Maximum Composite Likelihood Method Research Articles

Molecular Characterization and Screening of Protease Production from indigenous thermophilic fungi Malbranchea cinnamomea isolated from dairy farm compost soil

Compost environments represent a rich reservoir of microbial diversity, including thermophilic fungi with the potential to produce proteases. The dynamic and nutrient-rich nature of compost provides a conducive habitat for the growth and metabolism of diverse fungal species, including Malbranchea cinnamomea. In this study, Malbranchea cinnamomea was isolated from dairy farm compost soil and characterized based on its morphological characters and subsequently confirmed molecularly using internal transcribed spacer (ITS) sequencing. The Phylogenetic tree was inferred using Neighbour- joining method. The evolutionary distances were computed using the maximum composite likelihood method. The identified fungi was primarily screened for protease and further used for enzyme production in submerged fermentation for 4-6 days at 50OC. Culture filtrate obtained were used for the assessment of enzymatic activity. This study revealed thermophilic fungus Malbranchea cinnamomea holds significant potential for protease production. The enzyme activity was estimated at 50 °C, pH 7, six days incubation time and using 1.0 ml inoculum. Under submerged substrate cultivation, the best substrate lodge for maximal fungal biomass production was YPSs broth. Hence this isolate could be further exploited for numerous industrial applications that require thermophilic enzymes.

First Report of Eucalyptus Wilt Caused by Chalaropsis thielavioides in China.

Eucalyptus species are widely planted in the tropics and subtropics, and eucalyptus is among the most important cash crop in Southern China. One of the most important diseases on eucalyptus is Ceratocystis wilt, caused by the fungus Ceratocystis fimbriata Ellis & Halst., and the genus name Chalaropsis has been proposed for anamorphs of Ceratocystis species (de Beer et al. 2014). During April 2018, severely infected Eucalyptus robusta trees were observed in Kunming, Yunnan Province, China. Symptomatic trees initially exhibited yellowing and wilting of foliage on individual branches, then spread to the whole canopy, sometimes followed by death of the whole tree. Reddish-brown to dark-brown discoloration in the woody xylem of affected trees, sometimes a grayish white layer of fungal growth may be seen. The disease was observed on 16% of trees surveyed. The base of trunks with typical symptoms were collected, then the discolored xylem tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, a fungus was consistently observed growing from the tissue. Three isolates were obtained. In culture, colonies reaching 54mm diam within 15 days, mycelium initially white, then becoming celadon. Endoconidia unicellular, smooth, cylindrical, straight, biguttulate, 11.21 - 32.26 × 4.12 - 5.25 μm. Phialides produced on short, septate, aerial hyphae, lageniform and chain of phialoconidia (3.62 - 5.89 × 31.39 - 65.76 μm) were also observed. Chlamydospores (11.45 - 14.26 × 10.06 - 12.22 μm) were single, dark, thick-walled. Morphological characteristics of the fungus were consistent with the description of Chalaropsis thielavioides (Paulin-Mahady et al. 2002). The two of three isolates were used for molecular identification and genomic DNA was extracted from isolates (EKY2-2-1, EKY2-2-2) using the chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions MW242701, MW242702) showed that the isolates were 99% - 100% homologous to isolates of C. thielavioides from Hevea rubber, Monstera deliciosa L. and ants in China and Rosa sp. in Australia (GenBank accessions KT963172, KJ511482, KT963173 and KX954598) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis were performed using MEGA 6.06 based on ITS sequences (Fig 1), the evolutionary distances were computed using the Maximum Composite Likelihood method. Analyses showed that both isolates (EKY2-2-1, EKY2-2-2) located on the same clade with all C. thielavioides, and clustered with the C. thielavioides strains with high bootstrap support (97% - 100%). Therefore, the fungus was identified as C. thielavioides based on morphology and molecular evidences. Pathogenicity of C. thielavioides was tested by inoculation of six one-year-old pot grown Eucalyptus citriodora seedlings. The sterilized soil of six seedlings was inoculated by drenching with 20 ml spore suspension (2.0 × 106 spores / ml). Control plants were inoculated with 20 ml of sterile distilled water. The seedlings were kept in a controlled greenhouse at 25°C and watered weekly. After one month incubation, all the isolates produced wilt symptoms, whereas control trees showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. thielavioides according to the methods described above, and no fungal growth was observed in the controls, thus satisfying Koch's postulates. Although wilt and canker caused by Ceratocystis fimbriata on eucalyptus have been previously reported in Brazil, Uruguay, Uganda, China and Pakistan (Ferreira et al. 1999; Li et al. 2014; Alam et al. 2017), eucalyptus wilt caused by C. thielavioides has not been reported anywhere. Also, wilt of rubber tree and postharvest rot on carrot caused by C. thielavioides have been reported (Li et al. 2021; Xu et al. 2020). To our knowledge, this is the first report of eucalyptus wilt caused by C. thielavioides in China.

