PCR Products Of Gene Research Articles

A new molecular method for fast differentiation of cockles Cerastoderma edule and Cerastoderma glaucum

Cerastoderma edule and C. glaucum are two species of cockles that co-exist in European waters. They are morphologically similar but exhibit remarkable differences in biological, ecological, and genetic aspects, as well as in resistance to parasites (e.g., Martellia cochilia) and in disease incidence (e.g., disseminated neoplasia). Moreover, they differ in their economic significance; while C. edule represents a highly valuable marine resource, C. glaucum is only marginally fished. The aim of this work was to develop a simple and fast method that, for the first time, uses the sequence of a mitochondrial gene for the molecular differentiation of the two cockle species. A total of 304 partial sequences of the cytochrome c oxidase subunit I (COI) gene, retrieved from the Nucleotide database, were used to design two sets of species-specific primers to generate PCR products of different sizes (322 bp in C. glaucum and 247 bp in C. edule). The discriminatory ability of the PCR assay was tested in cockles from the Spanish, French, and Italian coasts with successful differentiation in all cases. This novel molecular identification method requires minimal technical equipment and can be carried out in one working day. For its simplicity, it can be very useful for conservation and sustainable management of the two cockle species, facilitating the assessment of distribution, abundance and relative sensitivity to viruses, parasites and diseases.

Epstein–Barr virus (Orthoherpesviridae: <i>Lymphocryptovirus</i>) among Russian ethnic groups: Prevalence of EBV types (EBV-1 and EBV-2), <i>LMP1</i> gene variants and malignancies

Introduction. The discovery of two EBV types (EBV-1 and EBV-2) has stimulated the study of their prevalence in populations and association with malignancies.
 Objective. To study the prevalence of EBV-1 and EBV-2 types among ethnic groups in Russia, to analyze PCR products of the LMP1 gene in virus isolates, and to evaluate the contribution of EBV types to the incidence of malignant neoplasms.
 Materials and methods. EBV isolates from oral lavages of the Republics Adygea, Kalmykia, Tatarstan and the Moscow Region (MR) representatives were studied by nested PCR for the belonging to EBV-1 and EBV-2 types. LMP1 amplicons obtained by real-time PCR from viral isolates DNA were classified and sequenced on an automatic DNA sequencer ABI PRISM 3100-Avant (USA). The sequencing results were analyzed using Chromas 230 and Vector NT programs (Invitrogen, USA). The reliability of the obtained data was assessed using statistical packages Statistica for Windows, 10.0.
 Results. The prevalence rates of EBV-1 and EBV-2 in representatives of four ethnic groups were compared with the incidence rates of some tumors in the population of three Republics and MR.
 The dominant persistence of the transforming in vitro EBV-1 type in representatives of the Republic of Tatarstan and MR correlated with a high incidence of gastric cancer and lymphomas in the population of these territories. On the contrary, predominant infection of the non-transforming in vitro EBV-2 type and both types of the virus in approximately the same percentage of representatives of Adygea and Kalmykia, respectively, correlated with a lower level incidence of above tumors in populations of these Republics. The differences between the incidence rates of neoplasms in the compared ethnic populations were statistically insignificant (p 0.05). LMP1 variants of viral isolates did not reflect either the level of EBV persistence types or the incidence of tumors.
 Conclusion. Infection of ethnic groups with EBV-1 and EBV-2 may vary significantly under the influence of various factors. The predominance of the in vitro transforming EBV-1 type in the population did not increase the incidence of tumors due to cases associated with the dominant virus type.

Ipomoea obscura Represents a New Host of Phytoplasma Belonging to 16SrII Group Associated with Witches'-Broom Symptoms in China.

