Nested PCR Assay Research Articles

Single - nucleotide polymorphisms of artemisinin resistance - related Pfubp1 and Pfap2mu genes in Bioko Island, Equatorial Guinea from 2018 to 2020

To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island. A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned. There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1-mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1gene mutation site in 19 Pfubp1-mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene (Pfap2mu-inner1, Pfap2mu-inner2, Pfap2mu-inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu-mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu-mutant P. falciparum isolates (10.06%). Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).

Torque teno sus virus k2a (TTSuVk2a) in wild boars from northeastern Patagonia, Argentina.

Torque teno sus virus k2a (TTSuVk2a) is a member of the family Anelloviridae that can establish persistent infections in both domestic pigs and wild boars. Its association with diseases has not been precisely elucidated, and it is often considered only as a commensal virus. This infectious agent has been reported in herds throughout the world. In this study, we investigated the detection rate and diversity of TTSuVk2a in free-living wild boars from northeastern Patagonia, Argentina. Total DNA was extracted from tonsil samples of 50 animals, nested PCR assays were carried out, and infection was verified in 60% of the cases. Sequence analysis of the viral non-coding region revealed distinct phylogenetic groups. These clusters showed contrasting patterns of spatial distribution, which presented statistically significant differences when evaluating spatial aggregation. In turn, the sequences were compared with those available in the database to find that the clusters were distinguished by having similarity with TTSuVk2a variants of different geographic origin. The results suggested that Patagonian wild boar populations are bearers of diverse viral strains of Asian, European, and South American provenance.

Distribution of maedi-visna virus in different organs of the spontaneously affected small ruminants in India.

Maedi-visna (MV) is a small ruminant lentiviral (SRLV) disease affecting sheep and goats, and causes pathological alterations in various organs including lungs, pulmonary lymph nodes, mammary glands, joints, and CNS. Aims: Present study was focused to detect the MV virus (MVV) nucleic acid and MVV p28 antigen in different organs of the spontaneously MVV affected sheep and goats. Total of 657 samples were collected from sheep and goats (169 blood, 136 lungs, 96 pulmonary lymph nodes, 74 brain, 54 mammary gland, 78 joints, and 50 spleen) and screened for MVV nucleic acid using nested PCR assay. Serum samples were screened for SRLV antibodies by cELISA. Immunolocalization of MVV was demonstrated by using the polyclonal antibody against p28 antigen by immunohistochemistry in lungs, lymph nodes, mammary glands, and joint tissues. Out of 657 samples, 10.7% (70) were found positive for MVV. Among different organs, lungs showed highest positivity (25.7%) followed by mammary glands (14.8%), blood (9.5%), joint tissues (7.7%), brain (5.4%), and pulmonary lymph node (1.0%). SRLV antibodies were detected in 29.2% of the serum samples of both sheep and goats by cELISA. MVV p28 antigen immunostaining was observed in lungs, lymph nodes, mammary glands, and joint tissues. However, the presence of MVV p28 antigen could not be demonstrated in the brain tissues. The highest positivity of MVV in lung tissues indicated higher predilection of the virus in the pulmonary tissue.

Toxoplasma gondii and Toxocara spp. contamination in university area.

Toxoplasma gondii and Toxocara spp. zoonotic infections may cause severe systemic and ocular illness in infected individuals. Cats play a significant role in environmental contamination and the transmission of parasites. The goal of the present study was to investigate the prevalence of Toxoplasma gondii (T. gondii) and Toxocara spp. infection among stray cats at Ahvaz Jundishapur University of Medical Sciences campus. The current descriptive study began with the collection of 170 fresh cat faecal samples from various sites in the Ahvaz Jundishapur University of Medical Sciences area. Sheather's sugar flotation method was applied to all specimens, and parasites were identified and examined microscopically. Next, a nested-PCR assay, sequencing, and real-time PCR with high-resolution melting curve (HRM) analysis were performed. In this study, out of 170 cat faecal samples microscopically evaluated, 8 (4.70%) and 37 (21.76%) were infected with T. gondii oocysts and Toxocara eggs, respectively. Using nested PCR, 8 out of 170 samples (4.70%) were found to be infected with T. gondii. HRM analysis showed that all isolates could be classified into three genetic lineages. Considerable prevalence, exceeding 50% for Toxocara and surpassing 25% for Toxoplasma in certain instances, along with genetic diversity, was observed in the present study. Hence, it is suggested that all individuals, including kindergarten children, students, employees, workers, and pregnant women who are in contact with their surroundings, take the necessary precautions.

