confirmed the molecular basis of the serotyping procedure and provided a method for determining serovars by PCR-RFLP. Although geno-variants have also been reported for most serovars the PCR-RFLP method for typing allows quick and objective identification of serovars of C. trachomatis. Antigenic variations of C. trachomatis were also considered among the strains from nasopharyngeal and conjunctival origins.

Characterization of a Chrysodeixis includens nucleopolyhedrovirus Isolate from Brazilian Cerrado and Assessment of its Co-Infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus

Abstract Chrysodeixis includens has become the major Lepidopteran pest of soybean crops, especially in the Brazilian Cerrado (savanna) region. A native isolate of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from this region, Buritis, MG, was assessed for its biological and molecular features. In addition, in vitro co-infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), another virus of an important soybean pest, was tested. The ChinNPV-Buritis isolate presented an average LC50 of 7,750 occlusion bodies (OBs)/ml of diet in C. includens larvae. Analysis of restriction endonuclease profiles of viral DNA revealed similarities with previously described ChinNPV isolates IE, IF, and IG from Brazil, although the presence of submolar bands indicates genetic heterogeneity. Optical microscopy analysis in conjunction with quantitative PCR (qPCR) demonstrated in vitro infection of this isolate in IPLB-SF-21AE, Sf9, and BTI-Tn-5B1-4 cell lines, but the amount of ChinNPV tends to decrease through serial passages. The qPCR method developed in this study successfully detected both AgMNPV and ChinNPV from cell culture and from infected larvae. The cell line Tn-5B1-4 is indicated for future development of in vitro production and co-infection studies.

Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus

ABSTRACTSpodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD50) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT50) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.

Detection and identification of lethal yellowing phytoplasma 16SrIV-A and D associated with Adonidia merrillii palms in Mexico

Adonidia merrillii is a palm species native to the Philippines and commonly known as the manila palm. Lethal yellowing-type symptoms were first observed for the manila palm in Florida, USA. Manila palms in Mexico have not been reported to carry the lethal yellowing-type phytoplasma and moreover we have documented other symptoms that are not commonly associated to lethal yellowing (LY) disease. Therefore, our objective was to use molecular techniques to identify and characterize the presence of phytoplasmas in manila palms from southeastern Mexico. Thirty-three manila palm plants were analyzed. In general, three syndromes were observed: mature leaf browning, spear leaf necrosis and spear leaf opening. The presence of phytoplasma DNA was detected by nested-PCR using universal and LY-specific primers. Of the 33 palms analyzed, 24 (73%) were positive for the presence of phytoplasmas using universal primers, and 18 (55%) were positive for phytoplasma DNA using the LY-specific primers. By a TaqMan/real-time PCR assay for LY detection, 82% of palms tested were positive for LY-specific phytoplasma DNA. The restriction enzyme profiles of the DNA tested showed two clear patterns. One pattern was similar to that of the phytoplasma 16SrIV-A strain associated with coconut LY, and the other corresponded to the Pritchardia pacifica 16SrIV-D strain. The 16SrIV-A strain is associated with mature leaf-browning syndrome. The palms infected with the 16Sr-IV-D phytoplasma exhibited spear leaf necrosis and opening. This is the first report of two phytoplasma strains belonging to the subgroups 16SrIV-A and 16SrIV-D in manila palms.

Solid-State Cultivation of Edible Oyster Mushrooms, <i>Pleurotus</i> spp. under Laboratory Conditions

