Positive Amplicons Research Articles

Molecular and serological detection of acute canine leptospirosis and associated predictive risk factors in and around Chennai, India.

Leptospirosis is a globally important re-emerging zoonotic disease affecting humans and many animal species including dogs. The present cross-sectional study aimed to diagnose acute leptospirosis among 210 suspected dogs using microscopic agglutination test (MAT) and polymerase chain reaction (PCR). Further, epidemiological risk factors were analyzed by univariate analysis and multivariate binomial logistic regression analysis. Out of the 210 dogs, anti-leptospiral antibody was detected in 123 (58.57%; 95% CI: 51.6-65.3) dogs by MAT (cut-off titer- ≥1:100), among which 67 (54.47%; 95% CI: 45.3-63.4) were seropositive to more than one serogroup. The predominant serogroups were Australis and Autumnalis in the study region. Whereas, acute leptospirosis was established in 49 (23.33%, 95% CI: 17.9-29.8) dogs based on ≥ 1:800 titer in unvaccinated dogs and ≥ 1:1600 titer in vaccinated dogs. The predictive risk factors of acute canine leptospirosis were adult dogs (1-5 years) (p=0.001), north-east monsoon season (p=0.011), outdoor management (p=0.047), history of rodent exposure (p=0.001) and history of contact with wet soil or stagnant water (p=0.046). Among 49 acutely infected dogs, thirteen dogs were positive for urine PCR and one dog was positive for both urine as well as plasma PCR. Positive PCR amplicons were identified as Leptospira interrogans based on secY gene sequencing and phylogenetic analysis. This study enlightened about the occurrence of acute leptospirosis among suspected dogs with the above important predictive risk factors, which should be taken into consideration while history taking so that proper treatment strategies can be adopted for early recovery of the animal.

Prevalence of <i>Wuchereria bancrofti</i> and <i>Plasmodium Spp.</i> infection in <i>Anopheles</i> mosquitoes and human blood sampled from Matayos in Busia County, Western Kenya

INTRODUCTION Lymphatic filariasis and malaria are mosquito-borne diseases and are co-endemic in coastal Kenya. We evaluated the occurrence of co-infections with Wuchereria bancrofti and Plasmodium falciparum, the causative agents of Lymphatic filariasis and malaria, respectively, in humans and known Anopheles vectors from Matayos constituency, Busia County, western Kenya. MATERIALS AND METHODS The samples were collected purposively based on clinical case reports. Members of the An. gambiae and An. funestus species complexes present and the presence of W. bancrofti and P. falciparum were detected using Polymerase Chain Reaction (PCR). Positive PCR amplicons were purified and Sanger sequenced. RESULTS In this study, 292 Anopheles mosquitoes were analyzed, and 288 of these (98.63%) were successfully identified by PCR. The majority of mosquitoes identified belonged to the An. funestus complex (85.76%) while An. gambiae complex mosquitoes comprised 14.24%. Two W. bancrofti infections were detected in the mosquitoes, one each in An. leesoni and An. funestus s.s. with an infection rate of 0.46% and 4.17% respectively. No P. falciparum was detected in the mosquito or human blood samples and only 2 blood samples (2.17%) were positive for W. bancrofti. Phylogenetic analysis based on the sequenced 18S rRNA gene showed that all W. bancrofti sequences from this study shared a close relationship with W. bancrofti sequences distributed in other regions. No case of co-infection of P. falciparum and W. bancrofti was found in the mosquito or blood samples analyzed. CONCLUSION This study confirms the presence of W. bancrofti infection in the human population and mosquito vectors in Matayos. The study also indicated that the majority of Anopheles mosquitoes from the study area belong to An. funestus complex. This study recommends additional screening before Mass Drug Administration, and that mosquito control programs be strengthened in the study area.

Microeukaryotes Associated with Freshwater Mussels in Rivers of the Southeastern United States.

Microeukaryotes are a diverse and often overlooked group of microbes that are important in food webs and other ecological linkages. Little is known about microeukaryotes associated with aquatic invertebrates, although filter feeders such as mussels are likely to take in and potentially retain microeukaryotes in their gut while feeding. Microeukaryotes such as apicomplexans have been reported in marine mussel species, but no studies have examined the presence of these microorganisms in freshwater mussels or how they relate to mussel host species or environmental conditions. In this study, microbial community DNA was extracted from the gut tissue of over 300 freshwater mussels, representing 22 species collected from rivers in the southeastern USA. Microeukaryote DNA was detected using PCR amplification, followed by the sequencing of positive amplicons. Microeukaryotes were found in 167 individual mussels (53%) of those tested. Amplicons included dinoflagellates/algae that differed between mussel species and are likely food sources that were distinct from those found in water and sediment samples analyzed concurrently. A total of 5% of the positive amplicons were non-photosynthetic alveolates that could represent parasitic microeukaryotes. Understanding the distribution of microeukaryotes in the freshwater mussel gut microbiome could further our understanding of the ongoing decline of mussel populations.