Phytoplasmas are phloem-limited plant pathogenic prokaryotes which can not be cultured in vitro. The pathogens could cause various plant symptoms such as witches'-broom, virescence, and leaf yellows. Ipomoea obscura is a valuable plant species belonging to the family Convolvulaceae, mainly used as a traditional Chinese medicine used to treat diseases such as dehydration and diuresis. In western countries it is commonly referred to as 'obscure morning glory'. During 2020 to 2021, plants showing abnormal symptoms including witches'-broom, internode shortening, and small leaves were found in Hainan Province, a tropical island of China. Approximately 30 % of I. obscura plants in the sampling regions which spanned 400 acres, showed symptoms. In order to identify the associated pathogen, six symptomatic samples and three asymptomatic samples were collected and total DNA were extracted from 0.10 g fresh plant leaf tissues using CTAB DNA extraction method. 16S rRNA and secA gene fragments, specific to phytoplasmas, were PCR amplified using primers R16mF2/R16mR1 and secAfor1/secArev3. The target PCR bands were obtained from the DNA of six symptomatic samples, whereas not from the DNA of the asymptomatic samples. The PCR products of phytoplasma 16S rRNA and secA gene obtained from the diseased samples were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The 16S rRNA and secA gene sequences identified in the study were all identical with the length of 1330 bp (GenBank accession: OR625212) and 720 bp (OR635662) respectively. According to methods and protocols of phytoplasma identification and classification (Wei and Zhao, 2022), the phytoplasma strain identified in the study was described as Ipomoea obscura witches'-broom (IoWB) phytoplasma, IoWB-hnld strain. The partial 16S rRNA gene sequence of IoWB showed 100 % sequence identity over the full 1330 bp sequence to phytoplasmas belonging to 16SrII group like cassava witches'-broom phytoplasma (KM280679). The BLAST search of the 720 bp partial secA gene fragment of IoWB showed 100% sequence identity for the full sequence to phytoplasmas belonging to 16SrII group like 'Sesamum indicum' phyllody phytoplasma (OQ420657). RFLP analysis based on the 16S rRNA gene using iPhyClassifier demonstrated that the IoWB strain was a member of 16SrII-A subgroup with the similarity coefficient 1.00 to the reference phytoplasma strain (L33765). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value indicated that IoWB-hnld was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value separately. To our knowledge, this is the first report that Ipomoea obscura can be infected by phytoplasmas belonging to 16SrII-A subgroup in China. This report adds to the host range of 'Ca. Phytoplasma aurantifolia', documenting the symptoms on I. obscura which will assist in monitoring and control of the associated pathogen.

Molecular Identification and Phylogenetic Analysis of Salmonella‎ species ‎Isolated from Diarrheal Children and Dogs in Baghdad Governorate, ‎Iraq‎

This work aimed to use conventional PCR to identify Salmonella‎ spp. that ‎were isolated from diarrheal children and healthy and diarrheic dogs based on four ‎virulence genes, hilA, stn, spvR‎, and marT. Sixteen Salmonella‎ isolates including: 9 ‎isolated from children's diarrhea from three species (S. Typhimurium, S. Enteritidis, S. ‎Typhi) and seven isolated from dogs including (S. Typhimurium, S. Enteritidis, S. ‎Muenchen), were identified primarily by several methods. The PCR products of the 16S ‎rRNA gene were sequenced and examined using BLAST analysis to find differences and ‎similarities between these Iraqi isolates and already-known global strains in order to ‎construct the phylogenetic tree of S. Muenchen which was detected for the first time in ‎dogs in Iraq. The results of the study revealed that all isolates of Salmonella‎ obtained ‎from children possess the hilA and stn genes. The marT gene was detected in 88% of the ‎Salmonella‎ serovars, and the spvR‎ gene was carried in 55% of the isolates. Among dog ‎Salmonella‎ isolates, the hilA gene was detected at 100%, the stn gene was at 85.7%, the ‎marT gene was present at 71.4%, while the spvR‎ gene was found at 57.1%. The result of ‎DNA sequencing and phylogenetic tree indicated that the local Iraqi S. Muenchen was ‎extremely close to the national strain and share the same ‎16S rRNA gene sequence, the ‎isolate was registered at NCBI and became a global reference with the accession number OQ999043.1. In conclusion, the presence of these important virulence genes among ‎Salmonella‎ serovars isolated from children and dogs alerted on the potential risk of ‎contamination of the environment and may lead to a community health crisis‎.

Rhodophyta DNA Barcoding: Ribulose-1, 5-Bisphosphate Carboxylase Gene and Novel Universal Primers.

Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the rbcL gene. The PCR products of the rbcL gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as Laurencia natalensis and Hypnea spinella, and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions.

Prevalence and clonal diversity of methicillin-resistant Staphylococcus aureus andmethicillin-resistant Staphylococcus pseudintermedius isolated from dogs andcatswith eye discharge.

