Identification of GR and TrxR systems in Setaria cervi: Purification and characterization of glutathione reductase

The glutathione reductase (GR) and thioredoxin reductase (TrxR) are important enzymes of the redox system that aid parasites to maintain an adequate intracellular redox environment. In the present study, the enzyme activity of GR and TrxR was investigated in Setaria cervi (S. cervi). Significant activity of both enzymes was detected in the somatic extract of adult and microfilariae stages of S. cervi. Both GR and TrxR were separated by partial purification using ammonium sulfate fractionation and DEAE ion exchange chromatography suggesting the presence of both glutathione and thioredoxin systems in S. cervi. The enzyme glutathione reductase (ScGR) was purified to homogeneity using affinity and ion exchange chromatography that resulted in 90 fold purification with a yield of 11.54%. The specific activity of the ScGR was 643 U/mg that migrated as a single band on SDS-PAGE. The subunit molecular mass was determined to be ~ 50 kDa while the optimum pH and temperature were found to be 7.0 and 35 °C respectively. The activation energy (Ea) was calculated from the slope of Arrhenius plot as 16.29 ± 1.40 kcal/mol. The Km and Vmax were determined to be 0.27 ± 0.045 mM; 30.30 ± 1.30 U/ml with NADPH and 0.59 ± 0.060 mM; 4.16 ± 0.095 U/ml with GSSG respectively. DHBA, a specific inhibitor for GR has completely inhibited the enzyme activity at 1 μM concentration. The inhibition of ScGR activity with NAI (IC₅₀ 0.71 mM), NEM (IC₅₀ 0.50 mM) and DEPC (IC₅₀ 0.27 mM) suggested the presence of tyrosine, cysteine and histidine residues at its active site. Further studies on characterization and understanding of these antioxidant enzymes may lead to designing of an effective drug against lymphatic filariasis.     

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.     

First genetic evidence for the presence of the rumen fluke Paramphistomum epiclitum in Pakistan

More than 70 species of the Superfamily Paramphistomoidea, have been identified in ruminants in different parts of the world. Most are pathogenic, causing amphistomosis. Adult flukes within this family have a predilection for the forestomach (rumen) or bile duct of the liver, where they may cause epithelial damage. Identification of adult Paramphistomum, Calicophoron, Gastrothylax and Fischoederius at the species level based on morphology requires specialised expertise, whereas molecular genetic marker analysis is more precise and transferable. In the present study, we performed molecular characterisation of twenty seven adult flukes collected from the forestomachs of buffalo, cattle and goats in the Punjab province of Pakistan. PCR and sequencing of the ITS-2 rDNA region revealed a single haplotype in all cases. Phylogenetic comparison of P. epiclitum ITS2-rDNA sequences with those from other Paramphistomum, Calicophoron, Gastrothylax and Fischoederius species was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for P. epiclitum identification. This work provides a validated species-specific marker of P. epiclitum and the first report of this parasite species from Pakistan. The results of this study also have implications for the diagnosis and control of rumen flukes in the region and the need for accurate species identification to understand parasite distribution and population genetics.     

Distribution and molecular phylogeny of biliary trematodes (Opisthorchiidae) infecting native Lutra lutra and alien Neovison vison across Europe

The recent identification of Pseudamphistomum truncatum, (Rudolphi, 1819) (Trematoda: Opisthorchiidae) and Metorchis bilis (Braun, 1790) Odening, 1962 (synonymous with Metorchis albidus (Braun, 1893) Loos, 1899 and Metorchis crassiusculus (Rudolphi, 1809) Looss, 1899 (Trematoda: Opisthorchiidae)) in otters from Britain caused concern because of associated biliary damage, coupled with speculation over their alien status. Here, we investigate the presence, intensity and phylogeny of these trematodes in mustelids (principally otters) across Europe (Czech Republic, Denmark, France, Germany, Norway, Poland and Sweden and Britain). The trematodes were identified to species using the internal transcribed spacer II (ITS2) locus. Both parasites were found across Europe but at unequal frequency. In the German state of Saxony, eight out of eleven (73%) otters examined were infected with P. truncatum whilst this parasite was not found in either mink from Scotland (n = 40) or otters from Norway (n = 21). Differences in the phylogenies between the two species suggest divergent demographic histories possibly reflecting contrasting host diet or competitive exclusion, with M. bilis exhibiting greater mitochondrial diversity than P. truncatum. Shared haplotypes within the ranges of both parasite species probably reflect relatively unrestricted movements (both natural and anthropogenic) of intermediate and definitive hosts across Europe.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.     

Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM +, IgG +, 10 IgM −, IgG +, and 50 IgM −, IgG −) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM +, IgG + samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM −, IgG + seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.     

Effect of artemether,artesunate, OZ78, praziquantel,and tribendimidine alone or in combination chemotherapy on the tegument of Clonorchis sinensis

We investigated the morphological effects of half-strength treatments with praziquantel, artemether, artesunate, OZ78 and tribendimidine as well as combinations of praziquantel with artemether, artesunate, OZ78 and tribendimidine and an artesunate–tribendimidine combination in rats harboring adult Clonorchis sinensis. Rats were infected with C. sinensis, dosed orally with single agents or combination treatments and flukes recovered at 3 or 5 days post-treatment. The number of flukes was counted, the viability recorded and surface changes monitored by scanning electron microscopy. Drug effects induced by the individual drugs at sub-curative doses 3 days post-treatment were minor with the exception of flukes recovered from rats treated with artemether and tribendimidine. Treatment with the praziquantel combinations of artesunate, OZ78 and tribendimidine did not produce a greater disruption of the tegument than the individual drugs 3 days post-treatment. On the other hand, at this time point many worms treated with artemether–praziquantel had died and eruptions, roughening or blebbing were observed on all worms examined. Five days post-treatment flukes exposed to any of the praziquantel combinations in rats had died. Rats treated with an artesunate–tribendimidine combination resulted in a rapid death of flukes, 3 days post-treatment all worms had been expelled.In conclusion, we have confirmed the promising clonorchicidal properties of different drug combinations in rats. Differences in the extent and time-scale of tegumental disruption have been observed. The effect of drug combinations against C. sinensis requires further scientific inquiry, e.g. in transmission electron microscopy studies and in the C. sinensis-rabbit model.     

A study on the association of TNF-α-308, IL-6-174, IL-10-1082 and IL-1RaVNTR gene polymorphisms with rheumatic heart disease in Pakistani patients

Inflammation is an important contributor to the pathogenesis of rheumatic heart disease (RHD), a disorder of heart valves caused by a combination of immune, genetic and environmental factors. Cytokines are important mediators of inflammatory and immune responses. The aim of this study was to investigate the role of cytokine gene polymorphisms and their potential usefulness as biomarkers in RHD patients from Pakistan. We screened 150 RHD patients and 204 ethnically matched controls for tumor necrosis factor (TNF)-α^-308G/A, interleukin (IL)-10^?1082 G/A, interleukin (IL)-6^-174 G/C and a variable number of tandem repeats (VNTRs) polymorphism of the IL-1Ra gene using polymerase chain reaction. The results showed that TNF-α^-308 A and IL-6^-174 G alleles were associated with susceptibility to RHD (p = 0.000; OR = 2.81; CI = 1.5–5.14 and p = 0.025; OR = 1.50; CI = 1.04–2.16 respectively). The TNF-α^-308 AA and GA genotypes were associated with susceptibility to RHD (p = 0.012; OR = 9.94; CI; 1.21–217.3 and p = 0.046; OR = 1.97; CI = 0.98–3.97 respectively) while the GG genotype seemed to confer resistance (p = 0.003; OR = 0.39; CI = 0.20–0.76). The GG genotype for IL-6^-174 was significantly associated with predisposition to RHD (p = 0.015; OR = 2.6; CI = 1.17–5.85). The A1 (four repeats) and A2 (two repeats) alleles at the IL-1Ra VNTR polymorphism were associated with resistance and susceptibility to RHD respectively. However, this polymorphism deviated from Hardy–Weinberg equilibrium in both patients and controls in our population. TNF-α^-308 and IL-6^-174 polymorphisms may be useful markers for the identification of individuals susceptible to RHD in Pakistan. These individuals could be provided aggressive prophylactic intervention to prevent the morbidity and mortality associated with RHD.     