Molecular detection of sub-microscopic infections and Plasmodium falciparum histidine-rich protein-2 and 3 gene deletions in pre-elimination settings of South Africa

South Africa’s efforts toward eliminating malaria have positioned the country in the pre-elimination stage. Imported and sub-microscopic cases still contribute to the persistence of malaria in regions of low transmission as identified in this study where diagnostics is built largely on the use of Rapid Diagnostic Test (RDT). However, the presence of Pfhrp2/3 gene deletion is known to interfere with the accuracy of diagnosis with the use of RDT. Malaria elimination and detection of Pfhrp2/3 gene deletion in the pre-elimination setting requires accurate molecular surveillance. With the core objective of this study being the determination of the presence sub-microscopic malaria cases and deleted Pfhrp2/3 gene markers, a total of 354 samples were collected from five districts of KwaZulu Natal, South Africa. These samples were prepared for molecular analysis using primers and PCR conditions specific for amplification of 18S rRNA and msp-1gene. Positive amplicons were analysed for the presence of Pfhrp2/3 and flanking genes, along with Sanger sequencing and phylogenetic studies. Out of 354 samples collected 339 were tested negative with PfHRP2 based RDTs. Of these Pfhrp2 and Pfhrp3 gene deletions were confirmed in 94.7% (18/19) and 100% (19/19) respectively. High migration rate (75%) among the study participants was noted and phylogenetic analysis of sequenced isolates showed close evolutionary relatedness with India, United Kingdom, Iran, and Myanmar and China isolates. Molecular-based test is recommended as an essential surveillance tool for malaria management programs as the target focuses on elimination.

Prevalence of Human Parvovirus B19 and Associated Risk Factors in Women with Bad Obstetric History in Babylon Province, Iraq

Abstract Background: Parvovirus infection is an important cause of fetal loss during all three trimesters of pregnancy. Objectives: This study was conducted to investigate the prevalence, susceptibility rate, and risk factors of B19-V in women aged 15 to 45 in the Al-Hamza region of Babil, Iraq, considering the negative effects of the infection. Materials and Methods: Between January 2022 and March 2023, blood samples were taken from 116 women, including those with and those without a history of bad obstetric history (BOH). Enzyme-linked immunosorbent assay (ELISA) was used to test sera taken from the study participants for B19 IgM and immunoglobulin G (IgG) antibodies. Those samples tested positive for IgG B19 were further subjected to polymerase chain reaction amplification and sequencing, targeting partial gene sequences of the nonstructural viral protein 1 (VP1) gene. Results: For human parvovirus IgG and IgM, the seropositivity was 44% and 16.5%, respectively. The following variables were associated in this study with greater seroprevalence rates of IgG and IgM, respectively: age 36–45 years (72.7%, 36.4%); abortion (80%, 32.5%); education (82.2%, 33.3%); unemployment (61%, 22.2%); and overcrowding (73.3%, 31.1%). In this case–control study, positive amplicons (700 bp) were detected from 27 samples out of 40 women of childbearing age who tested positive for IgG. Sequence analysis of the partial sequence of the VP1 gene in all positive amplicons was 100% identical with each other and with human parvovirus B19 (NC: 00088.3). According to the current study, about 66% of women are serologically negative and at risk of contracting parvovirus B19. Conclusion: Our findings conclude that pregnant women in Iraq, particularly those with BOH, must undergo frequent serological screening for B19-V during all three trimesters and be given the necessary advice to avoid B19-V infection to improve their pregnancy outcomes.