Methicillin-resistant Staphylococcus (MRS) has originated, spread extensively, and become a prominent source of bacterial infections in both human and animal. We report the prevalence, genetic diversity, and antimicrobial resistance pattern of Staphylococcus pseudintermedius and Staphylococcus aureus strains isolated from dogs and cats with eye discharges. A total of 12 (6.0%) coagulase-positives staphylococci were identified as (6/200, 3%) S. aureus and (6/200, 3%) S. pseudintermedius. The phenotypic methicillin resistance of S. aureus and S. pseudintermedius were 50.0% (3/6) and 16.7% (1/6), respectively. None of the isolates showed biofilm formation in the microtiter plate assay. The highest resistance (50.0%) for S. pseudintermedius strains was detected against clindamycin and tetracycline. 67.0% of S. aureus isolates were resistant to penicillin-G. The PCR analysis conducted for detection of mecA gene indicated that only one S. aureus isolated from a cat was mecA gene positive. Phylogenetic analysis based on repetitive sequence-based PCR (rep-PCR) showed that all strains were typable and generated PCR products ranging from 800 bp to 4,400 bp. The lineages ST241 and the novel ST2361 in multi-locus sequence typing (MLST) analysis were detected in one methicillin-susceptible S.pseudintermedius and methicillin-resistant S. pseudintermedius of dogs, respectively. Inaddition, the lineages ST4155 and ST7217 of two methicillin-resistant S. aureus strains of cats were connected epidemiologically to previously reported cases. These results indicate epidemiologically related strains (ST241, ST4155, and ST7217) transferring between animals and humans. Therefore, the strategies to combat the widespread MRS should be based on collaboration between human and veterinarymedicine under the One Health concept.

Characterizing the Wolbachia infection in field-collected Culicidae mosquitoes from Hainan Province, China

BackgroundMosquitoes are vectors of many pathogens, such as malaria, dengue virus, yellow fever virus, filaria and Japanese encephalitis virus. Wolbachia are capable of inducing a wide range of reproductive abnormalities in their hosts, such as cytoplasmic incompatibility. Wolbachia has been proposed as a tool to modify mosquitoes that are resistant to pathogen infection as an alternative vector control strategy. This study aimed to determine natural Wolbachia infections in different mosquito species across Hainan Province, China.MethodsAdult mosquitoes were collected using light traps, human landing catches and aspirators in five areas in Hainan Province from May 2020 to November 2021. Species were identified based on morphological characteristics, species-specific PCR and DNA barcoding of cox1 assays. Molecular classification of species and phylogenetic analyses of Wolbachia infections were conducted based on the sequences from PCR products of cox1, wsp, 16S rRNA and FtsZ gene segments.ResultsA total of 413 female adult mosquitoes representing 15 species were identified molecularly and analyzed. Four mosquito species (Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Culex gelidus) were positive for Wolbachia infection. The overall Wolbachia infection rate for all mosquitoes tested in this study was 36.1% but varied among species. Wolbachia types A, B and mixed infections of A × B were detected in Ae. albopictus mosquitoes. A total of five wsp haplotypes, six FtsZ haplotypes and six 16S rRNA haplotypes were detected from Wolbachia infections. Phylogenetic tree analysis of wsp sequences classified them into three groups (type A, B and C) of Wolbachia strains compared to two groups each for FtsZ and 16S rRNA sequences. A novel type C Wolbachia strain was detected in Cx. gelidus by both single locus wsp gene and the combination of three genes.ConclusionOur study revealed the prevalence and distribution of Wolbachia in mosquitoes from Hainan Province, China. Knowledge of the prevalence and diversity of Wolbachia strains in local mosquito populations will provide part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Hainan Province.Graphical

Development of multiplex species-specific PCR for the simultaneous identification of three closely related species in the genera Misgurnus and Paramisgurnus

We aimed to develop a multiplex PCR method for the simultaneous detection of the three most consumed commercial loaches in East Asia: Misgurnus mizolepis, Misgurnus anguillicaudatus, and Paramisgurnus dabryanus. Phylogenetic analysis of genetic divergence and a phylogenetic tree based on the cytochrome oxidase I gene enabled successful genetic identification of the 3 species of loach. Multiplex species-specific PCR (MSS-PCR) primers designed based on single nucleotide polymorphisms were able to differentiate the species and generate PCR products among species that differed in size by at least 150 bp. The lengths of the amplified fragments were 818 bp for P. dabryanus, 669 bp for M. mizolepis, and 486 bp for M. anguillicaudatus, which could be clearly distinguished by gel electrophoresis. The three sets of PCR primers were mixed and applied to simultaneously detect the three loaches. The MSS-PCR markers designed in this study will be useful for the detection and identification of M. mizolepis, M. anguillicaudatus, and P. dabryanus.

