This study was conducted to determine the feasibility of using three plastid DNA regions (matK, trnH-psbA, and rbcL) as DNA barcodes to identify the medicinal plant Clinacanthus nutans. In this study, C. nutans was collected at several different locations. Total genomic DNA was extracted, amplified by polymerase chain reaction (PCR), and sequenced using matK, trnH-psbA, and rbcL, primers. DNA sequences generated from PCR were submitted to the National Center for Biotechnology Information's (NCBI) GenBank. Identification of C. nutans was carried out using NCBI's Basic Local Alignment Search Tool (BLAST). The rbcL and trnH-psbA regions successfully identified C. nutans with sequencing rates of 100% through BLAST identification. Molecular Evolutionary Genetics Analysis (MEGA) 6.0 was used to analyze interspecific and intraspecific divergence of plastid DNA sequences. rbcL and matK exhibited the lowest average interspecific distance (0.0487 and 0.0963, respectively), whereas trnH-psbA exhibited the highest average interspecific distance (0.2029). The R package Spider revealed that trnH-psbA correctly identified Barcode of Life Data System (BOLD) 96%, best close match 79%, and near neighbor 100% of the species, compared to matK (BOLD 72%; best close match 64%; near neighbor 78%) and rbcL (BOLD 77%; best close match 62%; near neighbor 88%). These results indicate that trnH-psbA is very effective at identifying C. nutans, as it performed well in discriminating species in Acanthaceae.
Durian (Durio zibethinus) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity,H E = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10-3. Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.
This study was a co-operative investigation of myxosporean infections of Notopterus notopterus, the bronze featherback, which is a popular food fish in the South Asian region. We examined fish from Lake Kenyir, Malaysia and the River Ganga, Hastinapur, Uttar Pradesh, India, and observed infections with two myxosporeans: Myxidium cf. notopterum (Myxidiidae) and Henneguya ganapatiae (Myxobolidae), respectively. These species were identified by myxospore morphology, morphometry and host tissue affinity, and the original descriptions supplemented with small-subunit ribosomal DNA sequences and phylogenetic analysis. Free myxospores of M. cf. notopterum were found in the gallbladder, and measured 14.7 ± 0.6 μm long and 6.3 ± 0.6 μm wide; host, tissue and myxospore dimensions overlapped with the type, but differed in morphological details (spore shape, valve cell ridges) and locality (Malaysia versus India). Plasmodia and spores of H. ganapatiae were observed in gills, and myxospores had a spore body 9.7 ± 0.4 μm long, 4.5 ± 0.5 μm wide; sample locality, host, tissue, spore morphology and morphometry matched the original description. Small-subunit ribosomal DNA sequences were deposited in GenBank (M. cf. notopterum MT365527, H. ganapatiae MT365528) and both differed by >7% from congeneric species. Although the pathogenicity and clinical manifestation of myxozoan in humans are poorly understood, consumption of raw fish meat with myxozoan infection was reported to be associated with diarrhea. Identification of current parasite fauna from N. notopterus is an essential first step in assessing pathogen risks to stocks of this important food fish.
Short tandem repeat (STR) typing continues to be the primary workhorse in forensic DNA profiling. Therefore, the present review discusses the prominent role of STR marker in criminal justice system. All over the world, deoxyribonucleic acid (DNA) profiling provides evidence that may be used to convict criminals, as an irrefutable proof of wrongful convictions, invaluable links to the actual perpetrators of crimes, and could also deter some offenders from committing more serious offences. Clearly, DNA profiling tools have also aided forensic scientists to re-evaluate old cases that were considered closed as a result of inadequate evidence. In carrying out this review, a comprehensive electronic literature search using PubMed, ScienceDirect, Google Scholar and Google Search were used, and all works meeting the subject matter were considered, including reviews, retrospective studies, observational studies and original articles. Case reports presented here, further demonstrates the crucial role of forensic DNA profiling in mitigating and providing compelling evidence for the resolution of crimes. For case report 1, there was a 100% match between the DNA recovered from the items found in the crime scene, and the suspect's DNA sample collected via buccal swab following 15 STR loci examination. Case report 2 further highlights the indispensable contribution of DNA database in solving crime. Therefore, it has become very necessary for developing countries like Nigeria to develop a national DNA database and make policies and legislatures that will further expand and enable the practice of forensic genetics, particularly DNA profiling.
