Gene-specific Primers Research Articles

Prevalence and Molecular Identification of Cryptosporidium Species among Human Population in Zakho District, Duhok Province, Kurdistan Region, Iraq

This cross-sectional study was carried out to determine the prevalence and molecular identifications of Cryptosporidium species among the human population in Zakho district, Duhok Province, Kurdistan Region, Iraq. The study was performed during the period from August 2021 to March 2022 and involved 700 fecal specimens of both genders and different ages collected from Zakho General Hospital and pri¬mary schools located in the suburban area of Zakho City. The data were analyzed using statistical soft¬ware version SPSS (25). Cryptosporidium species oocyst was found in 87.57% (613/700) of the examined specimens using the Modified Ziehl Neelsen technique. Males showed a slightly higher infection rate than females (88.4% vs 85.5%) with a non-significant difference (P> 0.05). The highest infection rate (91.7%) was present in individuals between the ages of >30 to 40 years, with a statistically significant difference (P<0.05). Molecular identification of Cryptosporidium species was estimated using 18S-rRNA gene for Cryptosporidium genus using Nested PCR technique and genus-specific primers (Cry18 AS1 and Cry18 S2) that yielded 347 bps product indicating Cryptosporidium genus, then species-specific (Cry18 AS1 and Cry18 S1) were used for the detection of Cryptosporidium parvum and Cryptosporidium hominis. 45/50 samples were positive and revealed 240 bps. The acquired sequences were deposited into the GeneBank database under the accession numbers OP107010 for C. hominis, OP104444, OP153870, OP163188, OP163414, and OP164631 for C. parvum.

No Association between SARS-CoV-2 Infection and the Polymorphism of the Toll-like Receptor 7 (TLR7) Gene in Female Population

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus. Toll-like receptor 7 (TLR7) recognizes single-stranded RNA viruses. The TLR7 gene plays a critical role in the human innate and adaptive immune response to SARS-CoV-2 infections. Genetic factors probably affect SARS-CoV-2 infection susceptibility. In the current study, our aim was to search for genetic variations associated with COVID-19 patients in the TLR7 gene of a Korean population. We designed five gene-specific primers to cover the coding region of the human TLR7 gene. Using amplicon sequencing, we screened the genetic polymorphisms in the coding region of the TLR7 gene in COVID-19 patients and healthy controls. The genotype frequencies, allele frequencies, and Hardy–Weinberg equilibrium (HWE) were examined. We identified a low-frequency synonymous single nucleotide polymorphism (SNP) (rs864058) in the coding region of the TLR7 gene. There were no significant differences in the genotype or allele frequencies of the TLR7 rs864058 polymorphism between COVID-19 female patients and healthy controls (p = 1.0). In conclusion, TLR7 (rs864058) polymorphism is low frequency in Korean populations and is not associated with SARS-CoV-2 infection.

Validating post-enrichment steps in environmental RNA analysis for improving its availability from water samples.

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring.

Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as the double-stranded RNA Hubei chryso-like virus 1. Both viruses were found in the same Anopheles spp. homogenate extracted from a sample that showed CPE on both VeroB4 and C6/36 cells. The detection of both viruses in a single mosquito homogenate indicated coinfection. Phylogenetic analyses suggested that the Culex flavivirus sequence detected was closely related to a Culex flavivirus isolated from Uganda in 2008. All four Hubei chryso-like virus 1 segments clusters closely to Hubei chryso-like virus 1 strains isolated in Australia, China and USA. Two novel strains of insect-specific viruses in Anopheles mosquitoes were detected and characterized.