Expression of recombinant porcine reproductive and respiratory syndrome virus nucleocapsid protein with antigenicity and immunogenicity study

Porcine respiratory and reproductive syndrome virus (PRRSV) causes significant economic losses in the swine industry worldwide. Nucleocapsid protein (NP) encoded by ORF7 is the most abundant and high immunogenicity. However, there were only few studies on recombinant NP expression with antigenicity and immunogenicity from PRRSV in Thailand. The aim of this study was to express rNP from PRRSV found in Thailand and evaluate its antigenicity and immunogenicity. ORF7 gene was amplified by RT-PCR. The PCR products were cloned into pGEM®-T Easy vector, and subcloned into pET-24a (+) expression vector and transformed into E. coli. The rNP properties were proved by SDS-PAGE and Western blot. The antigenicity of rNP was evaluated by indirect ELISA comparing to a commercial test kit. The immunogenicity of rNP was proved by rabbit immunization. Then, the antiserum was tested with tissues from PRRSV-infected swine by immunohistochemistry (IHC). In addition, amino acid sequence of the rNP was analyzed and compared to PRRSV reference strains. The PCR products of a whole ORF7 gene were 388 bp. The rNP size was 18 kDa. The indirect ELISA assay showed high antigenicity of the rNP with 100% sensitivity and 93.33% specificity at 0.25 O.D. cutoff value. The rNP could induce specific antibody production in the rabbit, which reacted specifically to PRRSV in tissues by IHC. The amino acid sequence of the rNP had similarities to US, HP-PRRSV and EU PRRSV at 95.93-99.19%, 96.75%, and 57.03-61.47%, respectively. Our rNP can be used for PRRSV diagnosis and test kit development.

Development of a CAPS Marker and a LAMP Assay for Rapid Detection of Xylella fastidiosa Subsp. multiplex and Differentiation from X. fastidiosa Subsp. fastidiosa on Blueberry.

Bacterial leaf scorch (BLS), caused by Xylella fastidiosa (Xf), is a prevalent disease of blueberries in the southeastern United States. Initially, this disease was reported to be caused by X. fastidiosa subsp. multiplex (Xfm). However, a recent survey revealed the presence of another subspecies, X. fastidiosa subsp. fastidiosa (Xff), within naturally infected blueberry plantings in Georgia. Since knowledge regarding the origins of isolates causing Xf outbreaks can impact management recommendations, a routine method for identifying the pathogen at the subspecies level can be beneficial. Several detection strategies are available to identify Xf infection at the subspecies level. However, none of these have been developed for the routine and rapid differentiation of the blueberry-infecting Xf subspecies. Here, we developed two separate straightforward and rapid detection techniques, a cleaved amplified polymorphic sequence (CAPS) marker, and a loop-mediated isothermal amplification (LAMP) assay, targeting the RNA polymerase sigma-70 factor (rpoD) gene sequence of Xfm to discriminate between the two Xf subspecies infecting blueberry. With the CAPS marker, specific detection of Xfm isolates was possible from pure cultures, inoculated greenhouse-grown plant samples, and field infected blueberry samples by restriction digestion of the rpoD gene PCR product (amplified with primers RST31 and RST33) using the BtsI enzyme. The LAMP assay allowed for specific real-time amplification of a 204-bp portion of the Xfm rpoD gene from both pure bacterial cultures and infected plant material using the Genie® III system, a result further affirmed by gel electrophoresis and SYBR™ Green I DNA staining for visual observation. These detection strategies have the potential to greatly aid existing diagnostic methods for determining the distribution and prevalence of these Xf subspecies causing bacterial leaf scorch (BLS) in blueberries in the southeastern United States.