A survey of resistance mutations to reverse transcriptase inhibitors (RTIs) among HIV-1 patients in northeast of Iran.

The use of a combination of three-drug regimen has improved HIV-1 infected patients' life span and quality; however the emergence of drug-resistant strains remains a main problem. Reverse transcriptase inhibitors (RTIs) consist of a main part of highly active anti-retroviral therapy (HAART) regimen. The present study aimed to investigate resistant mutations to RTI drugs in both treatment naïve and under treatment HIV patients in Mashhad city, north-eastern Iran. RNA was extracted from sera of 22 treatment naïve and 22 under treatment patients. The mean age of under treated and treatment naive groups were 38.5±6.7 and 40.8±7.9 respectively. cDNA was synthesized and amplified with Nested PCR assay targeting specific sequences of RT gene. The PCR products were sent for sequencing. Bidirectional sequencing results were analysed using HIV drug resistance database supplied by Stanford University (HIV Drug Resistance Database, https://hivdb.stanford.edu). Among under treatment patients 10 out of 22 (45%) had at least one high-level resistance mutation which was higher than high level resistance mutation rate among treatment naive cases (P<0.01). Detected resistance mutations were as follows: K101E, K103N, K103E, V106M, V108I, E138A, V179T, Y181C, M184V, Y188L, Y188H, Y188F, G190A, L210W, T215F, T215Y, K219Q, and P225H. A high level of resistance mutations to RT inhibitors was observed that causes drug resistance especially against lamivudine (3TC). Such mutations should be considered as probable responsible for therapeutic failure. Serial surveillance studies of circulating drug resistance mutations are recommended.

Molecular detection and genetic characteristics of Giardia duodenalis in dairy cattle from large-scale breeding farms in Xinjiang, China.

Giardia duodenalis is an intestinal protozoan that can infect both humans and animals, leading to public health issues and economic losses in the livestock industry. G. duodenalis has been reported to infect dairy cattle, but there is limited information available on large-scale dairy farms in Xinjiang, China. The study collected 749 fresh faecal samples from five large-scale cattle farms in Xinjiang, China. The study used a nested PCR assay of the small subunit ribosomal RNA (SSU rRNA*) gene to determine the presence of G. duodenalis. The results showed that 24.0% (180/749) of dairy cattle were positive for G. duodenalis, with the highest infection rate observed in pre-weaned calves (45.1%, 69/153). Among the 180 G. duodenalis positive samples, three assemblages were identified: assemblage E (n = 176), assemblage A (n = 3) and assemblage B (n = 1). Sixty-nine, 67 and 49 sequences were obtained for the beta-giardin (bg*) gene, the glutamate dehydrogenase (gdh*) gene and the triose phosphate isomerase (tpi*) gene, respectively. Thirteen novel sequences of assemblage E were identified, including five sequences from the bg* gene, four sequences from the gdh* gene and four sequences from the tpi* gene. This study found that 32 G. duodenalis assemblage E isolates formed 26 MLGs, indicating genetic variation and geographic isolation-based differentiation in bovine-derived G. duodenalis assemblage E. These findings provide fundamental insights into the genetic diversity of G. duodenalis in dairy cattle and can aid in the prevention and control of its occurrence in large-scale dairy cattle farms.