A case of mistaken identity: a systematic review, meta-analysis, and reinvestigation of hemotropic Mycoplasma spp. infection in Ctenocephalides felis (cat flea)

Background Feline-associated hemotropic Mycoplasma (hemoplasmas) are believed to be transmitted by two primary mechanisms: (1) direct transmission via fighting and (2) vector-borne transmission by the cat flea (Ctenocephalides felis). While the efficiency of transmission by C. felis appears low, most manuscripts focus on the prevalence of hemoplasmas in wild-caught fleas and report either a very low (< 3%) or a high (> 26%) prevalence. Therefore, we aimed to assess the influence of sample processing and PCR methods on C. felis hemoplasma infection prevalence. MethodsA systemic review of PubMed articles identified 13 manuscripts (1,531 fleas/flea pools) that met the inclusion criteria (performed PCR for >1 hemoplasma on C. felis collected from cats). Risk of bias was assessed utilizing the ROBINS-E tool. Meta-analysis performed in R of these manuscripts found that not washing samples and a common set of 16S rRNA primers first published in Jensen et al. 2001 were associated with increased hemoplasma prevalence. To evaluate the influence of washing on newly collected fleas, we assessed the hemoplasma status of 20 pools of 5 C. felis each, half of which were washed and half not washed.ResultsFlea washing did not influence the detection of hemoplasma but instead amplified Spiroplasma. To assess non-specific amplification with the Jensen et al. 2001 primers, 67 C. felis samples (34% previously reported hemoplasma infected) were subject to PCR and sequencing. By this method, hemoplasma was detected in only 3% of samples. In the remaining “hemoplasma infected” fleas, PCR amplified Spiroplasma or other bacteria. ConclusionsTherefore, we concluded that hemoplasma infection in C. felis is rare, and future flea prevalence studies should sequence all positive amplicons to validate PCR specificity. Further investigation of alternative methods of feline-associated hemoplasma transmission and the ability of C. felis to maintain hemoplasma infection is necessary.Graphical

Molecular Surveillance, Prevalence, and Distribution of Cacao Infecting Badnavirus Species in Côte d'Ivoire and Ghana.

The cacao swollen shoot disease (CSSD) caused by a complex of badnavirus species presents a major challenge for cacao production in West Africa, especially Ghana and Côte d'Ivoire. In this study, CSSD species detection efficiency, diversity, and geographic distribution patterns in cacao plantations in Ghana and Côte d'Ivoire were investigated through field surveillance, PCR detection assays, sequencing of positive amplicons, and phylogeographic clustering. Cumulatively, the detection efficiency of the tested CSSD primer sets that were targeting the movement protein domain of the virus ranged from 0.15% (CSSD-3 primer) to 66.91% (CSSD-1 primer) on all the symptomatic cacao leaf samples assessed. The identified CSSD species differed phylogenetically and overlapped in distribution, with the cacao swollen shoot Togo B virus (CSSTBV) (n = 588 sequences) being the most prevalent and widely distributed compared to the other CSSD species that were encountered in both countries. Geographically, the cacao swollen shoot CE virus (CSSCEV) species (n = 124 sequences) that was identified was largely restricted to the bordering regions of Ghana and Côte d'Ivoire. These results provide updated knowledge of the geographic distribution of the key CSSD species and their diagnostic efficiency and, thus, provide guidance in identifying locations for structured testing of cacao germplasm and optimal diagnostics for the predominant CSSD species in Ghana and Côte d'Ivoire.

Spatial Distribution and Pathogen Profile of Dermacentor reticulatus Ticks in Southeastern Poland: A Genetic and Environmental Analysis

In recent years, significant changes have been observed in the distribution and abundance of local Dermacentor reticulatus populations. However, changes in D. reticulatus dynamics have not been studied in southeastern Poland. Our objective was to enhance our understanding of the environmental factors influencing the occurrence and density of D. reticulatus in this area. Additionally, we sought to investigate the genetic diversity of the tick population and the prevalence of tick-borne pathogens (TBPs). To this end, we established 45 study sites in the Subcarpathian province. Ticks were collected during their peak activity in both spring and autumn. A subset of randomly selected specimens underwent molecular analysis for TBPs screening, using high-throughput microfluidic real-time PCR. Positive amplicons were then sequenced, and phylogenetic analyses were conducted. Our findings confirmed the presence of D. reticulatus ticks in 24 surveyed sites, primarily concentrated in the northern and eastern parts of the region. The mean density of D. reticulatus ticks in their compact range was 5.8 ± 6.4 specimens/100 m2. Notably, air temperature and altitude emerged as significant factors influencing the species’ activity. We also identified a high prevalence of Rickettsia raoultii infections in adult D. reticulatus, reaching up to 84.21%. Additionally, 9.52% of ticks were found to be infected with R. helvetica and 4.76% with Anaplasma phagocytophilum. Furthermore, our genetic analyses confirmed the identity of D. reticulatus in the Subcarpathian region, aligning with haplotypes found in other regions of Poland, Czechia, Croatia, and Portugal. In conclusion, our study suggests that the surveyed region represents the current boundary of the compact range of D. reticulatus in Poland in which this tick species exhibits low genetic diversity and a narrow spectrum of detected TBPs.