ITS2 sequence variations among members of Diachasmimorpha longicaudata complex (Hymenoptera: Braconidae) in Thailand

The parasitoid Diachasmimorpha longicaudata complex in Thailand contains at least 3 cryptic species informally designated as species D. longicaudata A, B and BB. DNA sequence data of nuclear ITS2 (second internal transcribed spacer) were used to characterize members of this D. longicaudata complex. The polymerase chain reaction (PCR) amplicon of ITS2 region of D. longicaudata B (≈ 650 bp) clearly differentiated this species from A and BB (amplicon of ≈ 590 bp). Sequence alignment of individual parasitoids revealed that low intraspecies differences ranged from 0.457 to 3.991%, but interspecies differences ranged from 7.566 to 12.989%. Phylogenetic trees constructed using Neighbor-Joining (NJ) and Maximum Parsimony (MP) methods, taking the parasitoid Psyttalia concolor complex as an outgroup, revealed that D. longicaudata A, B and BB formed a monophyletic group, with species A and BB being more closely related than species B. ITS2 characterization of D. longicaudata complex has revealed an interesting divergence of the three cryptic sibling species in Thailand.     

Intraspecific variability of Pythium myriotylum isolated from cocoyam and other host crops

Intraspecific variability within 51 isolates of Pythium myriotylum from cocoyam (Xanthosoma sagittifolium) and other host crops was analysed using optimum growth temperature, esterase banding patterns, AFLPs, rDNA–ITS sequencing, and virulence to cocoyam. P. myriotylum isolates virulent to cocoyam could easily be differentiated from other isolates of P. myriotylum by their optimum growth temperature. Isolates from cocoyam grew best at 28 °C with no growth at 37 °C, while P. myriotylum isolates from other host crops had their optimum growth temperature at 37 °C. Esterases produced consistent zymograms with 18 discrete esterase markers, but no monomorphic markers were produced for isolates virulent to cocoyam. Isozyme profiles based on esterase analysis showed that isolates that infect cocoyam plantlets formed a related group, irrespective of their geographic origin. P. myriotylum isolates from other host plants also grouped together, but could clearly be distinguished from the cocoyam cluster. AFLPs produced 189 scorable bands for the cocoyam isolates, of which 77 % are monomorphic. Phenetic analysis of AFLP data grouped all isolates originating from cocoyam together except for the isolates C103-04, CMR17, CMR22, and CMR25. These isolates regrouped with isolates of Pythium myriotylum from other host crops or the outgroup and were found not to be pathogenic for cocoyam. ITS sequences of isolates of P. myriotylum from cocoyam were 99.1–99.7 % identical to sequences deposited in GenBank. However, alignments of ITS sequences revealed a base transition at position 824 from adenine in typical isolates of P. myriotylum to guanine in isolates that could infect cocoyam plantlets. In a limited pathogenicity test, all isolates from cocoyam having guanine at position 824 were able to infect tissue culture derived cocoyam but not those exhibiting adenine. This study demonstrates for the first time, molecular evidence that isolates of P. myriotylum that infect cocoyam are distinct from P. myriotylum isolates from other crops and have developed a certain degree of host adaptation.     

Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitological methods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1 + to 5 +. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥ 3, ≥ 4, and ≥ 5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.     

Primer caso de fungemia asociada a catéter por Candida nivariensis en la Península Ibérica