G894T of nos3 gene polymorphism and resistant arterial hypertension in Uzbek population

Abstract Aim To estimate the influence of G894T polymorphism of NOS3 gene on the risk of developing resistant arterial hypertension (AH) in the Uzbek population. Materials and Methods The study included 176 patients with AH I-III degree (ESH / ESC, 2018) of both sexes of Uzbek population. The mean age of the patients was 57.12 ± 11.03 years, the mean duration of AH was 9.58 ± 5.76 years. Resistant hypertension was defined in uncontrolled BP, even though ≥ 3 drugs of different classes were taken. Genotyping of the G894T polymorphism of NOS3 gene was performed using real-time PCR using gene-specific primers of allele-specific probes, followed by fluorescence detection of the corresponding alleles. Relative risk (RR) was calculated using genetic models. Calculations were performed using the online program "Calculator for calculating statistics in case-control studies". The results of all studies were considered statistically significant at p<0.05. Results All patients were divided into two groups: group 1 - patients with resistant hypertension (cases, n=61) and group 2 - patients with controlled hypertension (controls, n=115). Among patients of group 1, the following distribution of the G894T polymorphism of the NOS3 gene was revealed: GG genotype - determined in 45.9% of patients, GT genotype - in 44.3%, TT genotype - 9.8%, χ2=22.770, p=0.000 . The allelic distribution showed the predominance of carriage of the G allele: G allele - 68.0%, T allele - 32.0%, χ2=30.311, p=0.000. Among patients of group 2, the allelic distribution was with a significant prevalence of the G allele: G allele in 80.9%, T allele in 19.1%, respectively, χ2=172.878, p=0.000. The ratio of GG:GT:TT genotypes was as follows: 67.8% : 26.1% : 6.1%, χ2=102.704, p=0.000. An analysis using genetic models of inheritance revealed a protective effect of the G allele G894T of the NOS3 gene polymorphism on the risk of developing resistant hypertension in the Uzbek population: among 115 patients with controlled hypertension of group 2, the G allele was significantly more common than in patients with resistant hypertension of group 1 (80.9%; χ² = 7.29 P=0.007; OR=0.50, 95% CI 0.30-0.83) and only 19.1% of patients had the T allele (χ²=P=0.007; OR=1.99 95% CI 1.20-3.28). The general model of inheritance demonstrated a significant accumulation of the GG genotype among patients of group 2 67.8% (χ² = 8.01; P = 0.02; OR = 0.40, 95% CI 0.21-0.76), while the GT genotype was less common in 26.1% cases and the TT genotype is even rarer in 6.1% of cases. Whereas in 1st group of patients GG and GT genotypes were found in the same ratio: 45.9%: 44.3%, respectively. Conclusion The results obtained indicate a significantly greater accumulation of the G allele G894T of NOS3 gene polymorphism among patients with controlled hypertension. Carrying the G allele and GG genotype G894T of NOS3 gene polymorphism is associated with a low risk of developing resistant hypertension in Uzbek population.

Clinical and laboratory features of rickettsioses in Yucatan, Mexico

Rickettsia species are obligate intracellular bacteria that can cause mild to severe human disease. Based on phylogeny, clinical symptoms, and antigenic properties, rickettsiae are classified into four groups. Infections by these agents are characterized by clinical symptoms ranging from self-limited to severe and even fatal febrile illnesses, depending on the Rickettsia spp. involved, the patient's predisposition, and timely medical care.The present study aimed to characterize rickettsial diseases in Yucatan according to clinical and laboratory features appearing in medical records corresponding to 427 samples taken between 2015 and 2018.A study was conducted over the period 2015–2018 on 427 samples. Clinical and laboratory features were documented from the patients' medical records. For molecular diagnosis, blood was collected in 3.8 % sodium citrate as anticoagulant, and DNA was extracted. Single-step and nested PCR amplification was performed using genus-specific primers for the rickettsial 17kDa and ompB genes. The amplicons obtained were purified and sequenced.A total of 22.7 % (97/427) positive cases of Rickettsia spp. were identified by PCR from 14.15 % (15/106) of the municipalities in Yucatan. 75.2 % (73/97) of the cases were from the city of Merida during the autumn (September-December). The age groups with the highest frequency of confirmed cases were pediatric (5–14 years) (57.7 %) and adults (25-49 years) (42.2 %). There were six fatal cases in children, one associated with R. typhi and five with R. rickettsii. In non-fatal cases, 32.9 % (32/97) corresponded to the spotted fever group (SFG), and 60.8 % (59/97) to the typhus group (TG). Significant differences in signs, and laboratory data, were observed between the pediatric and adult populations. For the treatment of patients, oral and intravenous doxycycline was used in severe hospitalized cases.Typhus group and spotted fever group Rickettsiae are endemic pathological agents found in urban and rural areas of our region. Molecular identification allows for greater diagnostic accuracy and timely treatment and consequently a better prognosis. It is necessary to implement or reinforce measures focused on the dissemination of knowledge regarding rickettsial diseases and their prevention.