Oyster mushrooms of genus Pleurotus are well known as valuable edible mushrooms which are broadly cultivated in the world as well as wildly propagated in forests of the temperate climate zones including Russia. Indoor cultivation conditions adapted for a laboratory with non-specialized facilities were suggested for effective solid-state cultivation and fruit bodies’ production of Pleurotus species. Seven of 9 Pleurotus species of different origin produced fruit bodies successfully. The best fructification was shown for species P. sajor-caju, P. ostreatus, and P. pulmonarius with fruit body yields of 74%, 72%, and 61% by dry substrate weight in the first flushing cycle. Fruit bodies appeared in five to 10-day flushes. “Spawn run” stage was completed within 17 to 24 days, the longest colonization stage (26 days) being for the pink oyster, P. djamor. The cultivation parameters proposed in this study can be employed with ease for laboratory and “home” cultivation of oyster mushrooms. The species and strains’ identification was confirmed by restriction analysis of ITS region of rRNA gene cluster. Molecular barcodes based on restriction enzyme (AluI, and BsuRI) profiles of ITS sequences were shown to be applicable for molecular genotyping of Pleurotus species of different origin.

Temporal shifts of Bois Noir phytoplasma types infecting grapevine in South Tyrol (Northern Italy)

Results and Discussion: Two different restriction enzyme profiles of the Bois noir (BN) phytoplasma, corresponding to VK-Type I and VK-Type II (LANGER and MAIXNER 2004), were found in South Tyrol (Table). Both these BN-types were previously detected in German viticultural areas affected by grapevine yellows. While in Germany also a third BN-type was described (VK-Type III), which is exclusively restricted to the Mosel valley, this strain was not found in South Tyrol. Each BN-type has been associated with a different herbaceous host plant species of the vectoring insect H. obsoletus: VK-Type I with Urtica dioica, VK-Type II with Convolvulus arvensis, and VK-Type III with Calystegia sepium (LANGER and MAIXNER 2004). Consequently, it

Genetic and biological characterisation of a novel Plutella xylostella granulovirus, PlxyGV-SA

Plutella xylostella granulovirus (PlxyGV) has been isolated from insect populations in many countries and is considered a potential biopesticide for sustainable control of P. xylostella (L.) (Lepidoptera: Plutellidae). Several PlxyGV isolates have been genetically characterised, and the full genome sequence of PlxyGV-Japan is available for comparison with novel isolates. A South African PlxyGV was recently recovered from an overcrowded laboratory P. xylostella colony and identified as a genetically distinct isolate by sequencing of the granulin gene and restriction endonuclease (REN) analysis of genomic DNA. In this report, PlxyGV-SA was further characterised by PCR amplification and sequencing of egt, lef-8 and lef-9 genes, and several amino acid substitutions were observed. The PstI REN profile of PlxyGV-SA was different from that of PlxyGV-Japan in terms of band size and number, thereby confirming its novel genetic identity. Surface dose bioassays showed that PlxyGV-SA is pathogenic to neonate but not late instar larvae at the same and higher virus doses, indicating that a biopesticide should be targeted at early larval stages in the field.

Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates.

The differences or similarities among different isolates of Trypanosoma evansi through endonuclease profile was identified in the present study. The repetitive nuclear DNA of T. evansi isolated from infected cattle, buffalo and equine blood was initially amplified by PCR using specific primers. A panel of restriction enzymes, EcoRI, Eco91l, HindIII and PstI were for complete digestion of PCR products. Agarose gel electrophoresis of digested product did not show cleavage fragments and only single DNA band of the original size was visible in the ethidium bromide stained agarose gel. This indicated that the 227bp PCR product from repetitive sequence had no site-specific cleavage sites for the REs used in this study. No heterogeneity in the repetitive nuclear DNA restriction endonuclease profile among the different isolates was recorded.

Shiga Toxin 2A–Encoding Bacteriophages in EnteroaggregativeEscherichia coliO104:H4 Strains