Diversity of haemoparasites in migratory small ruminants of Himalayas, India

Vector-borne haemoparasitic diseases of migratory small ruminants have been less explored to date. Therefore, transhumant migratory sheep and goats of the Himalayas were molecularly screened for various vector-borne pathogens like Anaplasma, Theileria, Babesia, Anaplasma marginale, Theileria luwenshuni, Theileria orientalis, and Trypanosoma evansi using their specific primers. Out of the 171 animals screened, 73.09% of the animals were infected with Anaplasma spp. and 74.85% of the animals with Theileria spp., while no animals were found infected with Babesia spp. and Trypanosoma evansi. In a quest to identify the species, a few positive PCR amplicons representing Anaplasma spp., Theileria spp., and Theileria/Babesia spp. were sequenced. The sequences obtained were further BLAST analyzed to reveal maximum identity with Anaplasma capra (100%), Theileria luwenshuni (99.71%), and Theileria sinensis (98.73%). Among them Theileria luwenshuni was further confirmed with their species-specific PCR and the positive amplicons were sequenced and BLAST analysed to reveal their maximumsimilarity with Chinese isolates rather than Indian isolates. Further, phylogenetic analyses of the resulting sequences were conducted to identify their evolutionary history. It revealed that 1100 bp- amplicons of Theileria spp. were showing maximum likeness to Theileria sp. MK, a non-transforming Theileria. This is the first report of zoonotic Anaplasma capra and Theileria sp. MK in small ruminants from India and it could be mostly due to their transboundary migration history through the Indo-Tibetan border during summer seasons as a part of the traditional migratory system of livestock rearing practice in the Himalayas. The high prevalence of haemoparasites in migratory small ruminants calls for the adoption of effective control measures.

Unraveling the diversity of Trypanosoma species from Central Mexico: molecular confirmation on the presence of Trypanosoma dionisii and novel Neobat linages

Bats are one of the groups of mammals with the highest number of associated Trypanosoma taxa. There are 50 Trypanosoma species and genotypes infecting more than 75 species of bats across five continents. However, in Mexico, the inventory of species of the genus Trypanosoma associated with bats is limited to only two species (Trypanosoma vespertilionis and Trypanosoma cruzi) even though 140 species of bats inhabit this country. Specifically, 91 bat species have been recorded in the state of Veracruz, but records of trypanosomatids associated with this mammalian group are absent. Due to the complex Trypanosoma–bat relationship, the high diversity of bat species in Veracruz, as well as the lack of records of trypanosomatids associated with bats for this state, the aim of this work was to analyze the diversity of species of the genus Trypanosoma and their presence from a bat community in the central area of the state of Veracruz, Mexico. During the period of January to August 2022 in the Tequecholapa Environmental Management Unit where bats were collected using mist nets and blood samples were obtained from their thumbs. We extracted genetic material and amplified a fragment of 800 bp of the 18S ribosomal gene of the genus Trypanosoma by conventional PCR. The positive amplicons were sequenced, and phylogenetic reconstruction was performed to identify the parasite species. A total of 285 bats (149♀, 136♂) belonging to 13 species from 10 genera and a single family (Phyllostomidae) were collected. Twenty-three specimens from six species tested positive for the presence of Trypanosoma dionisii, Trypanosoma sp. Neobat 4, and a potential novelty species provisionally named as Trypanosoma sp. Neobat 6. The results of the present work increase the number of species of the genus Trypanosoma infecting bats in Mexico and in the Neotropical region.

White spot syndrome virus endogenous viral elements (EVE) revealed by circular viral copy DNA (cvcDNA) in shrimp