Role of ESAT-6 in pathogenicity of Beijing and non-Beijing Mycobacterium tuberculosis isolates

BackgroundMycobacterium tuberculosis Beijing genotype was associated with tuberculosis outbreaks and increased transmissibility. To understand the variation in virulence between Beijing and non-Beijing clinical isolates of M.tuberculosis genotypes, the esat-6 gene sequencing, and its expression was compared in the macrophage environment. Materials & methodsAmong 64 nonrepetitive, culture-positive M.tuberculosis, DNA extraction of 24 and 40 pure confirmed Beijing and non-Beijing isolates was accompanied by the boiling method. esat-6 gene PCR amplification and their sequencing were carried out by specific primers and its expression was performed on human macrophage cell line U937 after 6, 12, and 18 h of exposure to bacilli. The esat-6 mRNA transcription and expression in M. tuberculosis treated macrophage by Real-Time PCR and Western blot method. ResultsData analysis based on sequencing of the east-6 gene PCR product showed that this gene exists in all isolates and there are no changes or single nucleotide variation between the Beijing and non-Beijing isolates. In Beijing strains, the esat-6 expression was increased during the study times, but it was constant in non-Beijing isolates. esat-6 gene expression in Beijing isolates reached to about 44.9 times more than non-Beijing isolates after 18 h incubation on the macrophages cell line. Conclusionesat-6 is a conserved gene both in Beijing and non-Beijing isolates of M.tuberculosis. More expression of the east-6 gene in the macrophage model may indicate that this gene is likely to play a more important role in increasing the pathogenicity of Beijing strains.

The potential risk of enzootic Trypanosoma cruzi transmission inside four training and re-training military battalions (BITER) in Colombia

BackgroundColombia’s National Army is one of the largest military institutions in the country based on the number of serving members and its presence throughout the country. There have been reports of cases of acute or chronic cases of Chagas disease among active military personnel. These may be the result of military-associated activities performed in jungles and other endemic areas or the consequence of exposure to Trypanosoma cruzi inside military establishments/facilities located in endemic areas. The aim of the present study was to describe the circulation of T. cruzi inside facilities housing four training and re-training battalions [Battalions of Instruction, Training en Re-training (BITERs)] located in municipalities with historical reports of triatomine bugs and Chagas disease cases. An entomological and faunal survey of domestic and sylvatic environments was conducted inside each of these military facilities.MethodsInfection in working and stray dogs present in each BITER location was determined using serological and molecular tools, and T. cruzi in mammal and triatomine bug samples was determined by PCR assay. The PCR products of the vertebrate 12S rRNA gene were also obtained and subjected to Sanger sequencing to identify blood-feeding sources. Finally, we performed a geospatial analysis to evaluate the coexistence of infected triatomines and mammals with the military personal inside of each BITER installation.ResultsIn total, 86 specimens were collected: 82 Rhodnius pallescens, two Rhodnius prolixus, one Triatoma dimidiata and one Triatoma maculata. The overall T. cruzi infection rate for R. pallescens and R. prolixus was 56.1 and 100% respectively, while T. dimidiata and T. maculata were not infected. Eight feeding sources were found for the infected triatomines, with opossum and humans being the most frequent sources of feeding (85.7%). Infection was most common in the common opossum Didelphis marsupialis, with infection levels of 77.7%. Sylvatic TcI was the most frequent genotype, found in 80% of triatomines and 75% of D. marsupialis. Of the samples collected from dogs (n = 52), five (9.6%; 95% confidence interval: 3.20–21.03) were seropositive based on two independent tests. Four of these dogs were creole and one was a working dog. The spatial analysis revealed a sympatry between infected vectors and mammals with the military population.ConclusionsWe have shown a potential risk of spillover of sylvatic T. cruzi transmission to humans by oral and vectorial transmission in two BITER installations in Colombia. The results indicate that installations where 100,000 active military personnel carry out training activities should be prioritized for epidemiological surveillance of Chagas disease.Graphical abstract

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L.calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Differential Impact of Plant Secondary Metabolites on the Soil Microbiota.