Screening and Characterization of Soil Microbes Producing Antimicrobial Compounds in Makkah Province, Saudi Arabia

This study aimed to investigate a method to manage antimicrobial resistance (AMR) issues by exploring soil microorganisms that are capable of producing bioactive compounds. Eight different types of soil were selected from three locations to screen, isolate, and identify microorganisms that are capable of producing antimicrobial compounds. The multi-drug resistant strains are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans were selected for primary and secondary screening using the crowded plate method and the agar well diffusion method. Inhibition zones were measured, and data were assessed using statistical tests to check for normality and differences between parametric variables and nonparametric data. This was followed by biochemical characterization, DNA isolation, and polymerase chain reaction (PCR). Molecular identification was performed using 16S rRNA amplification and sequencing. Here, 86 isolates containing bacteria and fungi were successfully extracted from soil samples. Further, 49 of 86 microbes showed possible antimicrobial activity, but only 12 isolates resulted in distinct inhibition zones with the selected multi-drug resistant strains. The following different taxa were identified: Firmicutes (nine strains), Proteobacteria (one strain), Actinobacteria (one strain), and Azotobacter (one strain). Species are represented in a phylogenetic tree, which was constructed using the unweighted pair-group method with arithmetic mean (UPGMA) method. The evolutionary distances were computed using the Maximum Composite Likelihood method. The identified microorganisms showed antimicrobial activity, confirming that soil microorganisms have great potential to address AMR issues.

Composite likelihood inference for ordinal periodontal data with replicated spatial patterns.

Spatial ordinal data observed separately for multiple subjects are common in biomedical research, yet statistical methodology for such ordinal data analysis is limited. The existing methodology often assumes a single realization of spatial ordinal data without replications, a commonplace in disease mapping studies, and thus are not directly applicable. Motivated by a dataset evaluating periodontal disease (PD) status, we propose a multisubject spatial ordinal model that assumes a geostatistical spatial structure within a regression framework through an elegant latent variable representation. For achieving computational scalability within a classical inferential framework, we develop a maximum composite likelihood method for parameter estimation, and establish the asymptotic properties of the parameter estimates. Another major contribution is the development of model diagnostic measures for our dependent data scenario using generalized surrogate residuals. A simulation study suggests sound finite sample properties of the proposed methods. We also illustrate our proposed methodology via application to the motivating PD dataset. A companion R package clordr is available for easy implementation.

Bioinformatic analysis of chickpea acetohydroxyacid synthase gene

Aim. Analysis of homologues of chickpea gene encoding acetohydroxyacid synthase, by bioinformatics methods. Methods. Alignment of nucleotide sequences, UPGMA method, Maximum Composite Likelihood method, homologous modeling of three-dimensional structure of enzyme. Results. Homologues of the acetohydroxyacid syntase gene (AHAS) of chickpea were found among representatives of different families. A certain level of conservativeness of mRNA homologues sequences of AHAS gene within the families was noted, including legumes. The distribution of clusters corresponds to the taxonomic position of the investigated plant species. The single nucleotide polymorphism C / T at position 581, potentially associated with herbicide resistance, was detected. Based on the homologous modeling results, two models of the enzyme AHAS were constructed. The replacement of C / T, which leads to the replacement of the amino acids of alanine with valine, leads to a change in the conformation in the A chain of protein. Conclusions. Marker screening of the source breeding material by «real-time» polymerase chain reaction with the developed primers and the TaqMan probe to the polymorphic region of the AHAS gene will allow differentiating the herbicide «resistant» and «tolerant» alleles of the gene for the selection of target genotypes.
 Keywords: chickpea, acetohydroxyacid syntase gene, single nucleotide polymorphism, resistance to herbicides.