BackgroundIn recent years the incidence of candidemia caused by non-albicans Candida species has been increasing. Two cryptic species have been described within the Candida glabrata complex, Candida nivariensis and Candida bracarensis, which may be troublesome in laboratory identification and have lower susceptibility to fluconazole.AimsTo describe the first isolation of C. nivariensis in the Iberian Peninsula from a patient suffering from a catheter-related fungemia.Case reportAn 81-year-old man was hospitalized for surgical treatment of an intestinal fistula that was associated to a severe malnutrition. Cultures of the patient's central venous catheter tip and blood yielded white colonies in BD CHROMagar Candida^® medium, which could not be identified by conventional microbiological methods. Although intravenous fluconazole was administered, blood cultures continued being positive 5 days later. The MIC values of the isolate were as follows: 1 μg/ml for amphotericin B, 0.015 μg/ml for anidulafungin, 0.125 μg/ml for caspofungin, 0.015 μg/ml for micafungin, 4 μg/ml for fluconazole, 0.25 μg/ml for itraconazole, 0.25 μg/ml for posaconazole, and 0.03 μg/ml for voriconazole. Antifungal treatment was changed to intravenous caspofungin for 2 weeks. The intestinal fistula was surgically treated. There was no evidence of relapse during the following month, and the patient was discharged. The isolate was identified as C. nivariensis based on DNA sequencing of the ITS regions of rRNA.ConclusionsC. nivariensis should be regarded as an emerging pathogen which requires molecular methods for a definitive identification. Our patient was successfully treated with caspofungin.     

Secondary structural modeling of the second internal transcribed spacer (ITS2) from Pfiesteria-like dinoflagellates (Dinophyceae)

Pfiesteria piscicida is a harmful bloom-forming alga that has received a great deal of attention due to its potential association with large fish kills and neurological problems in humans. Since the discovery of Pfiesteria, several other Pfiesteria-like dinoflagellates (PLDs) have also been identified. Genetic identification and phylogenetic relationships among the PLDs commonly utilize sequence data from the genes and spacers of the ribosomal DNA (rDNA) operon. Of these, the internal transcribed spacers (ITSs) have been previously shown to fold into secondary structures that are critical for proper ribosomal processing. In this study, we modeled the secondary structure of the second internal transcribed spacer (ITS2) from 16 PLDs (as well as an outgroup taxon) using phylogenetic comparative methods and minimum free energy. The secondary structural models predicted for these dinoflagellates consisted of four paired helices separated by five unpaired regions, consistent with those reported from many eukaryotes. All of the structures were highly stable (ΔG = ?66.1 to ?122.3 kcal·mol at 37 °C) and several structural characters were found to be conserved either across the PLDs or were specific to monophyletic subgroups, strengthening previously inferred phylogenetic relationships among taxa. Additionally, an 18 bp motif was identified in the PLDs whose position corresponds to a ribosomal processing site described from other eukaryotes. Potential applications of these ITS2 secondary structures include utility in strain and species identification, phylogenetic inference and serving as a tool for identifying and excluding rDNA pseudogenes when assessing biodiversity within the PLDs.     

Sarcocystis tupaia,sp. nov., a new parasite species employing treeshrews (Tupaiidae,Tupaia belangeri chinensis) as natural intermediate hosts

The range of vertebrates that serve as intermediate hosts for parasites in the genus Sarcocystis remains incompletely defined. Here, we provide the first report of infections in treeshrews, describe the morphology of encysted parasites using light and transmission electron microscopy, and place this agent within a phylogenetic context by sequencing and comparing its 18 S ribosomal DNA to that of related parasites. Muscle infections were diagnosed in four of 45 wild treeshrews captured in the vicinity of Kunming, Yunnan Province, Mainland China. Thread-like cysts (10.773 ± 2.411 mm in length, 0.106 ± 0.009 mm in width) had walls (0.538–0.746 µm thick) that lacked perpendicular protrusions. The interior of the cyst was packed full with cyst merozoites, the shape of which was typical of Sarcocystis. The primary cyst wall consisted of a thin membrane supported by osmiophilic material, 31–60 nm in thickness. The ground substance was about 105–526 nm thickness. Cysts conformed to typical of ‘type 1’ sarcocysts. Freshly examined and frozen specimens did not differ in their cyst wall structure, however, the appearance of bradyzoites did differ: the conoid, rhoptries and micronemes were all visible in fresh bradyzoites; in stored bradyzoites, by contrast, the rhoptries appeared smaller, and although the conoid was visible, the micronemes were not. 18 S rRNA gene was distinct from any previously reported sequence in GenBank. Their genetic and morphological uniformity suggest that these parasites, derived from treeshrews, represent a single biological species, Sarcocystis tupaia, sp. nov.     