Plant metabolites can shape the microbial community composition in the soil. Two indole metabolites, benzoxazolinone (BOA) and gramine, produced by different Gramineae species, and quercetin, a flavonoid synthesized by many dicot species, were studied for their impacts on the community structure of field soil bacteria. The three plant metabolites were directly added to agricultural soil over a period of 28 days. Alterations in bacterial composition were monitored by next generation sequencing of 16S rRNA gene PCR products and phospholipid fatty acid analysis. Treatment of the soil with the plant metabolites altered the community composition from phylum to amplicon sequence variant (ASV) level. Alpha diversity was significantly reduced by BOA or quercetin, but not by gramine. BOA treatment caused a decrease of the relative abundance of 11 ASVs, while only 10 ASVs were increased. Gramine or quercetin treatment resulted in the increase in relative abundance of many more ASVs (33 or 38, respectively), most of them belonging to the Proteobacteria. Isolation and characterization of cultivable bacteria indicated an enrichment in Pseudarthrobacter or Pseudomonas strains under BOA/quercetin or BOA/gramine treatments, respectively. Therefore, the effects of the treatments on soil bacteria were characteristic for each metabolite, with BOA exerting a predominantly inhibitory effect, with only few genera being able to proliferate, while gramine and quercetin caused the proliferation of many potentially beneficial strains. As a consequence, BOA or gramine biosynthesis, which have evolved in different barley species, is accompanied with the association of distinct bacterial communities in the soil, presumably after mutual adaptation during evolution.

Tsetse blood-meal sources, endosymbionts and trypanosome-associations in the Maasai Mara National Reserve, a wildlife-human-livestock interface.

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.

Species-dependent variation of the gut bacterial communities across Trypanosoma cruzi insect vectors.

Triatomines (Hemiptera: Reduviidae) are the insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. The gut bacterial communities affect the development of T. cruzi inside the vector, making the characterization of its composition important in the understanding of infection development. We collected 54 triatomine bugs corresponding to four genera in different departments of Colombia. DNA extraction and PCR were performed to evaluate T. cruzi presence and to determine the discrete typing unit (DTU) of the parasite. PCR products of the bacterial 16S rRNA gene were pooled and sequenced. Resulting reads were denoised and QIIME 2 was used for the identification of amplicon sequence variants (ASVs). Diversity (alpha and beta diversity) and richness analyses, Circos plots, and principal component analysis (PCA) were also performed. The overall T. cruzi infection frequency was 75.9%, with TcI being the predominant DTU. Approximately 500,000 sequences were analyzed and 27 bacterial phyla were identified. The most abundant phyla were Proteobacteria (33.9%), Actinobacteria (32.4%), Firmicutes (19.6%), and Bacteroidetes (7.6%), which together accounted for over 90% of the gut communities identified in this study. Genera were identified for these main bacterial phyla, revealing the presence of important bacteria such as Rhodococcus, Serratia, and Wolbachia. The composition of bacterial phyla in the gut of the insects was significantly different between triatomine species, whereas no significant difference was seen between the state of T. cruzi infection. We suggest further investigation with the evaluation of additional variables and a larger sample size. To our knowledge, this study is the first characterization of the gut bacterial structure of the main triatomine genera in Colombia.

Identification of blood-feeding sources in Panstrongylus, Psammolestes, Rhodnius and Triatoma using amplicon-based next-generation sequencing

BackgroundTriatomines are hematophagous insects that play an important role as vectors of Trypanosoma cruzi, the causative agent of Chagas disease. These insects have adapted to multiple blood-feeding sources that can affect relevant aspects of their life-cycle and interactions, thereby influencing parasitic transmission dynamics. We conducted a characterization of the feeding sources of individuals from the primary circulating triatomine genera in Colombia using amplicon-based next-generation sequencing (NGS).MethodsWe used 42 triatomines collected in different departments of Colombia. DNA was extracted from the gut. The presence of T. cruzi was identified using real-time PCR, and discrete typing units (DTUs) were determined by conventional PCR. For blood-feeding source identification, PCR products of the vertebrate 12S rRNA gene were obtained and sequenced by next-generation sequencing (NGS). Blood-meal sources were inferred using blastn against a curated reference dataset containing the 12S rRNA sequences belonging to vertebrates with a distribution in South America that represent a potential feeding source for triatomine bugs. Mean and median comparison tests were performed to evaluate differences in triatomine blood-feeding sources, infection state, and geographical regions. Lastly, the inverse Simpsonʼs diversity index was calculated.ResultsThe overall frequency of T. cruzi infection was 83.3%. TcI was found as the most predominant DTU (65.7%). A total of 67 feeding sources were detected from the analyses of approximately 7 million reads. The predominant feeding source found was Homo sapiens (76.8%), followed by birds (10.5%), artiodactyls (4.4%), and non-human primates (3.9%). There were differences among numerous feeding sources of triatomines of different species. The diversity of feeding sources also differed depending on the presence of T. cruzi.ConclusionsTo the best of our knowledge, this is the first study to employ amplicon-based NGS of the 12S rRNA gene to depict blood-feeding sources of multiple triatomine species collected in different regions of Colombia. Our findings report a striking read diversity that has not been reported previously. This is a powerful approach to unravel transmission dynamics at microgeographical levels.