Phytocystatin, a type of protease inhibitor (PI), plays major roles in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance. Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine. It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty. In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species. A full-length cDNA of the phytocystatin gene, designated CypCl, was acquired using 5'/3' rapid amplification of cDNA ends method and it has been deposited in NCBI database (accession number KF545954.1). It has a 687 bp long open reading frame (ORF) which encodes 228 amino acids. BLAST result indicated that CypCl is similar to cystatin protease inhibitor from Cucumis sativus with 74% max identity. Sequence analysis showed that CypCl contains most of the motifs found in a cystatin, including a G residue, LARFAV-, QxVxG sequence, PW dipeptide, and SNSL sequence at C-terminal extension. Phylogenetic studies also showed that CypCl is related to phytocystatin from Elaeis guineensis.
The tertiary motifs in complex RNA molecules play vital roles to either stabilize the formation of RNA 3D structure or to provide important biological functionality to the molecule. In order to better understand the roles of these tertiary motifs in riboswitches, we examined 11 representative riboswitch PDB structures for potential agreement of both motif occurrences and conservations. A total of 61 unique tertiary interactions were found in the reference structures. In addition to the expected common A-minor motifs and base-triples mainly involved in linking distant regions the riboswitch structures three highly conserved variants of A-minor interactions called G-minors were found in the SAM-I and FMN riboswitches where they appear to be involved in the recognition of the respective ligand's functional groups. From our structural survey as well as corresponding structure and sequence alignments, the agreement between motif occurrences and conservations are very prominent across the representative riboswitches. Our analysis provide evidence that some of these tertiary interactions are essential components to form the structure where their sequence positions are conserved despite a high degree of diversity in other parts of the respective riboswitches sequences. This is indicative of a vital role for these tertiary interactions in determining the specific biological function of riboswitch.
Background. Central intermediary metabolism (CIM) in bacteria is defined as a set of metabolic biochemical reactions within a cell, which is essential for the cell to survive in response to environmental perturbations. The genes associated with CIM are commonly found in both pathogenic and non-pathogenic strains. As these genes are involved in vital metabolic processes of bacteria, we explored the efficiency of the genes in genotypic characterization of Burkholderia pseudomallei isolates, compared with the established pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) schemes. Methods. Nine previously sequenced B. pseudomallei isolates from Malaysia were characterized by PFGE, MLST and CIM genes. The isolates were later compared to the other 39 B. pseudomallei strains, retrieved from GenBank using both MLST and sequence analysis of CIM genes. UniFrac and hierachical clustering analyses were performed using the results generated by both MLST and sequence analysis of CIM genes. Results. Genetic relatedness of nine Malaysian B. pseudomallei isolates and the other 39 strains was investigated. The nine Malaysian isolates were subtyped into six PFGE profiles, four MLST profiles and five sequence types based on CIM genes alignment. All methods demonstrated the clonality of OB and CB as well as CMS and THE. However, PFGE showed less than 70% similarity between a pair of morphology variants, OS and OB. In contrast, OS was identical to the soil isolate, MARAN. To have a better understanding of the genetic diversity of B. pseudomallei worldwide, we further aligned the sequences of genes used in MLST and genes associated with CIM for the nine Malaysian isolates and 39 B. pseudomallei strains from NCBI database. Overall, based on the CIM genes, the strains were subtyped into 33 profiles where majority of the strains from Asian countries were clustered together. On the other hand, MLST resolved the isolates into 31 profiles which formed three clusters. Hierarchical clustering using UniFrac distance suggested that the isolates from Australia were genetically distinct from the Asian isolates. Nevertheless, statistical significant differences were detected between isolates from Malaysia, Thailand and Australia. Discussion. Overall, PFGE showed higher discriminative power in clustering the nine Malaysian B. pseudomallei isolates and indicated its suitability for localized epidemiological study. Compared to MLST, CIM genes showed higher resolution in distinguishing those non-related strains and better clustering of strains from different geographical regions. A closer genetic relatedness of Malaysian isolates with all Asian strains in comparison to Australian strains was observed. This finding was supported by UniFrac analysis which resulted in geographical segregation between Australia and the Asian countries.