To the Editor: In 2011, enteroaggregative Escherichia coli (EAEC) O104:H4 strains that produce Shiga toxins (EAEC-STEC) caused an outbreak of hemorrhagic disease affecting nearly 4,000 patients in Europe (1). During 2001–2013, several countries reported infections caused by EAEC O104:H4 and EAEC-STEC O104:H4 strains (1–9). Genomic analysis of EAEC and EAEC-STEC O104:H4 strains revealed high similarity, and it has been suggested that EAEC-STEC O104:H4 strains evolved from EAEC O104:H4 strains by uptake of Shiga toxin 2 (Stx2)–producing bacteriophages (3,4). We investigated Stx-2 subunit A (Stx-2A) bacteriophages in a group of epidemiologically unrelated EAEC-STEC O104:H4 strains isolated from animals and food in Germany (collection of the National Reference Laboratory for Escherichia coli). One phage genome ({type:entrez-protein,attrs:{text:P13374,term_id:138227,term_text:P13374}}P13374) was sequenced (2). The Stx-2A bacteriophages were highly similar in morphologic features, restriction endonuclease profiles, chromosomal integration sites, and superinfection immunity (2,3) and showed <65% similarity to Stx phages from non-O104 strains. Major genetic differences between the bacteriophages we investigated and other Stx phages were found in the genes for DNA replication, DNA metabolism, and in the immunity region (2,3). We identified 2 genes, orf15 and cIP13374, that were specific to Stx-2A bacteriophages found in EAEC-STEC O104:H4 strains (10). These genes were found in only 14 (5.8%) of 241 Stx-2A–positive non-O104 STEC strains. Viable Stx-2A bacteriophages isolated from 4 bovine non-O104 STEC strains were similar to Stx-2A bacteriophages from EAEC-STEC O104:H4 strains for all features described above (10). Similar to {type:entrez-protein,attrs:{text:P13374,term_id:138227,term_text:P13374}}P13374, one of the bovine phages ({type:entrez-protein,attrs:{text:P13803,term_id:122065179,term_text:P13803}}P13803) lysogenized an Stx-negative EAEC O104:H4 strain and converted it into an EAEC-STEC–producing Stx-2A bacteriophage (10). Our results provide experimental evidence that EAEC-STEC O104:H4 have evolved by uptake of a distinct type of Stx-2A bacteriophage. Bovine STEC harboring Stx-2A bacteriophages able to transduce Stx-2A genes to EAEC O104:H4 are found worldwide, and phage-mediated transfer of Stx-2A can occur in the environment (10). Thus, the emergence of EAEC-STEC O104:H4 does not appear to be the result of introduction of the strains from areas to which they are endemic. Instead, the process may have occurred spontaneously by phage transduction, which could explain why EAEC-STEC O104:H4 infections were found at different locations and at different times. Regardless of time or place, however, these strains show characteristic differences in their prophage and plasmid profiles, which may serve as indicators of epidemiologic origin (1–4). Investigation of EAEC-STEC O104:H4 strains from sporadic cases of human infection could reveal these markers and help differentiate between strains that were introduced from other areas and strains that were newly generated by phage transduction.

English

virus. Bluetongue virus is a member of the genus Orbivirus in the family Reoviridae. Its genome consists of ten double-stranded (ds) RNA segments coding for seven structural proteins (VP1-VP7) and four non-structural proteins (NS1-NS3 or NS3A, and NS4). At present, 26 serotypes have been reported throughout the world. Bluetongue can be diagnosed from several kinds of samples. For virus isolation, embryonated eggs, 9 to 12 days old, at intra­venous inoculation is best however, cell lines like KC line, the insect cell line C6-36 derived from Aedes spp., Baby hamster kidney (BHK-21) and African green monkey kidney (Vero) lines can be used. For antigen identification sandwich enzyme linked immunosorbent assay (ELISA), serotype specific reverse transcriptase Polymerase chain reaction (RT-PCR), Serogroup-specific RT-PCR, Real time quantitative PCR, Sequencing and phylogenetic analysis (targeting conserved genome segments), Restriction enzyme profile analysis (REPA), Molecular probes, RNA polyacrylamide gel electrophoresis (RNA-PAGE) are now available in an increasing number of laboratories for the identification of Bluetongue virus (BTV). For antibody identification, it is possible to use blocking ELISA, competition ELISA, indirect ELISA, agar-gel immunodiffusion test (AGID), Complement-fixation test (CFT) and Haemagglutination inhibition test (HI), serum or virus neutralisation test (VNT/SNT), Immunofluorescence, Immunoperoxidase, and Dot immunobinding assays (DIA). Key words: Antibody, antigen, bluetongue, bluetongue virus, diagnosis, structure, molecular technique.