Establishment of an ultrasensitive and visual detection platform for Neospora caninum based-on the RPA-CRISPR/Cas12a system

Neospora caninum is a protozoan parasite that causes neosporosis in cattle, and leads to a high rate of abortion and severe financial losses. Rapid and accurate detection is particularly important for preventing and controlling neosporosis. In our research, a highly effective diagnostic technique based on the RPA-CRISPR/Cas system was created to successfully identify N. caninum against the Nc5 gene, fluorescent reporter system and the lateral flow strip (LFS) biosensor were exploited to display results. The specificity and sensitivity of the PRA-CRISPR/Cas12a assay were evaluated. We discovered that it was highly specific and did not react with any other pathogens. The limit of detection (LOD) for this technology was as low as one parasite per milliliter when employing the fluorescent reporter system, and was approximately ten parasites per milliliter based on the LFS biosensor and under blue or UV light. Meanwhile, the placental tissue samples were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (59.4 %, 19/32). The canine feces were detected by our RPA-CRISPR/Cas12a detection platform were completely consistent with that of the nested PCR assay (8.6 %, 6/70). The RPA-CRISPR/Cas12a detection procedure was successfully finished in within 90 min and offers advantages of high sensitivity and specificity, speed and low cost. The technique was better suitable for extensive neosporosis screening in non-laboratory and resource-constrained locations. This study provided a new strategy for more rapid and portable identification of N. caninum.

Development of a molecular diagnostic to discriminate between Fomitiporia species and advancements in detection of the main grapevine decline‐related pathogens in propagating material and mature vines

AbstractA comprehensive study was conducted on the prevalence of grapevine trunk diseases in Greece, focusing specifically on contamination of grapevine propagation material by Phaeomoniella chlamydospora and black foot disease‐related species. Additionally, detection of esca pathogen Fomitiporia mediterranea, causing white rot in grapevine and other woody hosts, was assessed using a new PCR‐based assay that distinguishes F. mediterranea from F. punctata. Development of a nested PCR assay, combined with a cost‐effective DNA extraction protocol, revealed a high percentage of infection by P. chlamydospora (51%) and Ilyonectria species associated with black foot disease (28%) in different types of grapevine propagation material (dormant cuttings, field‐rooted benchgrafts and green‐growing plants). Interestingly, black foot disease‐related pathogens were more prevalent in nursery plants grown in the field for 6 months (57%) than in other types of propagation material and compared with P. chlamydospora (43%), indicating increased infection of propagation material during growth in the nursery field. The cost‐effective molecular method developed in this study could be used in mass inspections of propagation material for phytosanitary purposes. Finally, using primers specific for F. mediterranea and F. puncata developed in this study, combined with the universal ITS4 primer, a collection of Fomitiporia isolates from mature grapevine and other woody hosts from Greece and Italy (southern Europe) were characterized as F. mediterranea, whereas German and Swedish isolates from forest plants (central Europe) were classified as F. punctata. The developed primers can discriminate between the two Fomitiporia species, which are indistinguishable based on culture and morphological characteristics alone.

Detection of malaria in goats and sheep

Plasmodium species, as the causative agent of malaria, are a significant concern in livestock production. This study aims to investigate the presence of malaria parasites in small ruminants comprised of goats and sheep. Plasmodium infections in ungulates do not cause malaria in humans. However, they might have anopheline mosquitoes as the same vector of this parasite. This study uses molecular techniques, nested PCR, to detect Plasmodium infections in small ruminants, as traditional microscopic examination may lead to misdiagnosis. The results show that Plasmodium infections were found in goat samples from Sumba Barat Daya, Nusa Tenggara Timur, with a prevalence of 7.41% based on nested PCR assay. Notably, Plasmodium was not detected through microscopic examination, underscoring the sensitivity of molecular techniques. It should be noted that none of the goats tested positive for malaria based on microscopic examination, suggesting extremely low parasitemia levels. This study emphasizes the importance of understanding Plasmodium infections in small ruminants, shedding light on their impact on animal health and their potential role in local transmission patterns. The sample size in this study is limited. Further research with larger samples and expanded geographical scope is recommended to comprehensively understand Plasmodium prevalence in small ruminants.

Development of a Real-Time Quantitative PCR Based on a TaqMan-MGB Probe for the Rapid Detection of Theileria haneyi.

Equine piroplasmosis (EP) is a parasitic disease caused by Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi (T. haneyi). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of T. haneyi. In this study, we developed a new qPCR method to detect T. haneyi based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in T. equi or B. caballi. A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of T. haneyi infections in horses.