The earliest settlers in Peninsular Malaysia are the Orang Asli population, namely Semang, Senoi and Proto Malays. In the present study, we typed the HLA-A, -B and -DRB1 loci of the Kensiu and Semai Orang Asli sub-groups. Sequence-based HLA typing was performed on 59 individuals from two Orang Asli sub-groups. A total of 11, 18 and 14 HLA-A, -B and -DRB1 alleles were identified, respectively. These data are available in the Allele Frequencies Net Database under the population name "Malaysia Kedah Kensiu" and "Malaysia Pahang Semai".
A body of an unknown adult female was found within a shallow burial ground in Malaysia whereas the skull was exposed and visible on the ground. During autopsy examination, nine insect larvae were recovered from the interior of the human skull and subsequently preserved in 70% ethanol. The larvae were greyish in appearance, each with a posterior elongated breathing tube. A week after the autopsy, more larvae were collected at the burial site, and some of them were reared into adults. Adult specimens and larvae from the skull and from the burial site were sequenced to obtain DNA barcodes. Results showed all adult flies reared from the burial site, as well as the larvae collected from the skull were identified as Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae). Here, we report the colonization of E. arvorum larvae on a human corpse for the first time.
The ability to isolate and generate a DNA profile from human DNA recovered from tropical bed bugs (Cimex hemipterus) for identifying individuals can be useful for public health, forensic, and medical entomology. In this study, genomic DNA was recovered from both male and female bed bugs at every time interval tested (0, 1, 3, 5, 7, 14, 30, and 45 days post blood meal). The total DNA concentrations recovered from male bed bugs ranged from 12.93 to 65.97 ng/µL, while the total DNA concentrations from female bed bugs ranged from 8.93 to 44.53 ng/µL. However, based on the results from the BLAST search and PCR products, human DNA could be detected from female bed bugs at 0, 3, 5, 14, and 30 days post blood meal using the D18S51 marker. Concentrations of PCR products of the D18S51 locus from male bed bugs ranged from 4.20 to 35.50 ng/µL, whereas, for female bed bugs, concentrations ranged from 4.31 to 22.47 ng/µL. These were generally higher compared to the PCR products of the first hypervariable part (HVR1) marker. The results indicate the HVR1 locus was less sensitive than the D18S51 locus.
This study reports on the detection of additional expressed sequence tags (EST) derived simple sequence repeat (SSR) markers for the oil palm. A large collection of 19243 Elaeis guineensis ESTs were assembled to give 10258 unique sequences, of which 629 ESTs were found to contain 722 SSRs with a variety of motifs. Dinucleotide repeats formed the largest group (45.6%) consisting of 66.9% AG/CT, 21.9% AT/AT, 10.9% AC/GT and 0.3% CG/CG motifs. This was followed by trinucleotide repeats, which is the second most abundant repeat types (34.5%) consisting of AAG/CTT (23.3%), AGG/CCT (13.7%), CCG/CGG (11.2%), AAT/ATT (10.8%), AGC/GCT (10.0%), ACT/AGT (8.8%), ACG/CGT (7.6%), ACC/GGT (7.2%), AAC/GTT (3.6%) and AGT/ACT (3.6%) motifs. Primer pairs were designed for 405 unique EST-SSRs and 15 of these were used to genotype 105 E. guineensis and 30 E. oleifera accessions. Fourteen SSRs were polymorphic in at least one germplasm revealing a total of 101 alleles. The high percentage (78.0%) of alleles found to be specific for either E. guineensis or E. oleifera has increased the power for discriminating the two species. The estimates of genetic differentiation detected by EST-SSRs were compared to those reported previously. The transferability across palm taxa to two Cocos nucifera and six exotic palms is also presented. The polymerase chain reaction (PCR) products of three primer-pairs detected in E. guineensis, E. oleifera, C. nucifera and Jessinia bataua were cloned and sequenced. Sequence alignments showed mutations within the SSR site and the flanking regions. Phenetic analysis based on the sequence data revealed that C. nucifera is closer to oil palm compared to J. bataua; consistent with the taxanomic classification.