Comparison of IncL/M Plasmids Using the Neighbor-Joining Method on Basis repA and excA Genes

The aim of this study is to make comparison of IncL/M groups on basis of two genes candidates’ repA and excA genes. The sequences of 27 plasmids were compared using the neighbor-joining method. This method was used to construct a phylogenetic tree for the nucleotide sequences of two genes (repA and excA), using the program MEGA X software. The evolutionary distances were computed using the maximum composite likelihood method. The neighbor-joining method analysis showed different results based on the gene used for comparison. The repA gene was more accurate than excA gene to distinguish between different incL/M plasmids. This study suggested that IncL/M plasmids harboring different antibiotic resistance genes have evolved differently.

Bovine Leukemia Virus in Bovine Aborted Fetuses

We investigated the infection of Bovine Leukemia Virus (BLV) related to other pathogens [Neospora caninum, Bovine Herpesvirus-1 (BoHV-1), Bovine Viral Diarrhea Virus (BVDV), and pathogenic bacteria] in 80 bovine aborted fetuses. The materials comprised whole fetuses, fetal organs, and placenta. The BLV was diagnosed by nested-PCR (env gp51 BLV gene), the identification of viral genotypes by sequencing, and the phylogenetic analysis by neighborjoining and maximum composite likelihood methods. The other pathogens and diagnoses were, respectively: Neospora caninum (nested-PCR), BoHV-1 (nested-PCR), BVDV (PCR), Brucella spp. (isolation and identification), Leptospira spp. (PCR), aerobic bacteria [Enterobacteriaceae, Gram positive cocci, Trueperella (Arcanobacterium) pyogenes] and micro-aerophilic (Campylobacter spp., Histophilus somni, and Listeria monocytogenes) by isolation and identification. BLV fetal antibodies were identified by ELISA kit. Thirteen (16.25%) fetuses were positive by BLV nested-PCR. Phylogenetic analysis revealed BLV genotypes 1, 5, and 6, which are frequently found in cattle in Brazil, Argentina, and Uruguay. No fetuses were positive for BLV antibodies by ELISA. A single case of coinfection with BLV was found for each of the pathogens Trueperella (Arcanobacterium) pyogenes, Klebsiella spp., and Streptococcus spp. were isolated as a pure or representing the preponderance of bacteria in a pooled culture. In the 67 BLV-negative fetuses, pathogens identified were single cases of Trueperella (Arcanobacterium) pyogenes, Staphylococcus aureus, and Brucella abortus; 2 of Escherichia coli; 3 of bovine viral diarrhea virus; and 4 of Neospora caninum. No pathogens were found in 55 fetuses. The low number of BLV positive samples infected or no by other pathogens didn´t allow performing statistical analysis for understanding if there were significative differences among not infected and infected BLV fetuses. Because BLV is an immunosuppressive agent and predisposes the cow to other pathogens, its connection with Leukemia or abortions need additional studies with bigger sampling, for elucidating pathogenesis in the pregnant cow and in the fetus. The rates of BLV transplacental transmission show the necessity of prophylactic measures in Brazilian cattle herds, in order to avoid infection in utero.