Genomic analysis of bovine herpesvirus type 4 (BoHV-4) from Argentina: High genetic variability and novel phylogenetic groups

Bovine herpesvirus 4 (BoHV-4) is a γ-herpesvirus that has been isolated both from apparently healthy animals and from cattle with a variety of clinical signs, including post-partum endometritis and abortion. BoHV-4 causes either a persistent or a latent infection in cells of the monocyte/macrophage lineage. Two groups of BoVH-4 strains have been defined based on their restriction patterns: the Movar-like strains (European prototype) and the DN 599-like strains (American prototype). The purpose of the present study was to genetically characterize wild type BoHV-4 strains isolated from vaginal discharges of aborted cows in Argentina. The virus was identified by isolation and nested PCR in all vaginal discharge samples from aborted cows, either as a sole agent or in association with other pathogens. Restriction enzyme profiling and phylogenetic analysis demonstrated that there is a high genetic variability among the studied field isolates. The existence of three groups of strains, which were designated as genotypes 1, 2 and 3, is described. Genotypes 1 and 2 possibly correspond to the Movar-like and DN 599-like groups, respectively, whereas Genotype 3 corresponds to a novel group. Two viral strains did not cluster into any of these three groups, indicating that other genotypes could be circulating in Argentina. These results suggest a complex epidemiological background for the Argentinean BoHV-4 strains, probably influenced by independent events of genetic drift. This hypothesis cannot be rejected based on the available data. However, there is no direct evidence supporting this possibility. Thus, it seems speculative to suggest that interspecific jumps are responsible for the observed phylogenetic grouping. On the other hand, our analyses suggest a geographical structure for the observed viral genotypes, since genotypes 1 and 2 included the European (Movar-like) and American (DN599-like) reference strains, respectively. Geographic dispersion is known to be a driver of herpes viruses diversification, and independent evolution in geographical isolated places ensures the emergence of particular mutations in each location due to genetic drift (Compans, 2007; Zong et al., 1999). Therefore, at this point, the genetic drift hypothesis is the one that requires less ad-hoc considerations and thus, to our understanding, is the one that fits to the findings from this study. The involvement of this genetic variability in the detection and pathogenesis of BoHV-4 remains to be investigated.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFlP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Molecular characterization of Salmonella enterica serovar Saintpaul isolated from imported seafood, pepper, environmental and clinical samples

A total of 39 Salmonella enterica serovar Saintpaul strains from imported seafood, pepper and from environmental and clinical samples were analyzed for the presence of virulence genes, antibiotic resistance, plasmid and plasmid replicon types. Pulsed-field gel electrophoresis (PFGE) fingerprinting using the XbaI restriction enzyme and plasmid profiling were performed to assess genetic diversity. None of the isolates showed resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. Seventeen virulence genes were screened for by PCR. All strains were positive for 14 genes (spiA, sifA, invA, spaN, sopE, sipB, iroN, msgA, pagC, orgA, prgH, lpfC, sitC, and tolC) and negative for three genes (spvB, pefA, and cdtB). Twelve strains, including six from clinical samples and six from seafood, carried one or more plasmids. Large plasmids, sized greater than 50 kb were detected in one clinical and three food isolates. One plasmid was able to be typed as IncI1 by PCR-based replicon typing. There were 25 distinct PFGE-XbaI patterns, clustered to two groups. Cluster A, with 68.5% similarity mainly consists of clinical isolates, while Cluster C, with 67.6% similarity, mainly consisted of shrimp isolates from India. Our findings indicated the genetic diversity of S. Saintpaul in clinical samples, imported seafood, and the environment and that this serotype possesses several virulent genes and plasmids which can cause salmonellosis.

Identification of putative new Escherichia coli flagellar antigens from human origin using serology, PCR-RFLP and DNA sequencing methods

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.