Indian spinach (Basella rubra L.) is a red stem species of Basella that is cultivated worldwide as an ornamental and the aerial parts are also consumed as a vegetable. In May of 2011, symptoms of damping-off were observed on approximately 10% of the plants at the stem base around the soil line of seedlings in a greenhouse in Homestead, FL. Lesions were initially water soaked, grayish to dark brown, irregular in shape, and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Symptomatic stem tissue was surface sterilized with 0.6% sodium hypochlorite, rinsed in sterile distilled water, air dried, and plated on potato dextrose agar (PDA). Plates were incubated at 25°C in darkness for 3 to 5 days. A fungus was isolated in all six isolations from symptomatic tissues on PDA. Fungal colonies on PDA were light gray to brown with abundant growth of mycelia, and the hyphae tended to branch at right angles when examined under a microscope. A septum was always present in the branch of hyphae near the originating point and a slight constriction at the branch was observed. Neither conidia nor conidiophores were found from the cultures on PDA. The characteristics of hyphae, especially the right angle branching of mycelia, indicate close similarity to those of Rhizoctonia solani (2,3). The internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced (GenBank Accession No. JN545836). Subsequent database searches by the BLASTN program indicated that the resulting sequence had a 100% identity over 472 bp with the corresponding gene sequence of R. solani anastomosis group (AG) 4 (GenBank Accession No. JF701752.1), a fungal pathogen reported to cause damping-off on many crops. Pathogenicity was confirmed through inoculation of healthy India spinach plants with the hyphae of isolates. Four 4-week-old plants were inoculated with the isolates by placing a 5-mm PDA plug of mycelia at the stem base and covering with a thin layer of the soil. Another four plants treated with sterile PDA served as a control. After inoculation, the plants were covered with plastic bags for 24 h and maintained in a greenhouse with ambient conditions. Four days after inoculation, water-soaked, brown lesions, identical to the symptoms described above, were observed on the stem base of all inoculated plants, whereas no symptoms developed on the control plants. The fungus was isolated from affected stem samples, and the identity was confirmed by microscopic appearance of the hyphae and sequencing the ITS1/ITS4 intergenic spacer region, fulfilling Koch's postulates. This pathogenicity test was conducted twice. R. solani has been reported to cause damping-off of B. rubra in Ghana (1) and Malaysia (4). To our knowledge, this is the first report of damping-off caused by R. solani AG-4 on Indian spinach in Florida and the United States. With the increased interest in producing Asian vegetables for food and ornamental purposes, the occurrence of damping-off on Indian spinach needs to be taken into account when designing programs for disease management in Florida. References: (1) H. A. Dade. XXIX. Bull. Misc. Inform. 6:205, 1940. (2) J. R. Parmeter et al. Phytopathology 57:218, 1967. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St Paul, MN, 1991. (4) T. H. Williams and P. S. W. Liu. Phytopathol. Pap. 19:1, 1976.
Symptoms of water-soaked lesions and soft rot were first observed in June 2011 on bell pepper fruits (Capsicum annuum cv. Annuum) in the two main regions of pepper production in Malaysia (Cameron Highlands and Johor State). Economic losses exceeded 40% in severely infected fields and greenhouses with the estimated disease incidence of 70%. In pepper fruits damaged by insects, sunscald, or other factors, symptoms initially appeared in the peduncle and calyx tissues and entire fruits were turned into watery masses within 2 to 6 days. Fruits infected in the field tended to collapse and hang on the plant. When the contents leaked out, the outer skin of the fruit dried and remained attached to the plant. Field-grown transplants and infected soil were identified as probable sources of inocula. A total of 50 attached fruits were collected from 10 pepper fields and greenhouses located in the two growing regions. Tissue from the margins of water-soaked lesions was surface-sterilized in 1% NaOCl for 2 min, rinsed in sterile water, dried, and plated onto nutrient agar (NA) and eosin methylene blue agar (EMB) media (3). A similar bacterium was isolated from all samples. After 2 days, white to creamy bacterial colonies on NA and emerald green colonies on EMB developed. Five independent strains were subjected to further biochemical, molecular, and pathogenicity tests. Bacterial strains were gram-negative, motile rods, grew at 37°C, were facultatively anaerobic, oxidase-negative, phosphatase-negative, and catalase-positive. They degraded pectate, were sensitive to erythromycin, did not utilize Keto-methyl glucoside, were indole production-negative, and reduced sugars from sucrose (3). Acid production was negative from sorbitol and arabitol, but positive from melibiose and citrate. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment (2). Amplification of the intergenic transcribed spacer (ITS) region by G1 and L1 primers (4) gave two amplicons ca. 550 and 580 bp long. The expected amplicon was not produced with any of the strains using primers Br1f/L1r and Eca1f/Eca2r (1), whereas a 550-bp PCR product, typical of Pectobacterium carotovorum subsp. carotovorum, was obtained with primers EXPCCF and EXPCCR (1). Based on biochemical and molecular characteristics, and analysis of PCR-RFLP of 16S-ITS-23R rRNA genes using Rsa I enzyme (4), all five bacterial strains were identified as P. carotovorum subsp. carotovorum. BLAST analysis of the 16S rRNA sequence (GenBank Accession No KC189032) showed 100% identity to the 16S rRNA of P. carotovorum subsp. carotovorum strain PPC192. For pathogenicity tests, four mature pepper fruits of cv. Annuum were inoculated by injecting 10 μl of a bacterial suspension (108 CFU/ml) into pericarps and the fruits were incubated in a moist chamber at 80 to 90% relative humidity and 30°C. After 72 h, water-soaked lesions similar to those observed in the fields and greenhouses were observed and bacteria with the same characteristics were consistently reisolated, thereby fulfilling Koch's postulates. Symptoms were not observed on water-inoculated controls. References: (1) S. Baghaee-Ravari et al. Eur. J. Plant Pathol. 129:413, 2001. (2) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (3) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St Paul, MN, 2001. (4) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.
In July 2011, a severe outbreak of pod and stem blight was observed on lima bean (Phaseolus lunatus L.) plants grown in the Cameron Highlands, located in Pahang State, Malaysia. Disease incidence varied from 33 to 75% in different fields. Pods and stems exhibited withered, light brown to reddish brown necrotic areas. Sub-circular and brown lesions were produced on the leaves. These lesions varied in size, often reaching a diameter of 1 to 2 cm. After tissue death, numerous pycnidia were observed on the surface of the pod or stem. The pycnidia diameter varied from 155 to 495 μm, averaging 265.45 μm, and on the surface of the pod or stem, pycnidia were often arranged concentrically or linearly, respectively. Pycnidiospores were hyaline, 1-celled, usually straight, and rarely, slightly curved. The α-spores varied from 5.5 to 9.0 × 2.5 to 4.0 μm; averaging 7.3 × 3.5 μm. The β-spores found either alone or with pycnidiospores in pycnidia were slender, hyaline, nonseptate, and straight or curved. Size varied from 15.8 to 38.0 × 1.3 to 2.1 μm; averaging 25.86 × 1.8 μm. The colony characteristics were recorded from pure cultures grown on potato dextrose agar plates, and incubated in darkness for 7 days at 25 °C, then exposed to 16/8 h light and dark periods at 25°C for a further 14 to 21 days. Morphological characteristics of the colonies and spores on PDA matched those described for P. phaseolorum var. sojae (2). Colonies were white, compact, with wavy mycelium and stromata with pycnidia that contained abundant β-spores. Sequence analysis of the ribosomal DNA internal transcribed spacer obtained from the Malaysian isolate FM1 (GenBank Accession No. JQ514150) using primers ITS5 and ITS4 (1) aligned with deposited sequences from GenBank confirmed identity and revealed 99% to 100% DNA similarity with P. phaseolorum strains (AY577815, AF001020, HM012819, JQ936148). The isolate FM1 was used for pathogenicity testing. Five non-infected detached leaves and pods of 4-week-old lima bean were surface sterilized and inoculated by placing 10 μl of conidial suspension (106 conidia ml-1) on the surface of leaves and pods using either the wound/drop or non-wound/drop method and distilled water used as control (3). The inoculated leaves and pods were incubated at 25 °C and 98% RH, and the experiment was performed twice. Disease reactions and symptoms were evaluated after inoculation. After one week, typical symptoms of pod and stem blight appeared with formation of pycnidia on the surface of the tissues, but not on non-inoculated controls. P. phaseolorum var. sojae was consistently reisolated from symptoms. To our knowledge, this is the first report of P. phaseolorum var. sojae causing pod and stem blight of lima bean in Malaysia. References: (1) R. Ford et al. Aust. Plant Pathol. 33:559, 2004. (2) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. American Phytopathological Society, St. Paul, MN, 1999. (3) P. P. Than et al. Plant Pathol. 57:562, 2008.