Non‐stationary intensity‐duration‐frequency curves integrating information concerning teleconnections and climate change

Rainfall intensity‐duration‐frequency (IDF) curves are commonly used for the design of water resources infrastructure. Numerous studies reported non‐stationarity in meteorological time series. Neglecting to incorporate non‐stationarities in hydrological models may lead to inaccurate results. The present work focuses on the development of a general methodology that copes with non‐stationarities that may exist in rainfall, by making the parameters of the IDF relationship dependent on the covariates of time and climate oscillations. In the recent literature, non‐stationary models are generally fit on data series of specific durations. In the approach proposed here, a single model with a separate functional relation with the return period and the rainfall duration is instead defined. This model has the advantage of being simpler and extending the effective sample size. Its parameters are estimated with the maximum composite likelihood method. Two sites in Ontario, Canada and one site in California, USA, exhibiting non‐stationary behaviours are used as case studies to illustrate the proposed method. For these case studies, the time and the climate indices Atlantic Multi‐decadal Oscillation (AMO) and Western Hemisphere Warm Pool (WHWP) for the stations in Canada, and the time and the climate indices Southern Oscillation Index (SOI) and Pacific Decadal Oscillation (PDO) for the stations in United States are used as covariates. The Gumbel and the generalized extreme value distributions are used as the time‐dependent functions in the numerator of the general IDF relationship. Results show that the non‐stationary framework for IDF modelling provides a better fit to the data than its stationary counterpart according to the Akaike information criterion. Results indicate also that the proposed generalized approach is more robust than the common approach especially for stations with short rainfall records (e.g., R2 of 0.98 compared to 0.69 for duration of 30 min and a sample size of 27 years).

Max-Linear Competing Factor Models

Models incorporating “latent” variables have been commonplace in financial, social, and behavioral sciences. Factor model, the most popular latent model, explains the continuous observed variables in a smaller set of latent variables (factors) in a matter of linear relationship. However, complex data often simultaneously display asymmetric dependence, asymptotic dependence, and positive (negative) dependence between random variables, which linearity and Gaussian distributions and many other extant distributions are not capable of modeling. This article proposes a nonlinear factor model that can model the above-mentioned variable dependence features but still possesses a simple form of factor structure. The random variables, marginally distributed as unit Fréchet distributions, are decomposed into max linear functions of underlying Fréchet idiosyncratic risks, transformed from Gaussian copula, and independent shared external Fréchet risks. By allowing the random variables to share underlying (latent) pervasive risks with random impact parameters, various dependence structures are created. This innovates a new promising technique to generate families of distributions with simple interpretations. We dive in the multivariate extreme value properties of the proposed model and investigate maximum composite likelihood methods for the impact parameters of the latent risks. The estimates are shown to be consistent. The estimation schemes are illustrated on several sets of simulated data, where comparisons of performance are addressed. We employ a bootstrap method to obtain standard errors in real data analysis. Real application to financial data reveals inherent dependencies that previous work has not disclosed and demonstrates the model’s interpretability to real data. Supplementary materials for this article are available online.

First report of Stemphylium vesicarium on chilli pepper in Italy

In 2014 during a survey of fields of chilli pepper, grey leaf spot (GLS) symptoms were observed on leaves in two different Italian areas, Rieti (central Italy) and Cosenza (southern Italy). Diseased leaves were characterised by numerous tiny round spots (0.5 to 2.5 mm diameter, average 1.3 mm) that were white to grey with a sunken centre and brown edge, resulting in premature defoliation (Fig. 1). No other part of the plant was affected. The varieties ‘Fatalii White’ (Capsicum chinense) and ‘Diavolicchio Calabrese’ (C. annum) were the most susceptible in central and southern Italy, respectively. In both locations disease incidence on the two chilli pepper varieties was >40%. Tissue from leaves with typical GLS symptoms was plated on potato dextrose agar amended with streptomycin and ampicillin (100 ppm each) and incubated at 22 ±0.5°C with a 12 hour photoperiod. Single spore isolations were performed to obtain pure cultures (Fig. 2) and the morphological characteristics were consistent with the type description of Stemphylium vesicarium (Wallroth) Simmons (Simmons, 4). To confirm morphological identification, a representative isolate from each of the two Italian areas (ISPaVe2162 and ISPaVe2165) were subjected to molecular analysis. The internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (gpd) gene were amplified using universal primers ITS5 and ITS4 (White et al., 5) and gpd1 and gpd2 (Berbee et al., 2), respectively and sequences were deposited in the European Nucleotide Archive (Accession Nos. LN896692 and LN896693 for the ITS, and LN896694 and LN896695 for the gpd gene, respectively). A BLAST search in GenBank showed 100% identity with S. vesicarium in both the ITS region (JX424810) and gpd gene (DQ000654). To further support identification, phylogenetic analyses using the Maximum Composite Likelihood method (Kimura2/parameter model) was performed (MEGA 5.2). The isolates ISPaVe2162 and ISPaVe2165 clustered together with S. vesicarium isolates (Fig. 3). Pathogenicity tests of the two isolates were performed by artificial inoculations on detached leaves of the chilli pepper varieties ‘Diavolicchio Calabrese’ and ‘Fatalii White’. A 10 μl drop of a 1 × 105 conidia/ml suspension was placed on the abaxial leaf surface. A 10 μl drop of sterile distilled water was used as negative control. The inoculated leaves were incubated in sterile Petri plates, 20 mm in diameter, containing water-saturated sterile blotting paper at room temperature (24 ±2°C). Brown lesions started to be visible on detached leaves three days after inoculation. Subsequently, lesions enlarged in size and typical GLS were evident at all inoculation points on both chilli pepper varieties (Fig. 4). No symptoms developed on the leaf inoculated with sterile distilled water. In Italy, S. vesicarium has been reported as the causal agent of leaf spots on tomato (Porta-Puglia et al., 3) and wilting and root rot on radish sprouts (Belisario et al., 1). To our knowledge this is the first report of S. vesicarium on chilli pepper in Italy. The presence of S. vesicarium in two distinct geographic areas in Italy suggests an association with seed contamination and Stemphylium spp. are generally seed-borne. Consequently more strict control of seed health for chilli pepper production would be advisable.