Identification and specificity validation of unique and antimicrobial resistance genes to trace suspected pathogenic AMR bacteria and to monitor the development of AMR in non-AMR strains in the environment and clinical settings

The detection of developing antimicrobial resistance (AMR) has become a global issue. The detection of developing antimicrobial resistance has become a global issue. The growing number of AMR bacteria poses a new threat to public health. Therefore, a less laborious and quick confirmatory test becomes important for further investigations into developing AMR in the environment and in clinical settings. This study aims to present a comprehensive analysis and validation of unique and antimicrobial-resistant strains from the WHO priority list of antimicrobial-resistant bacteria and previously reported AMR strains such as Acinetobacter baumannii, Aeromonas spp., Anaeromonas frigoriresistens, Anaeromonas gelatinfytica, Bacillus spp., Campylobacter jejuni subsp. jejuni, Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumonia subsp. pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serovar Typhimurium, Thermanaeromonas toyohensis, and Vibrio proteolyticus. Using in-house designed gene-specific primers, 18 different antibiotic resistance genes (algJ, alpB, AQU-1, CEPH-A3, ciaB, CMY-1-MOX-7, CMY-1-MOX-9, CMY-1/MOX, cphA2, cphA5, cphA7, ebpA, ECP_4655, fliC, OXA-51, RfbU, ThiU2, and tolB) from 46 strains were selected and validated. Hence, this study provides insight into the identification of strain-specific, unique antimicrobial resistance genes. Targeted amplification and verification using selected unique marker genes have been reported. Thus, the present detection and validation use a robust method for the entire experiment. Results also highlight the presence ofanother set of 18 antibiotic-resistant and unique genes (Aqu1, cphA2, cphA3, cphA5, cphA7, cmy1/mox7, cmy1/mox9, asaI, ascV, asoB, oxa-12, acr-2, pepA, uo65, pliI, dr0274, tapY2, and cpeT). Of these sets of genes, 15 were found to be suitable for the detection of pathogenic strains belonging to the genera Aeromonas, Pseudomonas, Helicobacter, Campylobacter, Enterococcus, Klebsiella, Acinetobacter, Salmonella, Haemophilus, and Bacillus. Thus, we have detected and verified sets of unique and antimicrobial resistance genes in bacteria on the WHO Priority List and from published reports on AMR bacteria. This study offers advantages for confirming antimicrobial resistance in all suspected AMR bacteria and monitoring the development of AMR in non-AMR bacteria, in the environment, and in clinical settings.

Characterization of Trichoderma strains for novel species-specific markers by multiplex PCR and antagonistic property against Alternaria ricini in castor (Ricinus communis L.)

The study aimed at characterizing six Trichoderma strains isolated from the rhizospheric soil of castor growing region of Telangana, India. Microscopic examination and sequencing of ribosomal DNA (rDNA) using internal transcribed spacer (ITS) and D1-D2 region-specific primers revealed them as members of Trichoderma sp. Whereas the phylogenetic analysis revealed four of those strains as Trichoderma asperellum, two strains were confirmed as Trichoderma harzianum with a high sequence similarity of >99%. Further, multiplex PCR results with Trichoderma-specific primers; TEF1 (translation elongation factor 1) and rpb2 (the second largest subunit of RNA polymerase II) gene-specific primers confirmed the findings resulting from the phylogenetic analysis. To develop a set of sequence characterized amplified region (SCAR) markers, these strains were screened with 185 random amplified polymorphic DNA (RAPD) primers. A pairwise comparison of correlation of coefficient values determined from the RAPD-based genetic similarity analysis revealed that TV3 and TS4 strains were closely related to each other (0.98) confirming them as T. harzianum while N13, 7316, TV5, and TV2B16 strains formed a different cluster with T. asperellum strains. A set of novel SCAR markers were developed to distinguish these two species, T. asperellum and T. harzianum including the reported Trichoderma strains as Ex-type strains. T. harzianum strains had extremely low sporulation ability to be tested for biocontrol properties. Of the four T. asperellum strains, 7316 followed by N13 demonstrated high antagonistic ability against the fungal pathogen, Alternaria ricini indicating these strains as potential biocontrol agents to limit Alternaria infection in castor.