Warneckea parvifolia (Melastomataceae–Olisbeoideae), a new “sand-forest” endemic from northeastern KwaZulu-Natal (South Africa) and southernmost Mozambique,and a phylogenetic analysis of eastern and southern African representatives of W. section Warneckea

Warneckea populations from “sand-forest” or “sand-thicket” habitats in Tembe Elephant Park, South Africa, and Licuati Forest Reserve in adjacent southern Mozambique were previously thought to be a small-leaved form of W. sousae, which typically includes larger-leaved plants ranging from central Mozambique northward to Tanzania. We examine this hypothesis using molecular and morphological evidence. Maximum-likelihood phylogenetic analysis of combined nrDNA ETS and ITS sequence data failed to resolve W. sousae and the Maputaland populations as an exclusively monophyletic group. Instead, the Kenyan endemic W. mouririifolia was strongly supported as the sister species of W. sousae, and the Maputaland plants were resolved in a separate, strongly supported clade together with populations of an as-yet undetermined Warneckea species from northern Mozambique. A hypothesis of exclusive monophyly for the plants from Tembe and Licuati had moderate support in separate ETS and ITS1 analyses (bootstrap proportions of 88% and 81%, respectively). Statistically significant differences in leaf dimensions and internode length were found between the Maputaland plants and typical W. sousae. We conclude that the populations from Tembe and Licuati represent a distinct species, which we describe as W. parvifolia. The species differs from W. sousae in having shorter internodes (mostly 5–25 mm not 10–60 mm long), smaller leaves (mostly 14–32 × 8–19 mm not 40–76 × 22–52 mm), shorter petioles (mostly 1–1.5 mm not 1.5–6 mm long), smaller flowers (hypanthium 1 × 1.5–1.75 mm not 1.5–2 × 2 mm; calyx lobes 0.5 mm not 0.75 mm long; staminal filaments 3–4 mm not 5 mm long; style 4–5 mm not 9 mm long), and globose fruit (not obovoid). An IUCN conservation status of Endangered (EN) B1a, b(ii, iii) is indicated for W. parvifolia, due to its limited distribution and projected declines in its habitat quality and area of occupancy.     

The prevalence and intensity of external parasites in domestic pigeons (Columba livia domestica) in Egypt with special reference to the role of deltamethrin as insecticidal agent

This study aimed to investigate the prevalence and intensity of external parasites in domestic pigeons in Giza, Egypt, from January 2020 to December 2020. A total of 300 domestic pigeons (25 pigeons per month) were examined. The birds were divided into groups based on their age. The oxidative stress parameters; serum zinc concentration, serum malondialdehyde (MDA), and serum Nitric oxide were evaluated in single and mixed external parasitic infestations. The prevalence of external parasites in examined pigeons was 80.3%. The detected parasites were Pseudolynchia canariensis (P. canariensis), Hippobosca equina (H. equina), Columbicola columbae (C. columbae), Menopon gallinae (M. gallinae), Knemidocoptes species (spp.) and Dermanyssus gallinae (D. gallinae); their incidences were 41.6, 26, 7, 5,0.33 and 0.33%, respectively. The highest infestation was recorded in both spring and summer. . The incidence of disease was higher in squabs and young birds than in adults. The mixed external parasitic infestation was recorded in this study. The infected birds showed decreased serum zinc concentration and elevated MDA and serum Nitric oxide levels. In conclusion, regular monthly treatment with deltamethrin is recommended as an effective drug in the treatment of the infested birds and succeeded in reducing the incidence of externalparasites in the treated birds; in addition, pigeon management measures must be implemented to reduce the risk of external parasites.     