Genetic identification of marine eels through DNA barcoding from Parangipettai coastal waters

Anguilliformes, also known as “true eels”, are an ecologically diverse group of predominantly marine origin whose members were easily recognized by their extremely elongated bodies with reduced cross-sectional areas and universal lack of pelvic fins. The Marine Eels were collected from landing centres of Parangipettai coastal waters and identified based on their morphometric and meristic characters. The newly recorded species were used for the barcoding analysis. Information on molecular taxonomy of marine eels was very meagre and hence, the present study was aimed to study the barcoding of marine eels which were present along the southeast coast of India. The cube of lateral muscle was exercised for DNA isolation followed by its amplification. Cluster IX 2.06 was used to align the nucleotide sequences (Thomson, 1997). The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004). The barcodes sequences were submitted in NCBI (National centre for Biotechnological Information). The species within genera of Muraenidae, Muraenesocidae and Ophichthidae family were clustered in a same clade with high bootstrap value. The evolutionary relationships of six species were analyzed using neighbor joining method. This results of phylogenetic tree showed maximum genetic relatedness with the sequenced results which were submitted in gene bank.

Differentiation of the Chinese minority medicinal plant genus Berchemia spp. by evaluating three candidate barcodes.

The genus Berchemia comprises important Chinese plants with considerable medicinal value; however, these plants are often misidentified in the herbal medicinal market. To differentiate the various morphotypes of Berchemia species, a proficient method employing the screening of universal DNA barcodes was used in this work. Three candidate barcoding loci, namely, psbA-trnH, rbcL, and the second internal transcribed spacer (ITS2), were used to identify an effective DNA barcode that can differentiate the various Berchemia species. Additionally, PCR amplification, efficient sequencing, intra- and inter-specific divergences, and DNA barcoding gaps were employed to assess the ability of each barcode to identify these diverse Berchemia plants authentically; the species were differentiated using the Kimura two-parameter and maximum composite likelihood methods. Sequence data analysis showed that the ITS2 region was the most suitable candidate barcode and exhibited the highest interspecific divergence among the three DNA-barcoding sequences. A clear differentiation was observed at the species level, in which a maximum distance of 0.264 was exhibited between dissimilar species. Clustal analysis also demonstrated that ITS2 clearly differentiated the test species in a more effective manner than that with the two other barcodes at both the hybrid and variety levels. Results indicate that DNA barcoding is ideal for species-level identification of Berchemia and provides a foundation for further identification at the molecular level of other Rhamnaceae medicinal plants.

The distribution of Blastocystis subtypes in isolates from Qatar.