Detection of Aminoglycoside Modifying Enzyme (AME) genes in Acinetobacter baumannii isolates and the inhibitory effect of efflux pump activity on drug susceptibility pattern

IntroductionThe development of aminoglycoside modifying enzymes (AMEs) and increased efflux activity are considered important aminoglycosides resistance mechanisms. Aim.This study is focused on the detection of the AMEs gene and assessing the effect of efflux pump inhibitor on the reversal of A. baumannii drug susceptibility. MethodologyBacterial DNA was amplified using AMEs gene-specific primers. Isolates were also investigated for efflux pump activity using efflux pump inhibitor (EPI) i.e. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the impact of both mechanisms was analyzed. ResultsAmong A. baumannii isolates, 55% isolates (n ​= ​22/40) were identified to have aminoglycoside modifying enzymes genes; ant(3′)-I gene (50%, 11/22), aac(6′)-Ib gene (45.4%, 10/22), aph(3′)-I gene (18.1%, 4/22) and aac(3)-I (9.1%, 2/22). Total 70% isolates have shown MIC alteration in different classes of drugs in response to EPI-CCCP. Such alteration was found in 100% amikacin sensitive and 58.6% amikacin resistant, 93.7% and 57.1% gentamicin sensitive and resistant isolates respectively. ConclusionThe presence of aminoglycosides modifying enzymes was frequent among aminoglycosides resistant A. baumannii isolates and the coexistence of efflux pumps activity also plays an important role to increase drug resistance. RepositoriesGenbank and their accession numbers are MT903331[aac(3)-I], MT903332 MT903333 [ant(3′)-I], MT903334, MT903335 [aph(3′)-I)] and MT903336, MT940242 [ aac(6’)-Ib].

Identification of Buxtonella sulcata with phylogenetic analysis in cattle in Egypt

Protozoan diseases are of great significance in ruminants. In Egypt, the published data for Buxtonella sulcata (B. sulcata) in cattle is limited. This is the first study on B. sulcata in Assiut governorate via PCR and phylogenetic analysis. This study investigated the B. sulcata positive rate in cattle and its association with diarrhea. Out of 100 cattle fecal samples (50 males and 50 females) with different ages were collected from Assiut governorate, Egypt. Microscopically, the positive rate of B. sulcata was 69% (69/100). The cysts were oval or round with a size range of (82.23×78.63 to 53.65×48.62 μm) with a mean of (58.46×54.35 μm). While, its trophozoite appeared oval with characteristic grooves with a size range from (80.9×40.2 to 41.8×30.64 μm) with a mean of (58.78×41.26 μm). Females are slightly higher infected 72% (36/50) than male 66% (33/50). While, the highest positive rate recorded in age less than 1 year 78.6% (22/28), followed by age more than 3 years 68.4% (13/19), then the lowest in age 1-3 years 64.2% (34/53). The positive rate of B. sulcata was 92.2% (47/51) and 44.9% (22/49) of diarrheic and non-diarrheic feces, respectively, with very high significant differences. Diarrhea was established in all fecal samples with more than 1000 cysts per gram of faeces. Three fecal samples were positive for B. sulcata SSrDNA gene specific primer (1047 bp) by PCR. After DNA sequencing and phylogenetic analysis, all nucleotide sequences showed high similarity to B. sulcata. We advised regular examination for B. sulcata in the predominant areas with excessive studies to aid in planning effective control strategies against it.