Circular, viral copy DNA (cvcDNA) can reveal the existence of endogenous viral elements (EVE) in shrimp genomic DNA. Here we describe isolation and sequencing of cvcDNA from a breeding stock of the whiteleg shrimp Penaeus vannamei. The stock was developed by onward breeding and selection for white spot syndrome virus (WSSV)-free individual survivors of white spot disease outbreaks. The stock exhibits high tolerance to WSSV. A pool of DNA extracted from 10 shrimp from this stock was subjected to cvcDNA isolation and amplification followed by high throughput sequencing. This revealed DNA fragments corresponding to locations covering much of the 300,000 bp WSSV genome. However, high frequency-read-fragments (HFRF) mapped to a surprisingly small region of approximately 1400 bp. We hypothesized that the HFRF reflected their selection due to provision of tolerance to WSSV. Four PCR primer sets were designed to cover the 1365 bp mapped region. One pair (Set 1) targeted the whole 1365 bp mapped region, while another 3 primer sets (Set 2–4), targeted regions within the 1365 bp target. All 4 primer sets were used with DNA samples from each of 36 shrimp from the same breeding stock (including the 10 used for cvcDNA preparation). Individual positive PCR results varied from shrimp to shrimp, ranging from only 1 primer set up to 4 primer sets. Only 1 specimen gave an amplicon of 1365 bp, while others gave single to multiple positive amplicons in both continuous and discontinuous regions. This indicated that the amplicons did not arise from contiguous targets and might vary in insertion length. The results also confirmed that none of the shrimp were infected with WSSV. Sequencing of the PCR amplicons of expected sizes revealed sequence identity to extant WSSV genomes. This data will be used to screen the breeding stock for EVE that provide WSSV tolerance.

Evidence of zoonotic rickettsiae in ixodid ticks of domestic animals in some communal farms in the Eastern Cape Province, South Africa.

The abundance of tick populations in South Africa represents a probable risk for both animal and human health. Rickettsia spp. and Borrelia spp. are well-known agents of emerging human tick-borne infectious diseases worldwide. Nevertheless, the epidemiology of their infections has been underreported in South Africa. Therefore, this study aimed to profile zoonotic Rickettsia and Borrelia species from ticks infesting domesticated animals in the Eastern Cape, South Africa. Morphological and molecular identification techniques were conducted on 1,200 tick samples collected from domestic animals before screening for the target bacterial pathogens. The molecular identification of the tick samples was based on the amplification of the 12S rRNA mitochondrial Deoxyribonucleic acid. At the same time, those of Rickettsia and Borrelia species were carried out by amplifying fragments of gltA and ompB genes for Rickettsia and flaB gene for Borrelia spp. Thereafter, the positive amplicons for Rickettsia ompB were sequenced and further analyzed. Borrelia PCRs were negative; therefore, sequencing could not be performed. Eight species of ticks belonging to three genera; Rhipicephalus, Amblyomma, and Haemaphysalis, were identified. A total of 27% (320/1,200) samples were confirmed positive for Rickettsia, of which 23% (74/320) were positive for ompB genes. Phylogenetic analysis of ompB revealed a high homology to rickettsial reference strains from GenBank, with no positive result for Borrelia. The generated sequences showed homology with R. africae-KX227790 (100%), R. parkeri-KY113111 (99.8%), R. peacockii (99.3%), and R. slovaca-JX683122 (99.1%) representative sequences in GenBank. The findings from this study revealed that ticks harbored Rickettsia species with possible zoonotic potential.

Detection of Immune Escape and Basal Core Promoter/Precore Gene Mutations in Hepatitis B Virus Isolated from Asymptomatic Hospital Attendees in Two Southwestern States in Nigeria.

Several mutations in the surface (S), basal core promoter (BCP), and precore (PC) genes of the hepatitis B virus have been linked to inaccurate diagnosis and the development of immune escape mutants (IEMs) of the infection, which can lead to chronic infection. Understanding the prevalence and spread of these mutations is critical in the global effort to eliminate HBV. Blood samples were collected from 410 people in Osun and Ekiti states, southwest Nigeria, between 2019 and 2021. Participants were drawn from a group of asymptomatic people who were either blood donors, outpatients, or antenatal patients with no record of HBV infection at the medical outpatients' unit of the hospital. DNA was extracted from plasma using a Qiagen DNEasy kit, followed by nested PCR targeting HBV S and BCP/PC genes. The Sanger sequencing method was used to sequence the positive PCR amplicons, which were further analyzed for IEMs, BCP, and PC mutations. HBV-DNA was detected in 12.4% (51/410) of individuals. After DNA amplification and purification, 47.1% (24) of the S gene and 76.5% (39) of the BCP/PC gene amplicons were successfully sequenced. Phylogenetic analysis showed that all the HBV sequences obtained in this study were classified as HBV genotype E. Mutational analysis of the major hydrophilic region (MHR) and a-determinant domain of S gene sequences revealed the presence of three immune escape mutations: two samples harbored a T116N substitution, six samples had heterogenous D144A/N/S/H substitution, and one sample had a G145E substitution, respectively. The BCP/PC region analysis revealed a preponderance of major BCP mutants, with the prevalence of BCP double substitutions ranging from 38.5% (A1762T) to 43.6% (G1764A). Previously reported classical PC mutant variants were observed in high proportion, including G1896A (33.3%) and G1899A (12.8%) mutations. This study confirms the strong presence of HBV genotype E in Nigeria, the ongoing circulation of HBV IEMs, and a high prevalence of BCP/PC mutants in the cohorts. This has implications for diagnosis and vaccine efficacy for efficient management and control of HBV in the country.