BackgroundBlastocystis is a common single-celled intestinal parasite of humans and other animals comprising at least 17 genetically distinct small subunit ribosomal RNA lineages (subtypes (STs)), nine of which have been found in humans. The geographic distribution of Blastocystis subtypes is variable, but the subtypes present in Qatar are at present unknown.MethodsStool samples were collected from randomly selected, apparently healthy subjects arriving in Qatar for the first time. Blastocystis subtypes were determined by sequencing of the small subunit rRNA gene (SSU rDNA) PCR products. Phylogenetic analyses were done using Maximum Composite Likelihood method.Results71.1 % of samples were positive for Blastocystis infection based on PCR-detection methodology compared to only 6.9 % by microscopy. Prevalence of Blastocystis did not differ between the sexes nor between age classes. However, there was a regional difference in prevalence with subjects arriving from Africa showing the highest (87.6 %), those from Western Asia intermediate (68.6 %) and from Eastern Asia the lowest prevalence (67.6 %). Genetic analysis detected only three STs. ST3 was the most common (69.3 %) and ST2 was the rarest (3.5 %), while ST1 had a prevalence of 27.2 %. ST2 showed a regional variation, being absent from the 64 Western Asian Blastocystis-positive subjects. Both ST1 and ST3 showed significant differences in prevalence between the sexes.ConclusionsThis is the first report exploring the distribution of Blastocystis subtypes in our region. We recommend that stool screening via microscopy for the presence of Blastocystis should be abandoned since it is extremely insensitive. In future, the prevalence of Blastocystis infections should be based on PCR methodology and we predict that in the years ahead diagnostic PCR will become the tool of choice. More work is needed to identify the full range of Blastocystis subtypes that circulate in our region.

The equine arteritis virus isolate from the 2010 Argentinian outbreak.

A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular-based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA-trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2-parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA-trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species-level identification of the genus Ocimum.

Hepatitis B virus subgenotype A1, occurrence of subgenotype D4, and S gene mutations among voluntary blood donors in Kenya

Kenya is one of the high endemic zones for hepatitis B virus (HBV) infection. The consensuses on prevalence of the HBV genotypes and the existence of their variants have not been fully established in Kenya. Hence, there is a need to further monitor the diversity of HBV. This study aimed to extend the current molecular and epidemiological information about the geographical distribution of HBV genotypes and subgenotypes, as well as to describe the hepatitis B surface antigen (HBsAg) variants circulating in different Regional Blood Transfusion Centres of Kenya. A total of 32 HBsAg positive blood units from five different blood transfusion centers in Kenya were used in the study. The HBV DNA preS/S-gene was amplified and sequenced. Alignments of S gene were applied using reference sequence from GeneBank. Phylogenetic analysis was performed using the MEGAv4.0 software with the neighbor-joining and maximum composite likelihood methods. Twenty-one plasma samples (65.6%) were DNA positive and were successfully sequenced. Eighteen out of the twenty-one isolates (85.7%) belonged to subgenotype A1 Afro-Asian: six were from Nairobi, four from Kisumu, two from Embu, and three each from Eldoret and Mombasa. The other three strains (14.3%, 3/21) belonged to subgenotype D4 from Mombasa. The HBsAg mutations were detected in nine isolates (42.9%, 9/21). The HBV/A1 and HBV/D4 are dominant among blood donors in Kenya. This demonstrates that continuous monitoring of the HBV diversity would help reveal circulating genotypes and subgenotypes as well as mutants of clinical significance in Kenya.

BartonellaSpecies in Raccoons and Feral Cats, Georgia, USA

<i>Bartonella</i>Species in Raccoons and Feral Cats, Georgia, USA

Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies

Bovine leukemia virus (BLV) was investigated in the central nervous system (CNS) of cattle with neurological syndrome. A total of 269 CNS samples were submitted to nested-PCR (BLV env gene gp51), and the viral genotypes were identified. The nested-PCR was positive in 4.8% (13/269) CNS samples, with 2.7% (2/74) presenting at histological examination lesions of nonpurulent meningoencephalitis (NPME), whereas 5.6% (11/195) not presenting NPME (P > 0.05). No samples presented lymphosarcoma. The PCR products (437 bp) were sequenced and submitted to phylogenetic analysis by neighbor-joining and maximum composite likelihood methods, and genotypes 1, 5, and 6 were detected, corroborating other South American studies. The genotype 6 barely described in Brazil and Argentina was more frequently detected in this study. The identity matrices showed maximum similarity (100%) among some samples of this study and one from Argentina (FJ808582), recovered from GenBank. There was no association among the genotypes and NPME lesions.