Mitochondrial genome amplification of avian haemosporidian parasites from single-infected wildlife samples using a novel nested PCR approach

Haemosporidian parasites that infect birds (Apicomplexa: Haemosporida) are blood parasites that require an invertebrate host (vector) and a vertebrate host for their lifecycle and cause malaria-like diseases. This group of parasites has provided valuable insights into host specificity, virulence, and parasite dispersal. Additionally, they have played a significant role in reshaping our understanding of the evolutionary history of apicomplexans. In order to accurately identify species and to address phylogenetic questions such as the timing of the haemosporidian radiation, the use of a sufficiently large genetic data set is crucial. However, acquiring this genetic data poses significant challenges. In this research, a sensitive nested PCR assay was developed. This assay allows for the easy amplification of complete mitochondrial genomes of haemosporidian parasites in birds, even during the chronic stage of infection. The effectiveness of this new nested PCR assay was evaluated using blood and tissue samples of birds with verified single parasite infections from previous studies. The approach involves amplifying four overlapping fragments of the mitochondrial genome and requires DNA extracts from single-infected samples. This method successfully amplified the complete mitochondrial genomes of 24 distinct haemosporidian parasite lineages found in various bird species. This data is invaluable for conducting phylogenetic analyses and accurately defining species. Furthermore, this study proposes the existence of at least 15 new haemosporidian parasite species based on the genetic information obtained. Data regarding pGRW04, previously categorized as Plasmodium relictum like pSGS1 and pGRW11, indicates that the pGRW04 lineage is actually a separate, hidden Plasmodium species.

First report of filarial nematodes in the genus Onchocerca infecting black flies (Diptera: Simuliidae) in Iran

Black flies are blood-sucking insects of public health importance, and they are effective vectors of pathogens and parasites, such as filarial nematodes of the genus Onchocerca. Our previous surveys have shown that individuals of Simulium turgaicum are annoying pests of humans and livestock in the Aras River Basin of Iran. In the present study, adult black flies of S. turgaicum were trapped from different ecotopes of five villages in Khoda-Afarin County, Iran. By using a sensitive nested PCR assay and targeting the nuclear 18S rDNA-ITS1 marker, filarial infections were found in 38 (1.89%) of 2005 black flies. Homology exploration of 360 bp of the sequences indicated that the filarial worms are members of the family Onchocercidae, with maximum alignment scores of 93–95%. Phylogenetic analysis showed that two Iranian Onchocerca isolates were clustered in the O. fasciata–O. volvulus lineage and were well separated from other filarial nematodes. Both the entomological evidence (empty abdomen of the specimens) and climatologic data (adequate accumulated degree days for development) suggest that the filarial DNA was probably that of infective larvae of vertebrates. This is the first report of an infection by Onchocerca species in S. turgaicum and the first record of onchocercids in black flies in Iran; however, more research is required to demonstrate transmission of these filarial worms by black flies in nature.

Changing Molecular Profiles of Human Cryptosporidiosis Cases in Scotland as a Result of the Coronavirus Disease, COVID-19 Pandemic.