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species.

Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E+2-13/M+2-13Hr158 generated PCR products for just H. harpax, while E+3-14/M+3-2HhHr151 and E+2-13/M+2-13Hh150 generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E+2-13/M+2-13Hr158, E+3-14/M+3-2HhHr151, and E+2-13/M+2-13Hh150 were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.

Identification of cholix toxin gene in Vibrio cholerae non-O1/non-O139 isolated from diarrhea patients in Bushehr, Iran.

Background and Objectives:Cholixin (cholix toxin) is a novel exotoxin in Vibrio cholerae identified as an elongation factor II specific ADP-ribosyltransferase which inhibits protein synthesis in the eukaryotic cell. Previous researches have suggested that cholixin probably is an important virulence factor in non-O1/non-O139 V. cholerae (NAG) serotypes that could be related to extra-intestinal rather than intestinal infections. This study was aimed to investigate the frequency and genetic diversity of colixin gene (chxA) in clinical V. cholerae NAG isolates.Materials and Methods:The presence of chxA gene in 44 clinical V. cholerae NAG isolates were screened using PCR through specific primers designed for the receptor-binding domain (RBD) of chxA gene. The five PCR products of chxA gene were sequenced.Results:This study showed that chxA gene presented in 19 V. cholerae NAG isolates. The sequences analysis of 5 out of 19 the partial chxA genes amplicon showed that 4 of them belonged to chxA I and the other one belonged to chxA II subtypes. Two distinct clusters were revealed for these isolates by phylogenic analysis, too.Conclusion:The chxA gene contained high frequency among V. cholerae NAG isolates in Bushehr, Iran. The polymorphism study on RBD of cholixin gene is suggested as an appropriate method for phylogenic characterization of the various chxA gene subtypes.

Understandingleptospirosiseco-epidemiologyby environmental DNA metabarcoding of irrigation water from two agro-ecological regions of Sri Lanka.

BackgroundLeptospirosis is one of the most significant zoonoses across the world not only because of its impact on human and animal health but also because of the economic and social impact on agrarian communities. Leptospirosis is endemic in Sri Lanka where paddy farming activities, the use of draught animals in agriculture, and peridomestic animals in urban and rural areas play important roles in maintaining the infection cycle of pathogenic Leptospira, especially concerning animals as a potential reservoir. In this study, an environmental DNA (eDNA) metabarcoding methodology was applied in two different agro-ecological regions of Sri Lanka to understand the eco-epidemiology of leptospirosis.Methodology/Principal findingsIrrigation water samples were collected in Kandy District (wet zone mid-country region 2) and Girandurukotte, Badulla District (intermediate zone low-country region 2); and analysed for the presence of pathogenic Leptospira, associated microbiome and the potential reservoir animals. Briefly, we generated PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. The analysis of eDNA showed different environmental microbiomes in both regions and a higher diversity of Leptospira species circulating in Kandy than in Girandurukotte. Moreover, the number of sequence reads of pathogenic Leptospira species associated with clinical cases such as L. interrogans was higher in Kandy than in Girandurukotte. Kandy also showed more animal species associated with pathogenic bacterial species than Girandurukotte. Finally, several pathogenic bacterial species including Arcobacter cryaerophilus, responsible for abortion in animals, was shown to be associated with pathogenic Leptospira.Conclusions/SignificanceLeptospirosis has been considered to be endemic in wet regions, consistently, leptospiral sequences were detected strongly in Kandy. The great Leptospira species diversity in Kandy observed in this study shows that the etiological agents of leptospirosis in Sri Lanka might be underestimated. Furthermore, our eDNA metabarcoding can be used to discriminate bacterial and animal species diversity in different regions and to explore environmental microbiomes to identify other associated bacterial pathogens in the environment.