Molecular and phylogenetic analysis of matrix gene of avian influenza viruses isolated from wild birds and live bird markets in the USA

Please cite this paper as: Chander et al. (2012) Molecular and phylogenetic analysis of matrix gene of avian influenza viruses isolated from wild birds and live bird markets in the USA. Influenza and Other Respiratory Viruses 7(4), 513–520. Background Wild birds are the natural hosts for influenza A viruses (IAVs) and provide a niche for the maintenance of this virus. Objectives This study was undertaken to analyze nucleotide sequences of the matrix (M) gene of AIVs isolated from wild birds and live bird markets (LBMs) to index the changes occurring in this gene. Methods M‐gene of 229 avian influenza virus (AIV) isolates obtained from wild birds and LBMs was amplified and sequenced. Full‐length sequences (∼900 nt.) thus obtained were analyzed to identify changes that may be associated with resistance to adamantanes. Phylogenetic analysis of all sequences was performed using clustalw, and evolutionary distances were calculated by maximum composite likelihood method using mega (ver. 5.0) software. Results Twenty‐seven different viral subtypes were represented with H3N8 being the most dominant subtype in wild birds and H7N2 being the predominant subtype among isolates from LBMs. Phylogenetic analysis of the M‐gene showed a high degree of nucleotide sequence identity with US isolates of AIVs but not with those of Asian or European lineages. While none of the isolates from wild birds had any antiviral resistance–associated mutations, 17 LBM isolates carried polymorphisms known to cause reduced susceptibility to antiviral drugs (adamantanes). Of these 17 isolates, 16 had S31N change and one isolate had V27A mutation. Conclusions These results indicate independent evolution of M‐gene in the absence of any antiviral drugs leading to mutations causing resistance indicating the need for continued active surveillance of AIVs.

Molecular characterization of subcutaneous and muscular coenurosis of goats in United Arab Emirates

Coenurosis is a parasitic disease caused by the larval stage of Taenia multiceps, which affects various ruminants species, particularly sheep and goats, and occasionally humans. In this note, non-cerebral coenurosis in goats with the goal to compare morphological and biomolecular characteristics is focused in order to determine if the non-cerebral specimens should be considered a new specie or a strain of T. multiceps. Three hundred goats slaughtered with macroscopic lesions due to the presence of metacestodes were examined in an abattoir in Dubai, United Arab Emirates in order to evaluate the presence of extra-cerebral coenuri. Forty-eight coenuri were found under the skin, between fasciae of the skeletal muscles, diaphragm and in the abdominal cavities. Morphologically, the examined non-cerebral coenuri recovered showed the same features reported by other authors for Coenurus cerebralis, but their location outside the central nervous system suggests that they may be a different strain or genetic variants of T. multiceps. Nine caprine coenuri were then processed for sequencing of mitochondrial partial COI (396 bp) and ND1 (471 bp) genes indicating that they had a pairwise distance of 1.0–1.3% and 2.4–4.1% compared with parasite’ COI sequences from Italy (Tm1–Tm3 strains) and Erzurum strains of ovine origin, respectively; whereas it had 0.6–1.3% and 0.4–1.1% pairwise distance for ND1. Phylogenetic trees of their ND1 and COI sequences using the maximum composite likelihood method with MEGA showed that these nine parasites recovered in Emirates abattoir could be grouped into a new strain. In conclusion, morphological and molecular characterization of caprine coenurosis from Dubai, United Arab Emirates, shows that the specimens are significantly different from other strains or genotypes in COI sequence, which suggests that they should belong to different genotypes or strains of T. multiceps.