Unveiling the Potential of OsRuvB DNA Helicases in Enhancing Salinity Stress Tolerance in Chickpea

Chickpea (Cicer arietinum L.) is a self-pollinated true diploid (2n=2x=16) cool season leguminous crop that ranks second among food grain legumes after soybean. It grows under a wide range of climatic conditions and is highly sensitive to salt stress. In the present study, transgenic chickpea plants (var. HC-1) carrying OsRuvB gene were screened for salt stress. Putative transformants were screened at an early stage through PCR amplification using gene specific primers and a transformation frequency of 36.2% was observed. Physio-biochemical analysis of selected T2 transgenic plants subjected to 100 mM salt stress showed that transgenic plants were able to maintain higher chlorophyll content, relative water content, cell viability, proline content, Na+/K+ content, catalase and peroxidase activity compared to the wild type plants. Whereas electrolytic leakage and lipid peroxidation were relatively less as compared to the wild type plants under 100 mM stress. Among all transgenic lines, line 8 performed well with respect to all the parameters studied and can be taken further for the development of transgenic chickpea plants for salt stress tolerance.

Ixodid tick infections of horses, and seasonal dynamics and infection of pasture ticks with piroplasmids in Gorny Altai

The purpose of the research is the characterization of species composition and abundance of ixodid ticks that parasitize on horses, seasonal dynamics of ticks inhabiting natural pastures and their piroplasmid infection. Materials and methods. The studies were performed in four Gorny Altai physiographic provinces. Ticks were manually collected from horses (1183 specimens). Ticks were counted on natural pastures every ten days with subsequent determination of the species composition (1109 specimens). Collected ticks of the genus Dermacentor (443 specimens) were examined by nested PCR in the presence of genus-specific primers for Babesia spp. and Theileria spp.; the species identification of discovered piroplasmas was determined by 18S rRNA gene sequencing. Results and discussion. Ixodid ticks of three genera (Dermacentor, Ixodes, Haemaphysalis) and 6 species: D. nuttalli, D. silvarum, D. reticulatus, D. marginatus, H. concinna and Ix. Persulcatus, were recorded in the collections from horses and pastures. The most numerous species was D. nuttalli (56.1%), and the most common, Ix. persulcatus, that was recorded in all districts except Kosh-Agachsky in South-Eastern Altai. D. silvarum accounted for 14.9%, and other species were represented to a lesser extent. The highest species diversity, 5 species, was observed in the Northern Altai pastures and only 1 species, D. nuttalli, was recorded in the South-Eastern Altai. The seasonal dynamics of ticks in the pasture was characterized by two peaks in numbers, namely, the spring peak in the 3rd decade of April and the autumn peak in the 2nd decade of October. The spring-summer period (March-June) had 87.1% of recorded ticks, and the autumn period (September-October) had 7.2%. Three species of the examined ticks (D. silvarum, D. marginatus, D. nuttalli) were found to have DNA of three piroplasmid species (Babesia caballi, Theileria equi and Babesia sp). Piroplasmids infection rate in ticks was 2.7-25.0%, and 2.7% on average in Gorny Altai.

Vasconcellea cauliflora – a new natural host of cucumber mosaic virus

Vasconcellea cauliflora plants exhibiting chlorotic spots, mottling and mosaic with yellowing on leaves were observed in Pune, India in October 2020. The disease incidence was 16%. The samples were screened for cucumber mosaic virus (CMV), papaya ringspot virus, tobacco mosaic virus and zucchini yellow mosaic virus using ELISA. Sap inoculation on healthy V. cauliflora plants using CMV positive DAS-ELISA samples reproduced field symptoms. The transmission upon mechanical inoculation was 100%. For further confirmation, RT-PCR was carried out using CMV coat protein gene specific primers. Amplicons of expected size (c. 650 bp) were obtained in all symptomatic and sap inoculated samples. No amplification was observed in the asymptomatic samples. BLASTn analysis revealed that the amplicon sequence shares more than 99% identity with CMV isolates and in phylogenetic analysis it clustered with CMV IB isolates. This is the first record of V. cauliflora as a natural host of CMV worldwide.

Discovery and Comprehensive Characterization of Novel Circular RNAs of the Apoptosis-Related BOK Gene in Human Ovarian and Prostate Cancer Cells, Using Nanopore Sequencing.