In-silico Analysis of Newcastle Disease Virus Strains from Outbreaks in Zaria and Kano of Nigeria

Introduction: Newcastle Disease (ND) is the major constraint to poultry production in developing country like Nigeria. Numerous outbreak cases of ND have being reported frequently even in vaccinated farms. The large antigenic disparity of the Newcastle Disease Virus (NDV) and potential for significant evolutionary divergence between field strains and widely used vaccinations are thought to be the root of the issue.&#x0D; Aim/Objectives: A cross-sectional studies was carried-out to identify a discrepancy between the vaccinal strains and circulating NDV field strains in Zaria and Kano so as to provide solution for the control of ND.&#x0D; Materials and Methods: In this study, molecular characterization and pathotyping of the field strains of NDV and strains of ND vaccines was carried out to evaluate their relatedness. Using RT-PCR with primers targeting partial fusion gene, 19 out of the (n= 120) samples purposefully collected for the detection of NDV in Zaria (n= 44) and Kano (n= 76) were positives. Twelve of the 19 positive amplicons were successfully sequenced and analyzed.&#x0D; Results: Only few number of the study’s sample have similarity with some of the vaccine strains.&#x0D; Conclusion: This study has raised concerns about the methods used to prevent NDV in Nigeria.&#x0D; Recommendation: It is crucial to develop an ND vaccine that matches the field strains within the country, along with ongoing surveillance of outbreaks for the effective control of the disease.

Seromolecular surveillance of rabbit haemorrhagic disease virus in Nigeria.

Following the first 2020 rabbit haemorrhagic disease virus (RHDV) outbreak in Nigeria which caused massive mortalities in several rabbitries, there was a need to know the spread and strains circulating in the affected states. Over 100 rabbitries still existing post-RHDV outbreak in Ogun and Kwara States were investigated. A commercial enzyme-linked immunosorbent assay kit was used to screen for RHDV immunoglobulin G in 192 rabbit sera, while RHDV VP60 gene was amplified in RNA extracted from these sera and tissues (liver and/or spleen harvested from 37 carcasses necrotized) by reverse transcription-polymerase chain reaction (RT-PCR). Sequences obtained from the amplicons were subjected to phylogenetic analysis. The results revealed a seroprevalence of 82.3% (158/192). RHDV VP60 gene was detected in 15/17 (88.2%) and 2/20 (10.0%) carcasses from Ogun and Kwara States, respectively, while none of the sera was positive. Sequences of the two positive amplicons selected (one from each states) shared 98.95% nucleotide identity and belonged to RHDV 2/GI.2 strain. Also, nBLAST of these sequences revealed 98.43-99.55% homology with the prototype Nigerian RHDV strain RHDV/NGR/ILN/001 (MT996357.1). Furthermore, these strains clustered with this prototype and a German RHDV strain (LR899166.1). Pathologic lesions affecting the respiratory, cardiovascular, renal, lymphatic, and digestive systems were observed in necropsied carcasses. This study indicated that RHDV 2/GI.2 strain was the cause of 2020 RHD outbreak in Nigeria. Thus, while continuous public sensitization about RHD especially among rabbit farmers in Nigeria is important, efforts aimed at design and implementation of RHD vaccination policy, preferably using indigenous seed, should be expedited.

First detection and genetic identification of Rickettsia spp. from ticks collected from rodents in north India