Cryptosporidium, the most frequently reported parasite in Scotland, causes gastrointestinal illness resulting in diarrhoea, nausea and cramps. Two species are responsible for most cases: Cryptosporidium hominis (C. hominis) and Cryptosporidium parvum (C. parvum). Transmission occurs faecal-orally, through ingestion of contaminated food and water, or direct contact with faeces. In 2020, the COVID-19 pandemic led to global restrictions, including national lockdowns to limit viral transmission. Such interventions led to decreased social mixing, and reduced/no local and international travel, which are factors associated with transmission of multiple communicable diseases, including cryptosporidiosis. This report assessed the impact of the pandemic on Scottish cryptosporidiosis cases, and identified changes in circulating molecular variants of Cryptosporidium species. Molecular data generated using real time PCR and GP60 nested-PCR assays on laboratory-confirmed cryptosporidiosis cases reported during 2018-22 were analysed. The Scottish Microbiology Reference Laboratories (SMiRL), Glasgow, received 774 Cryptosporidium-positive faeces during 2018-22, of which 486 samples were successfully subtyped. During this time period, C. hominis (n = 155; 21%) and C. parvum (n = 572; 77%) were the most commonly detected species. The total number of cases during 2020, which was greatly affected by the pandemic, was markedly lower in comparison to case numbers in the 2years before and after 2020. The most predominant C. hominis family detected prior to 2020 was the Ib family which shifted to the Ie family during 2022. The most common C. parvum variant during 2018-22 was the IIa family, however a rise in the IId family was observed (n = 6 in 2018 to n = 25 in 2022). The dominant C. hominis subtype IbA10G2, which accounted for 71% of C. hominis subtypes in 2018-19 was superseded by three rare subtypes: IeA11G3T3 (n = 15), IdA16 (n = 8) and IbA9G3 (n = 3) by 2022. Frequently reported C. parvum subtypes in 2018-19 were IIaA15G2R1 and IIaA17G1R1, accounting for 59% of total C. parvum subtypes. By 2022, IIaA15G2R1 remained the most common (n = 28), however three unusual subtypes in Scotland emerged: IIdA24G1 (n = 7), IIaA16G3R1 (n = 7) and IIaA15G1R2 (n = 7). Continuous monitoring of Cryptosporidium variants following the pandemic will be essential to explore further changes and emergence of strains with altered virulence.

Diagnostic validity of molecular diagnostic assays for white spot syndrome virus at different severity grades

The high virulence of the white spot syndrome virus (WSSV) in acute infections and the lack of effective treatments underscore the necessity for a rapid, accurate, and efficient diagnostic process to control white spot disease. The analytical sensitivity of diagnostic assays (polymerase chain reactions; PCRs and rapid diagnostic kit) at different severity grades of WSSV infection were determined using the 95% limit of detection (LOD95%). The LOD95% of nested, real-time, and one-step PCRs and rapid diagnostic kit were 0.70, 4.67, 1.19, and 79,434.65 viral genome copies/reactions, respectively. From the intramuscular challenge tests conducted under different dose and temperature conditions, the WSSV severity grades of time-course collected whiteleg shrimp (Litopenaeus vannamei) and dead or live shrimps were determined based on the viral loads of the pleopod. By applying the WSSV severity grades, a total of 92 shrimps were classified as G0. Furthermore, 92, 66, 199, and 79 shrimps were classified as G1, G2, G3, and G4, respectively. Additionally, 222 shrimps were classified as negative as WSSV was not confirmed in the nested PCR assay. Diagnostic sensitivity (DSe) and specificity (DSp) values of molecular diagnostic assays were compared with those of nested PCR in artificial WSSV-infected shrimp to evaluate diagnostic performance. The real-time and one-step PCRs exhibited a DSe >92.4% for G0 grade (approximately 101–102 copies/mg), indicating WSSV detection at low copy numbers. The rapid diagnostic kit presented a DSe >92.4% for G2 grade (approximately 104–105 copies/mg), suggesting the detection of WSSV-infected shrimp with clinical signs during the endemic period. These results suggest that the strategy of presumptive diagnosis using one-step PCR and rapid diagnostic kit at different seasonal periods followed by confirmatory diagnosis using real-time PCR assay could aid in controlling WSD with rapidity, accuracy, and cost-effectiveness.

Genetic diversity and characterization of Wolbachia endosymbiont in canine filariasis

Canine filariasis is caused by nematodes from the family Onchocercidae, which is transmitted by arthropod vectors. The disease is commonly found in Southeast Asia and exists worldwide. Some filarial nematodes are associated with intracellular bacteria of the genus Wolbachia, which plays an important role in embryogenesis, molting, and the long-term survival of adult worms. This study aims to characterize Wolbachia sp. and determine the association between Wolbachia and canine filarial nematode species in Thailand. A total of 46 dog blood samples that were naturally infected with filarial nematodes were obtained to identify filarial nematode species by Giemsa stained under a light microscope and confirmed using the molecular technique. In order to characterize Wolbachia sp., the nested PCR assay targeting the 16S rRNA gene showed that all samples of Dirofilaria immitis and fifteen samples of Candidatus Dirofilaria hongkongensis were grouped into Wolbachia supergroup C. In addition, all samples of Brugia spp. and five samples of Candidatus Dirofilaria hongkongensis were classified into Wolbachia supergroup D. The genetic diversity analysis conducted using the 16S rRNA gene revealed a similar result when analyzed through phylogenetic tree analysis. This is the first genetic diversity study of Wolbachia of Candidatus Dirofilaria hongkongensis in infected dogs in Thailand.