CircRNAs have become a novel scientific research hotspot, and an increasing number of studies have shed light on their involvement in malignant progression. Prompted by the apparent scientific gap in circRNAs from apoptosis-related genes, such as BOK, we focused on the identification of novel BOK circRNAs in human ovarian and prostate cancer cells. Total RNA was extracted from ovarian and prostate cancer cell lines and reversely transcribed using random hexamer primers. A series of PCR assays utilizing gene-specific divergent primers were carried out. Next, third-generation sequencing based on nanopore technology followed by extensive bioinformatics analysis led to the discovery of 23 novel circRNAs. These novel circRNAs consist of both exonic and intronic regions of the BOK gene. Interestingly, the exons that form the back-splice junction were truncated in most circRNAs, and multiple back-splice sites were found for each BOK exon. Moreover, several BOK circRNAs are predicted to sponge microRNAs with a key role in reproductive cancers, while the presence of putative open reading frames indicates their translational potential. Overall, this study suggests that distinct alternative splicing events lead to the production of novel BOK circRNAs, which could come into play in the molecular landscape and clinical investigation of ovarian and prostate cancer.

Molecular Identification of Tomato Leaf Curl New Delhi Virus Associated with Mosaic Disease of Pumpkin from Central India

The Tomato leaf curl New Delhi virus is an emerging disease of pumpkin associated with mosaic disease in central, India. Symptomatic pumpkin leaf samples from different locations in the Bhopal district were collected and confirmed for begomovirus disease by PCR using a begomovirus coat-protein gene-specific primer. All five samples were amplified, expecting ~800 bp of amplicons of associated begomovirus; these amplicons were purified and sequenced for identification of begomovirus species associated with mosaic disease on the pumpkin. The consensus nucleotide sequence data were submitted to the NCBI database under the accession numbers OQ320768, OQ320770, OQ320774, OQ116977, and OQ116978. The understudy begomovirus isolates showed the highest 95–97% nucleotide sequence identities and close phylogenetic relationships with several isolates of Tomato leaf curl New Delhi virus. To our knowledge, this was the first record of the Tomato leaf curl New Delhi virus associated with the mosaic disease of pumpkin in Central India.

Surveillance of Escherichia coli in different types of chicken and duck hatcheries: one health outlook

Escherichia coli is an important zoonotic bacterium that significantly impacts one health concept. E. coli is normally detected in the gut of warm-blooded animals, but some serotypes can cause diseases in humans and animals. Moreover, it can continue for a long time in different environments, replicate in water, and survive outside different hosts. In this study, 171 samples collected from 10 different types of poultry hatcheries (automatic, semiautomatic, and manual "traditional" types) were examined for the prevalence of E. coli. PCR was applied to verify the E. coli isolates via 16S rRNA gene-specific primers. From the gathered samples, 62 E. coli isolates were recovered (36.3%). The highest prevalence was met with the manual "traditional" hatcheries (57.1%) with no significance difference (P=0.243) in the 3 types of hatcheries. The incidence of E. coli varied significantly in different tested avian types and breeds. The prevalence was 35.7% in duck hatcheries and 37% in chicken hatcheries, with significant differences between breeds of both species (P=0.024 and 0.001, respectively). The identification of zoonotic E. coli serotypes in this study is concerning, highlighting the need for collaborative efforts across various sectors, including social, environmental, and governance, to promote the adoption of the one health principle in the chicken business. Periodical surveillance, biosecurity measures at the hatcheries and farm levels, and boosting the immunity of birds were recommended to limit the risk of E. coli spread from avian sources to humans.

Effective Agrobacterium-mediated genetic transformation of okra (Abelmoschus esculentus L.) and generation of RNAi plants resistant to Begomovirus infecting okra

ABSTRACT Okra is an important vegetable crop of the Malvaceae family and is infected by varying numbers of viruses of the genus Begomovirus. Regardless of the importance of the crop, very little consideration has been given to its genetic improvement. RNA interference (RNAi), a potent biotechnological tool, is known to control Begomovirus in many crops. For the implementation of successful RNAi, there is a need for an efficient genetic transformation system in okra. In the present study, we developed a procedure for Agrobacterium-mediated tissue culture–dependent regeneration of okra plants for the application of RNAi. Eleven transgenic okra RNAi plants were regenerated by utilising hypocotyls as explants. Transformed plants were screened with hygromycin at the regeneration stage and the presence of transgenes (AC1, AC2 & AC4 codes for replication-associated protein, transcriptional activator protein and suppressor of PTGS) in putative transformed plants was confirmed by polymerase chain reaction (PCR). Wild and transgenic lines were challenged with a dimeric Begomovirus clone or viruliferous whiteflies and the level of resistance was estimated with quantitative real-time reverse transcriptase PCR (qRT-PCR) by utilising viral gene–specific primers. The resistant transgenic lines accumulated very low titres of viral gene products according to the qRT-PCR assays compared to the control plants. This is the first report of tissue culture–mediated RNAi-derived resistance in okra against Begomovirus infection.