BackgroundChanging climatic conditions and invasion of ticks in urban areas have led to a greater number of cases of tick-borne diseases, thus, becoming a matter of increasing concern. Tick borne rickettsioses are one of the important emerging diseases worldwide. Knowledge of epidemiology of the vector and pathogen in the community is essential in order to understand and prevent the transmission of the disease to healthy population. MethodsIn our present study, we trapped rodents in selected areas of Chandigarh and Punjab in north India. The rodents were screened for the presence of ticks which were further screened for the presence of rickettsial agents. PCRs targeting 17 ​kDa and gltA genes were carried out followed by Sanger sequencing of the positive amplicons followed by phylogenetic analysis of the sequences. ResultsA total of 17 ticks were collected out of which one (Rhipicephalus sanguineus) was found to be harboring a Rickettsia sp. PCR targeting gltA and 17 ​kDa genes of rickettsia were put up and Sanger sequencing was performed. Phylogenetic analysis revealed the sequences to be closely related to Rickettsia rhipicephali. ConclusionThe current study establishes the presence of rickettsial agents in the community. Although Rickettsia rhipicephali is a non-pathogenic agent, the study encourages more vigorous community surveillance should be carried out in order to determine the exact burden of rickettsial agents in our community. To our knowledge, this is the first study reporting Rickettsia rhipicephali in India.

Application of loop-mediated isothermal amplification combined with lateral flow assay visualization of Plasmodium falciparum kelch 13 C580Y mutation for artemisinin resistance detection in clinical samples

Resistance to the antimalarial drug artemisinin (ART) has emerged in Greater Mekong Subregion. The molecular marker predominantly used to identify ART resistance is the C580Y mutation in Pfkelch13 of Plasmodium falciparum. Rapid and accurate detection of ART resistance in the field is necessary to guide malaria containment and elimination interventions. Our study evaluates the PfC580Y by using the loop-mediated isothermal amplification and single nucleotide polymorphism analysis visualization using a lateral flow assay (LAMP-SNP-LFA) method for detecting ART resistance in clinical samples collected from Thailand between 2014 and 2019. The optimized incubation condition for the reaction was determined as 45 min at 56 °C, followed by visual detection of positive amplicons using LFA. The assay demonstrated high analytical sensitivity and specificity, with a limit of detection of 16.8 copies of C580Y plasmid/µL of and 100% accuracy for C580Y mutation detection. The PfC580Y LAMP-SNP-LFA method is faster and simpler than conventional polymerase chain reaction/DNA sequencing and has the potential to support antimalarial management policies, malaria control, and global elimination efforts.

Emergence of equine-like G3 and porcine-like G9 rotavirus strains in Sarawak, Malaysia: 2019-2021.

Rotavirus is the leading causative viral agent of pediatric acute gastroenteritis globally, infecting mostly children 5 years old and below. Data on rotavirus prevalence in Malaysia is scarce, despite the WHO's recommendation for continuous rotavirus surveillance, and has underestimated the need for national rotavirus vaccination. Characteristics of the current rotavirus strains in Malaysia have to be determined to understand the rotavirus epidemiology and vaccine compatibility. This study sought to determine the genetic relatedness of Sarawak rotavirus strains with global strains and to determine the antigenic coverage and epitope compatibility of Rotarix and RotaTeq vaccines with the Sarawak rotavirus strains via in silico analysis. A total of 89 stool samples were collected from pediatric patients (<5 years old) with acute gastroenteritis at private hospitals in Kuching, Sarawak. Rotavirus was detected using reverse transcription-polymerase chain reaction. Positive amplicons were analyzed using nucleotide sequencing before phylogenetic analyses and assessment of epitope compatibility. Genotyping revealed G1P[8] (1/13; 7.7%), G3P[8] (3/13; 23%), G9P[4] (1/13; 7.7%), and G9P[8] (3/13; 23%), G9P[X] (1/13; 7.7%), GXP[4] (1/13; 7.7%), and GXP[8] (3/13; 23%) in samples. All wild-type Sarawak rotavirus strains, with the exception of G1, showed variations in their phylogenetic and antigenic epitope characteristics.

Phylogenetic identification of Anaplasma phagocytophilum in horses in Baghdad, Iraq

This study aimed to detect Anaplasma phagocytophilum in horses through hematological and molecular tests. The 16S rRNA gene of the Anaplasma phagocytophilum parasite was amplified by polymerase chain reaction (PCR), then sequenced, and subjected to phylogenetic analysis to explore "Equine Granulocytic Anaplasmosis" (EGA) infection in three important gathering race horses areas in Baghdad governorate, Iraq. Blood samples were obtained from 160 horses of varying ages, three breeds, and both sexes, between January and December 2021. Prevalence and risk variables for anaplasmosis were analyzed using statistical odds ratio and chi-square tests. Results demonstrated that clinical anaplasmosis symptoms comprised jaundice, weight loss, paleness of mucus membrane with petechial hemorrhage in the third elides, and edema in extremities; There was no tick infestation. The hematological test did not significantly reveal decreases in red and white blood cells and platelet count. Microscopically found 11 from 160 smears (6.88%) had morulae within granulocytes, PCR results of Anaplasma spp primers was 32 positive amplicons (20%), and molecular sequencing results of “16S ribosomal RNA genes” confirmed 21 horses (13.13%) infected by Anaplasma phagocytophilum for the first time in Iraq horses. The results of the phylogenetic analysis revealed compatibility values similarity 98.81-99.76% with worldwide isolates. Mares occurred not significantly riskier; also age and breed were not illustrated risks of any group. This study is the first molecular detection of Anaplasma phagocytophilum in racehorses reared in Baghdad in Iraq. The outcomes of this study provide genetic data for early identification of Anaplasma phagocytophilum infection, treatment, and management of the illness in Iraq horses, as well as monitoring its transmission to the human population.