We explored and constructed haplotype network for simian malaria species: Plasmodium knowlesi, P. cynomolgi and P. inui aiming to understand the transmission dynamics between mosquitoes, humans and macaques. Mosquitoes were collected from villages in an area where zoonotic malaria is prevalent. PCR analysis confirmed Anopheles balabacensis as the main vector for macaque parasites, moreover nearly 60% of the mosquitoes harboured more than one Plasmodium species. Fragments of the A-type small subunit ribosomal RNA (SS rRNA) amplified from salivary gland sporozoites, and equivalent sequences obtained from GenBank were used to construct haplotype networks. The patterns were consistent with the presence of geographically distinct populations for P. inui and P. cynomolgi, and with three discrete P. knowlesi populations. This study provides a preliminary snapshot of the structure of these populations, that was insufficient to answer our aim. Thus, collection of parasites from their various hosts and over time, associated with a systematic analysis of a set of genetical loci is strongly advocated in order to obtain a clear picture of the parasite population and the flow between different hosts. This is important to devise measures that will minimise the risk of transmission to humans, because zoonotic malaria impedes malaria elimination.
This preliminary study aimed to identify a commercial gamat species, Stichopus horrens Selenka, 1867, and a timun laut species, Holothuria (Mertensiothuria) leucospilota (Brandt, 1835), from Pangkor Island, Perak, Malaysia, employing morphological techniques based on the shape of the ossicles and molecular techniques based on the cytochrome c oxidase I (COI) mitochondrial DNA (mtDNA) gene. In Malaysia, a gamat is defined as a sea cucumber species of the family Stichopodidae with medicinal value, and timun laut refers to non-gamat species. S. horrens is very popular on Pangkor Island as a main ingredient in the traditional production of air gamat and minyak gamat, while H. leucospilota is the most abundant species in Malaysia. In contrast to previous studies, internal body parts (the respiratory tree and gastrointestine) were examined in this study to obtain better inferences based on morphology. The results showed that there were no ossicles present in the gastrointestine of H. leucospilota, and this characteristic is suggested as a unique diagnostic marker for the timun laut species. In addition, the presence of Y-shaped rods in the respiratory tree of S. horrens subsequently supported the potential to use internal body parts to identify the gamat species. Phylogenetic analysis of the COI mtDNA gene of the sea cucumber specimens using the neighbour-joining method and maximum likelihood methods further confirmed the species status of H. leucospilota and S. horrens from Pangkor Island, Perak, Malaysia. The COI mtDNA gene sequences were registered with GenBank, National Center for Biotechnology Information (NCBI), US National Library of Medicine (GenBank accession no.: KC405565-KC405568). Although additional specimens from various localities will be required to produce more conclusive results, the current findings provide better insight into the importance of complementary approaches involving morphological and molecular techniques in the identification of the two Malaysian sea cucumber species.
The (R)-3-hydroxyacyl-ACP-CoA transferase catalyses the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serves as the ultimate precursor for polyhydroxyalkanoate (PHA) polymerisation from unrelated substrates in pseudomonads. PhaG was found to be responsible for channelling precursors for polyhydroxyalkanoate (PHA) synthase from a de novo fatty acid biosynthesis pathway when cultured on carbohydrates, such as glucose or gluconate. The phaG gene was cloned from Pseudomonas sp. USM 4-55 using a homologous probe. The gene was located in a 3660 bp Sal I fragment (GenBank accession number EU305558). The open reading frame (ORF) was 885 bp long and encoded a 295 amino acid protein. The predicted molecular weight was 33251 Da, and it showed a 62% identity to the PhaG of Pseudomonas aeruginosa. The function of the cloned phaG of Pseudomonas sp. USM 4-55 was confirmed by complementation studies. Plasmid pBCS39, which harboured the 3660 bp Sal I fragment, was found to complement the PhaG-mutant heterologous host cell, Pseudomonas putida PhaGN-21. P. putida PhaGN-21, which harboured pBCS39, accumulated PHA that accounted for up to 18% of its cellular dry weight (CDW). P. putida PhaGN-21, which harboured the vector alone (PBBR1MCS-2), accumulated only 0.6% CDW of PHA.