Agave attenuata and Agave amica, new hosts of Tuberose mild mosaic virus in Mexico.

Agave attenuata is a Mexican wild plant originally from highlands in the central and occidental mountains of Mexico. This species, known as "swan´s neck agave", is used only as an ornamental plant in public and private gardens. No virus had previously been reported from A. attenuata before this study. In a survey conducted in a commercial greenhouse in Cuautla, Morelos, in 2018, several plants were observed with symptoms of green mosaic and streaks, consistent with a putative viral infection. Sap inoculation from symptomatic A. attenuata plants to herbaceous indicator plants (Nicotiana benthamiana and N. tabacum) failed to produce symptoms in the mechanically inoculated plants. ELISA specific test to CMV, TEV, AMV, TMV and Potyvirus Group (Agdia, Inc.), was positive only for the last one (Chen and Chang, 1998). To determine the identity of the potyvirus involved, total nucleic acid extracts from 100 mg of symptomatic leaves (Trizol reagent; Gibco BRL Life Technologies, England) were used as template in RT-PCR with genus-specific potyvirus primers POT1-POT2, which targeted the variable 5´ terminal half of the coat protein gene of potyviruses (Colinet et al. 1998). The expected 900 bp amplicon was consistently detected in 10 symptomatic A. attenuata plants whereas no PCR products were obtained from 15 asymptomatic A. attenuata plants collected from the "Agaves de México" section at the 'Botanic Garden' of the Instituto de Biología de la UNAM, México. The amplicons were sequenced by the Sanger´s method and the obtained nucleotide (nt) sequences (Acc. No KY190217.1; OP964597-598) and their derived amino acid (aa) sequences were 94.68% to 95.80% similar to an isolate of Tuberose mild mosaic virus (TuMMV; Potyvirus; (Acc. No ON116187.1) characterized from Agave amica in India (Raj et al. 2009). Interestingly, A. amica (formerly Poliantes tuberose) is also a wild Mexican plant that is geographically distributed in the central and south regions of Mexico and is currently being commercially cultivated as an ornamental plant. Plants of A. amica (n=10) showing yellow mild streak were collected from commercial greenhouse and tested positive for TuMMV by RT-PCR and Sanger sequencing (No Acc. OP964599-601 levels) described above. The derived TuMMV sequences from A. attenuata and A. amica were 99-100% similar to each other at the nt/aa level. To exclude the involvement of additional viral agents in the disease, high-throughput sequencing analysis was performed separately for each species of Agave on total RNA extracts from a composite sample of symptomatic leaf tissues using Illumina´s Next Seq 500 platform. Analysis of the obtained 13,260,700 reads (each 75 nt) by the Trinity software, with a total number of sequences of 22,793, resulted in the identification of a single viral contig of 9500 nt for A. attenuata (Acc. No OP964595). Similarly, for A. amica, 27,262,248 reads were obtained, with a total number of sequences of 23,269, resulting in the identification of a single viral contig of 8500 nt (ACC. No OP964602). These contigs showed an identity percentage of 96%/88% and 98%/96% for nucleotides and amino acids, respectively, compared to an isolate of TuMMV from India (Acc. OM293939). Mexico is a center of origin for numerous species of genus Agave which have high economic, social, and ecological impact. TuMMV could be a threat to these plants and potentially to other unknown susceptible crops. To our knowledge, this is the first report of TuMMV in A. attenuata and A. amica in Mexico. REFERENCE Chen, C. C., and Chang, C. A. 1998. Characterization of a potyvirus causing mild mosaic on tuberose. Plant Dis. 82:45-49. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F. Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82:223-229. Raj, S.K., Snehi, S.K., Kumar, S., Ram, T. and Goel, A.K. 2009. First report of Tuberose mild mosaic potyvirus from tuberose (Polianthes tuberosa L.) in India. Australasian Plant Dis. Notes 4, 93-95.