First report of Colletotrichum siamense Causing anthracnose on Cinnamomum camphora in Malaysia.

Cinnamomum camphora (Lauraceae), commonly known as camphor tree, is widely grown as an ornamental and is used as a source of camphor in Malaysia. In June 2021, leaves of three camphor trees with anthracnose symptoms were collected from a park (6°02'00.8"N, 116°07'18.5"E) at the Universiti Malaysia Sabah in Sabah province. The average disease severity across diseased plants was about 60% with 30% incidence on 10 surveyed plants. The disease severity on disease area of 10 leaves from each three diseased plants was estimated using ImageJ software. The disease incidence was determined based on Sharma et al. (2017). Gray spots were observed primarily on the surface of the leaves. After a week, the spots coalesced into larger patches, and anthracnose developed. Small pieces (5 x 5 mm) of symptomatic leaf tissue from three camphor trees were excised from the margin between healthy and symptomatic tissue. The pieces were surface-sterilized with 75% ethanol for 1 minute, washed with 2% sodium hypochlorite solution for 1 minute, rinsed, and air dried before plating in three Petri dishes with Potato dextrose agar, and incubated for 7 days at 25°C in the dark. After 7 days, all the PDA plates had abundant gray-white fluffy hyphae. Mycelium was dark brown when observed from the underside of the plate. The isolates UMS02, UMS04 and UMS05 were characterized morphologically and molecularly. The conidia were one-celled, cylindrical, hyaline, and smooth, with blunt ends, and ranged in size from 13.9 to 16.3 x 3.8 to 6.1 μm (n = 20). Appressoria were round to irregular in shape and dark brown in color, with size ranging from 7.8 to 9.8 μm x 5.3 to 6.8 μm (n= 20). Genomic DNA was extracted from fresh mycelium of the isolates based on Khoo et al. (2022a). Amplification of the internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of the isolate was performed using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF1/GDR1 (Weir et al. 2012). PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. for sequencing. Sequences of the isolates were deposited in GenBank as OK448747, OM501094, OM501095 (ITS), OL953034, OM513908, OM513909 (CAL), OL953031, OM513910, OM513911 (ACT), OL953037, OM513912, OM513913 (CHS-1), and OL953040, OM513914, OM513915 (GAPDH). They were 100% identical to ITS (MN296082), CAL (MN525840), ACT (MW341257, MN525819), CHS-1 (MT210318), and GAPDH (MT682399, MN525882) sequences of Colletotrichum siamense. Phylogenetic analysis using maximum likelihood on the concatenated ITS, CAL, ACT, CHS-1 and GAPDH sequences indicated that the isolates formed a clade (82% bootstrap support) to C. siamense. Morphological and molecular characterization matched the description of C. siamense (Huang et al. 2022). Koch's postulates were performed by spraying a spore suspension (106 spores/ml) on leaves of three healthy two-month-old camphor trees, while water was sprayed on three additional camphor trees which served as control. The inoculated camphor trees were covered with plastics for 48 h at 25°C in the dark, and then placed in the greenhouse. Monitoring and incubation were performed based on Chai et al. (2017) and Iftikhar et al. (2022). Symptoms similar to those observed in the field occurred 8 days post-inoculation. No symptoms occurred on controls. The experiment was repeated two more times. C. siamense has been reported causing anthracnose on camphor tree in China (Liu et al. 2022), Citrus spp. in Mexico (Pérez-Mora et al. 2021), and Crinum asiaticum and eggplant in Malaysia (Khoo et al. 2022b, 2022c). To our knowledge, this is the first report of C. siamense causing anthracnose on C. camphora in Malaysia. Our findings expand the geographic range of C. siamense and indicate it could be a potential threat limiting the camphor production of C. camphora in Malaysia.