Molecular Survey of Piroplasmids and Hemosporidians in Vampire Bats, with Evidence of Distinct Piroplasmida Lineages Parasitizing Desmodus rotundus from the Brazilian Amazon

Although bats can serve as reservoirs for several viruses and bacteria, there is limited knowledge regarding the diversity of apicomplexan protozoan belonging to the Piroplasmida and Haemosporida orders within this group of mammals. The present study aimed to investigate the occurrence and phylogenetic assessment of piroplasmids and hemosporidians in spleen samples collected from 229 vampire bats (228 Desmodus rotundus and 1 Diaemus youngii) in the states of Pará, Roraima, Amapá, and Amazonas, northern Brazil. Out of 229 bat spleen samples, 43 (18.77%) tested positive in a nested PCR for piroplasmids based on the 18S rRNA gene. Thirteen sequences (ranging from 474 to 828 base pairs) of the partial 18S rRNA gene showed 91.04–100% identity to Theileria sp., Babesia sp., and Piroplasmida previously detected in deer, tapirs, opossums, and crab-eating raccoons. The phylogenetic analysis based on the near-complete 18S rRNA gene positioned the obtained sequences from three D. rotundus in distinct clades (Theileria sensu stricto, Tapirus terrestris, and “South America Marsupialia”). All bat spleen DNA samples tested negative in a nested PCR assay for hemosporidians based on the cytB gene. The present study reported, for the first time, the presence Babesia sp. and Theileria sp. DNA in D. rotundus. The distinct positioning of the 18S rRNA gene sequences within different clades demonstrates the occurrence of different piroplasmid species in vampire bats.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

Exploring the presence of bovine leukemia virus among breast cancer tumors in a rural state.

The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry. We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle. The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls. We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.

Molecular Detection and Characterization of Ehrlichia canis Isolates from Three Geographic Regions in Mexico: A Retrospective Study.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

The development of a novel quantitative assay for the detection of convert mortality nodavirus (CMNV) in Litopenaeus vannamei

Due to the viral covert mortality disease (VCMD), a new disease brought on by covert mortality nodavirus (CMNV), the Litopenaeus vannamei aquaculture industry in China and Southeast Asian countries have experienced major economic losses over the past ten years. A rapid and accurate on-site pre-detection plays as the first step and a critical part in the efficient control of VCMD spreads and outbreaks. For the rapid and accurate on-site detection of CMNV, a novel real-time quantitative assay based on quantitative recombinase polymerase amplification (qRPA) was developed in this study. By optimizing the optimum primer pairs based on the conserved RNA-dependent RNA polymerase (RdRp) genes, appropriate reaction temperatures, and probe dosages. It was possible to perform the CMNV-qRPA test at 42 °C in 20 min without exhibiting any cross-reactivity with the four major aquatic infections. Then its ability was compared to real-time quantitative PCR (qPCR) as well as nested PCR assays for detection of CMNV. As results of the sensitivity tests, the limit of detection (LOD) for the CMNV-qRPA, CMNV nested PCR, and CMNV qPCR assays were 5.5 × 102 copies μL−1, 5.5 copies μL−1 and 5.5 × 102 copies μL−1, respectively. Based on these results, it is demonstrated that the CMNV-qRPA assay developed in this study is both sensitive and specific for the rapid on-site pre-diagnosis of CMNV. Those findings were also demonstrated the application potential of CMNV-qRPA assay for efficiently preventing and controlling the spread and outbreak of VCMD in the aquaculture industry and application prospect of this diagnosis method in other related fields.