Co-occurrence of Carbapenemase Genes blaNDM, blaVIM, blaKPC and blaOXA-48 in Pseudomonas aeruginosa Clinical Isolates

Pseudomonas aeruginosa, a gram-negative bacterium, is notorious for its innate resistance to many antibiotics. Carbapenems are broad-spectrum antibiotics often used to treat severe P. aeruginosa infections. However, the emergence and proliferation of carbapenem-resistant P. aeruginosa (CRPA) strains have become a grave global health concern. This study examined the co-occurrence of four major carbapenemase genes, namely blaNDM, blaVIM, blaKPC, and blaOXA-48, in clinical isolates of P. aeruginosa. Using standard microbiological methods,150 P. aeruginosa clinical isolates were collected and identified, and antimicrobial susceptibility testing was conducted following Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) with gene-specific primers was used to detect the presence of carbapenemase genes. Among the 150 P. aeruginosa clinical isolates, 62(41.3%) were found to be carbapenem-resistant. The most detected carbapenemase genes were blaKPC (49%), blaNDM (31%), blaOXA-48 (22%), and blaVIM (9%). Notably 13(12.9%) isolates carried two carbapenemase genes. The combination of blaKPC and blaNDM genes was found in eight isolates, two isolates carried blaKPC and blaVIM, and three isolates carried blaOXA-48 and blaNDM. Four isolates (6.5%) harbored three carbapenemase genes. Co-occurrence of blaNDM, blaVIM, blaKPC, and blaOXA-48 was observed in four isolates (2.8%). Our findings highlight the alarming prevalence of carbapenemase genes, particularly blaNDM and blaKPC, in clinical isolates of P. aeruginosa. The co-occurrence of multiple carbapenemase genes in the same isolate raises concerns about the potential for horizontal gene transfer and dissemination of multidrug-resistant P. aeruginosa strains in clinical settings. Further research is needed to elucidate the molecular mechanisms underlying the co-occurrence of carbapenemase genes and their impact on the clinical outcomes of P. aeruginosa infections. Urgent measures, such as enhanced surveillance, infection control protocols, and antibiotic stewardship programs, are imperative to combat the emergence and spread of CRPA strains.

Molecular Mimicry of NodA Genes by an N-acetylmuramoyl-L-alanine Amidases Gene in a Nodule Forming Staphylococcus cohnii [RESEARCH NOTE

A rare, nodule-forming, Gram-positive bacterium (Sub4) was isolated from the forage and cover crop Pueraria phaseoloides, which aligned at 99.26% sequence identity to a partial 16S rDNA sequence of Staphylococcus cohnii sp. This was the first record of a strain/species of Staphylococcus capable of independent, unassisted nodule formation in a legume host. When a nodA gene was sought by PCR using a pair of gene-specific primers synthesized using a related (Firmicutes) Paenibacillus sp. nodA gene, the reaction yielded a PCR product of similar size but a distinct identity. The resulting ~400bp PCR product coded for a likely N-acetylmuramoyl-L-alanine amidase and not an acyltransferase. It may be inferred that the N-acetylmuramoyl-L-alanine amidase gene was amplified due to sequence similarities/mimicry between 2 cosmetically unrelated loci. There was also an analogy in protein backbones between homology models of the Sub4 N-acetylmuramoyl-L-alanine amidase and a Paenibacillus nodA gene product, which also suggests that the 2 protein families may have an evolutionary relationship. When the phylogeny of the sequenced locus was inferred, a unique clade with high bootstrap support was formed by the sequence under study with an Azorhizobium nodA gene. Azorhizobia were distinct in relation to their nodule formation on stems and their ability to fix nitrogen in the free-living and the symbiotic forms. N-acetylmuramoyl-L-alanine amidases may have roles to play in remodeling peptidoglycan for LysM receptor interactions, likely without nod factors.