Enrichment,isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6

The objectives of this research were to isolate pure phenol-degrading strains from enriched mixed cultures, monitoring the variations of species during the enrichment period. Two strains were isolated from the acclimated mixed culture. They were identified as Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. DGGE indicated that strain P. resinovorans appeared at the beginning, and maintained well during the enrichment period. The second strain, Brevibacillus sp., did not appear in the initial stage, but showed up after 2 weeks of enrichment. The optimum growth temperatures for P. resinovorans and Brevibacillus sp. were 31 and 39 °C, respectively. P. resinovorans could degrade phenol completely within 57.5 h, when the initial phenol concentration was lower than 600 mg l⁻¹. If the initial phenol concentration was lower than 200 mg l⁻¹, Brevibacillus sp. could remove phenol completely within 93.1 h. It was obvious that the phenol-degrading ability of P. resinovorans was much better than that of Brevibacillus sp. The metabolic pathway for P. resinovorans phenol degradation was assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

The morpho-agronomic characterization study of Lens culinaris germplasm under salt marsh habitat in Swat,Pakistan

The present research study evaluate and identify the most suitable and high yielding genotypes of Lens culinaris for the salt marsh habitat of Swat in moist temperate sort of agro climatic environment of Pakistan. A total of fourteen genotypes were cultivated and analyzed through Randomized Complete Block Design (RCBD). These genotypes were AZRC-4, NL-2, NL4, NL-5, NL-6, NARC-11-1, NARC-11-2, NARC-11-3, NARC-11-4, 09503, 09505, 09506, P.Masoor-09 and Markaz-09. Different parameters i.e., germination rate, flowering, physiological maturity, plant height, biological grain yield, seed weight, pods formation and its height, pods per plants and protein content were focused specially throughout the study. Preliminary the Lentil genotypes have significant variability in all the major morpho-agronomic traits. The days to germination, 50% flowering and 100 seed weight ranged from 7 to 9, 110 to 116 days, and from 5.4 to 7.3 gm respectively. Biological yield and grain yield ranged from 5333 to 9777 kg ha⁻¹ and 1933 to 3655 kg ha⁻¹ respectively. Whereas, protein contents ranged from 23.21% to 28.45%. It was concluded that the genotype AZRC-4 is better varity in terms of grain yield plus in 100 seed weight and moreover, 09506 genotype was significant under salt marsh habitat in early maturing for the Swat Valley, Pakistan.     

DNA barcoding species in Alexandrium tamarense complex using ITS and proposing designation of five species

Alexandrium species can be very difficult to identify, with A. catenella, A. tamarense, and A. fundyense that compose “Alexandrium tamarense species complex” (Atama complex) as a distinct example. DNA barcoding is promising to offer a solution but remains to be established. In this study, we examined the utility of ITS in resolving the Atama species complex, by analyzing previously studied strains plus unstudied Chinese strains within the LSU- and SSU-rDNA based group/clade frameworks recently established. We further investigated the presence of intragenomic polymorphism and its implications in species delimitation. Similar to the previous SSU and LSU results, our ITS-based phylogenies divided the complex to five clusters, but with longer and evener branch lengths between the clusters. Based on the ITS region, the inter-cluster genetic distances (p = 0.134–0.216) were consistently and substantially greater than intra-cluster genetic distances (p = 0.000–0.066), with an average inter-cluster (species) distance (p = 0.167) 7.6-fold of the average intraspecific difference (p = 0.022), qualifying the approximately 510–520 bp ITS as a DNA barcode for Atama complex. We detected varying levels of intragenomic polymorphism in ITS but found that this did not impact the taxon-resolving power of this gene. With this DNA barcode, the new East and South China Sea strains and one Antarctic strain were placed in Clade IIC/Group IV, even though there were 7–10 polymorphic sites in their ITS, in contrast to none in SSU. Furthermore, our results suggest that the five clusters are recognizable as distinct species according to the phylogenetic species concept. Based on the phylogenetic placements of the type-locality strains of the existing three morphospecies and the dominant localities of other strains, we propose that Group I/Clade I be designated as A. fundyense, Group III/Clade IIB as A. tamarense, Group IV/Clade IIC as A. catenella, Group II/Clade IIA as A. mediterranis, and Group V/Clade IID as A. australis.     

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380 bp for An. minimus, 180 bp for An. harrisoni, 150 bp for An. aconitus, 310 bp for An. varuna, 276 bp for P. falciparum, and 300 bp for P. vivax. The sensitivities were 0.5 pg/μl (25 sporozoites/μl) for P. falciparum DNA and between 0.5 and 5 pg/μl (25–250 sporozoites/μl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.