Staphylococcus kloosii, an orange pigment-producing bacterium, was isolated from the respiratory tree of Holothuria (Mertensiothuria) leucospilota (Brandt 1835) from Teluk Nipah, Pangkor Island, Perak, Malaysia. This report is the first documentation of this Gram-positive strain, referred to as Strain 68 in Malaysia. A partial 16S ribosomal RNA gene sequence of the mesophilic strain has been registered with GenBank (National Center for Biotechnology Information, US National Library of Medicine) with accession number JX102547. Phylogenetic analysis using the neighbour-joining method further supported the identification of Strain 68 as S. kloosii. The circular strain produced orange pigments on tryptone glucose yeast extract agar (TGYEA) and in nutrient broth (NB) at approximately pH 7. The visible spectra of ethanolic and methanolic pigment extracts of the bacterial strain were considered identical with λmax at 426, 447 and 475 nm and λmax at 426, 445 and 473 nm, respectively. Both visible spectra resemble the visible spectra of lutein, which is a commercial carotenoid; however, further analyses are required to confirm the identity of this pigment. The methanolic extracts of the intracellular pigments comprised at least three pigment compounds: an orange pigment compound (major compound), a yellow pigment compound (the least polar) and a pink pigment compound (the most polar). These findings are the first documentation of the pigment composition of S. kloosii as no such record could be found to date.
Trypanosomiasis is a disease caused by a flagellate protozoon called Trypanosoma and can be mechanically transmitted by vectors to humans and animals. Various species of Trypanosoma are found in livestock and poultry, which include Trypanosoma evansi, T. brucei, T. vivax and T. congolense. The camel is the most sensitive livestock for T. evansi, so the exact identification of infection is very important for epidemiological studies and the design of control programs. The present study was conducted with the aim of molecular detection of camel trypanosomiasis in the Sistan region in 2015. Previous studies have shown that internal transcribed spacer one (ITS1) of the ribosomal DNA is a reliable genetic marker for carrying out systematic molecular studies of trypanosomes. In order to investigate infections of camels with T. evansi, a total of 113 blood samples were collected randomly and the presence of parasites in each sample was evaluated using the microscopic method and polymerase chain reaction (PCR) test. Genomic DNA was extracted and the ITS-1 was amplified by PCR. In comparison to the nucleotide sequence obtained with the sequences recorded in GenBank, it was determined that there is a 99% homology with the recorded sequence of T. evansi. The obtained sequence was registered in Gen Bank with kx900449 code. The T. evansi sequences from different countries such as India, Taiwan, Thailand, the Philippines, China and Argentina and etc., were extracted from the Gene bank and aligned using the ClustalW2 sequence alignment tool and MEGA software. In this study the prevalence of T. evansi infection using the molecular method was 6.19% and no positive samples were found by microscopic observation.
Knowledge of molecular identification of tick-borne pathogens in camels in Saudi Arabia is very limited; few molecular epidemiological studies have been under taken. This study was to detect Anaplasma spp. and Piroplasma spp. in camels from Asir Province, Saudi Arabia. A total of 150 blood samples were collected from camels in Asir Province and investigated by polymerase chain reaction (PCR) that targeted 18S rRNA and 23S rRNA to detect the DNA of Piroplasma spp. and Anaplasma spp., respectively. The positive samples for 23S rRNA were assayed again by PCR targeting the 16S rRNA. All the blood samples were free from Piroplasma spp. infection. Three camels (2%) were found to be positive for Anaplasma infection through use of PCR that targeted the 23S rRNA gene. There were no significant differences between ages or sexes in the camels that tested positive for Anaplasma. All positive Anaplasma infections were recorded in camels that were infested by ticks. Two Anaplasma sequences for the16S rRNA gene were deposited in GenBank with accession numbers MN882724 and MN882725. They recorded 99.16% and 99.34% similarities (respectively) with KF843825.1 (Candidatus Anaplasma camelii reported in Unizah, Saudi Arabia). Phylogenetic analyses revealed that the two sequences recorded in this study were close to each other; both were located in one cluster with Candidatus Anaplasma camelii isolates that were recorded before in the adjacent areas of Unizah in Saudi Arabia and Iran. In conclusion: two new Anaplasma genotypes close to Candidatus Anaplasma camelii were found in camels in Asir Province, Saudi Arabia for the first time. The camels in this province were found to be free of Piroplasma infection.