Clinical and laboratory diagnosis of Mayaro virus (MAYV): Current status and opportunities for further development.

The recent outbreaks related to Mayaro virus (MAYV) infection in the Americas have brought this neglected virus as a potential threat to global public health. Given the range of symptoms that can be associated with MAYV infection, it can be challenging to diagnose individuals based on clinical signs, especially in countries with simultaneous circulation of other mosquito-borne viruses, such as dengue virus (DENV) and chikungunya virus (CHIKV). With this challenge in mind, laboratory-based diagnosis assumes a critical role in the introduction of measures to help prevent virus dissemination and to adequately treat patients. In this review, we provide an overview of the clinical features reported in infected patients and currently available laboratory tools that are used for MAYV diagnosis, discussing their advances, advantages, and limitations to apply in the field. Moreover, we explore novel point-of-care (PoC) diagnostic platforms that can provide de-centralised diagnostics for use in areas with limited laboratory infrastructure.

Single-molecule optofluidic microsensor with interface whispering gallery modes.

Label-free sensors are highly desirable for biological analysis and early-stage disease diagnosis. Optical evanescent sensors have shown extraordinary ability in label-free detection, but their potentials have not been fully exploited because of the weak evanescent field tails at the sensing surfaces. Here, we report an ultrasensitive optofluidic biosensor with interface whispering gallery modes in a microbubble cavity. The interface modes feature both the peak of electromagnetic-field intensity at the sensing surface and high-Q factors even in a small-sized cavity, enabling a detection limit as low as 0.3 pg/cm2 The sample consumption can be pushed down to 10 pL due to the intrinsically integrated microfluidic channel. Furthermore, detection of single DNA with 8 kDa molecular weight is realized by the plasmonic-enhanced interface mode.

Enhanced Molecular Interaction of 3D Plasmonic Nanoporous Gold Alloys by Electronic Modulation for Sensitive Molecular Detection.

Three-dimensional gold and its alloyed nanoporous structures possess high surface areas and strong local electric fields, rendering them ideal substrates for plasmonic molecular detection. Despite enhancing plasmonic properties and altering molecular interactions, the effect of alloy composition on molecular detection capability has not yet been explored. Here, we report molecular interactions between nanoporous gold alloys and charged molecules by controlling the alloy composition. We demonstrate enhanced adsorption of negatively charged molecules onto the alloy surface due to positively charged gold atoms and a shifted d-band center through charge transfer between gold and other metals. Despite similar EM field intensities, nanoporous gold with silver (Au/Ag) achieves SERS enhancement factors (EF) up to 6 orders of magnitude higher than those of other alloys for negatively charged molecules. Finally, nanoporous Au/Ag detects amyloid-beta at concentrations as low as approximately 1 fM, with SERS EF up to 10 orders of magnitude higher than that of a monolayer of Au nanoparticles.

A rapid molecular detection tool for toxigenic M1UK Streptococcus pyogenes.

BACKGROUND: The gradual replacement of the Streptococcus pyogenes M1global genotype by a newly emergent M1UK variant is a global public health threat warranting increased surveillance. M1UK differs from progenitor M1global genotype by 27 single nucleotide polymorphisms (SNPs) and is characterised by increased speA superantigen expression in vitro. METHODS: An allele-specific real-time PCR assay was developed for the rapid detection of M1UK strains. The assay was used in combination with whole-genome sequencing to determine emm (sub)type distribution for 51 invasive (n = 9) and non-invasive (n = 42) S. pyogenes clinical isolates. RESULTS: Emm1 was the most prevalent S. pyogenes emm serotype (n = 11) in this set of clinical isolates, with M1UK being the dominant emm1 genotype (4/5 invasive, 3/6 non-invasive isolates). The assay accurately detected M1UK strains. Whole genome sequencing revealed continued presence of Australian M1UK sub-lineages associated with epidemic scarlet fever-causing S. pyogenes in Asia. CONCLUSIONS: Our study establishes a suitable target for detection of the toxigenic M1UK, and confirms the maintenance of M1UK strains in Queensland, Australia. This assay can be deployed in laboratories and provides a valuable, cost-effective tool to enhance surveillance of the expanding M1UK clone.

Effects of Degreasing Pretreatment on Immunohistochemistry and Molecular Analysis of Gastrointestinal and Breast Cancer Samples.

Lymph node status is a key factor in determining stage, treatment, and prognosis in cancers. Small lymph nodes in fat-rich gastrointestinal and breast cancer specimens are easily missed in conventional sampling methods. This study examined the effectiveness of the degreasing pretreatment with dimethyl sulfoxide (DMSO) in lymph node detection and its impact on the analysis of clinical treatment-related proteins and molecules. Thirty-three cases of gastrointestinal cancer specimens from radical gastrectomy and 63 cases of breast cancer specimens from modified radical mastectomy were included. After routine sampling of lymph nodes, the specimens were immersed in DMSO for 30 minutes for defatting. We assessed changes in the number of detected lymph nodes and pN staging in 33 gastrointestinal cancer specimens and 37 breast cancer specimens. In addition, we analyzed histologic characteristics, Masson trichrome special staining, and immunohistochemistry (gastrointestinal cancer: MMR, HER2, and PD-L1; breast cancer: ER, PR, AR, HER2, Ki-67, and PD-L1). Molecular status was evaluated for colorectal cancer (KRAS, NRAS, BRAF, and microsatellite instability) and breast cancer (HER2) in gastrointestinal cancer specimens and the remaining 26 breast cancer specimens. Compared with conventional sampling, DMSO pretreatment increased the detection rate of small lymph nodes (gastrointestinal cancer: P < .001; breast cancer: P < .001) and improved pN staging in 1 case each of gastric cancer, colon cancer, and rectal cancer (3/33; 9.1%). No significant difference in the morphology, special staining, protein, and molecular status of cancer tissue after DMSO treatment was found. Based on these results and our institutional experience, we recommend incorporating DMSO degreasing pretreatment into clinical pathologic sampling practices.

Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens.

OBJECTIVES: The performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed. METHODS: Prospectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method. RESULTS: The overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively. CONCLUSIONS: The FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.

Diaporthe phaseolorum causing dieback disease on Melia dubia cav. in Karnataka state (India).

Melia dubia is an important tree species grown worldwide for its medicinal and timber values. It is widely used in timber and pulp industry and also as an organic pesticide, fertilisers, agro-forestry and herbal formulations. During 2019-2022, a dieback disease in plantations of M. dubia was recorded in Mysore, Mandya, Chamarajanagar, Hassan and Tumkur districts of Karnataka state (India) with disease incidence of 26.25%. The associated pathogen was isolated on PDA medium and its morpho-cultural characteristics were studied. The genomic DNA of the pathogen was isolated, and rDNA was amplified and sequenced using universal primers. Based on the microscopic, morpho-cultural, sequence data and phylogenetic analysis, the pathogen was identified as Diaporthe phaseolorum (Cooke & Ellis) Sacc. Koch's postulates were performed both in vitro and in vivo and the typical symptoms of dieback disease were recorded on post-inoculated saplings. The dieback disease is responsible for the poor growth of Melia species in the region, and hence, there is an urgent need to manage the disease in plantations using integrated management practices. This is the first report of the occurrence of D. phaseolorum on M. dubia plantations in India.

Advances, challenges, and opportunities for food safety analysis in the isothermal nucleic acid amplification/CRISPR-Cas12a era.

Global food safety stands out as a prominent public concern, affecting populations worldwide. The recurrent challenge of food safety incidents reveals the need for a robust inspection framework. In recent years, the integration of isothermal nucleic acid amplification with CRISPR-Cas12a techniques has emerged as a promising tool for molecular detection of food hazards, presenting next generation of biosensing for food safety detection. This paper provides a comprehensive review of the current state of research on the synergistic application of isothermal nucleic acid amplification and CRISPR-Cas12a technology in the field of food safety. This innovative combination not only enriches the analytical tools, but also improving assay performance such as sensitivity and specificity, addressing the limitations of traditional methods. The review summarized various detection methodologies by the integration of isothermal nucleic acid amplification and CRISPR-Cas12a technology for diverse food safety concerns, including pathogenic bacterium, viruses, mycotoxins, food adulteration, and genetically modified foods. Each section elucidates the specific strategies employed and highlights the advantages conferred. Furthermore, the paper discussed the challenges faced by this technology in the context of food safety, offering insightful discussions on potential solutions and future prospects.

An On-Farm Workflow for Predictive Management of Paralytic Shellfish Toxin-Producing Harmful Algal Blooms for the Aquaculture Industry.

Paralytic shellfish toxins (PSTs) produced by marine dinoflagellates significantly impact shellfish industries worldwide. Early detection on-farm and with minimal training would allow additional time for management decisions to minimize economic losses. Here, we describe and test a standardized workflow based on the detection of sxtA4, an initial gene in the biosynthesis of PSTs. The workflow is simple and inexpensive and does not require a specialized laboratory. It consists of (1) water collection and filtration using a custom gravity sampler, (2) buffer selection for sample preservation and cell lysis for DNA, and (3) an assay based on a region of sxtA, DinoDtec lyophilized quantitative polymerase chain reaction (qPCR) assay. Water samples spiked with Alexandrium catenella showed a cell recovery of >90% when compared to light microscopy counts. The performance of the lysis method (90.3% efficient), Longmire's buffer, and the DinoDtec qPCR assay (tested across a range of Alexandrium species (90.7-106.9% efficiency; r2 > 0.99)) was found to be specific, sensitive, and efficient. We tested the application of this workflow weekly from May 2016 to 30th October 2017 to compare the relationship between sxtA4 copies L-1 in seawater and PSTs in mussel tissue (Mytilus galloprovincialis) on-farm and spatially (across multiple sites), effectively demonstrating an ∼2 week early warning of two A. catenella HABs (r = 0.95). Our tool provides an early, accurate, and efficient method for the identification of PST risk in shellfish aquaculture.

A real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of highly pathogenic H5 clade 2.3.4.4b avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIV) are a major threat to the global poultry industry and public health due to their zoonotic potential. Since 2016, Europe and France have faced major epizootics caused by clade 2.3.4.4b H5 HPAIV. To reduce sample-to-result times, point-of-care testing is urgently needed to help prevent further outbreaks and the propagation of the virus. This study presents the design of a novel real-time colourimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of clade 2.3.4.4b H5 HPAIV. A clinical validation of this RT-LAMP assay was performed on 198 pools of clinical swabs sampled in 52 poultry flocks during the H5 HPAI 2020-2022 epizootics in France. This RT-LAMP assay allowed the specific detection of HPAIV H5Nx clade 2.3.4.4b within 30 min with a sensitivity of 86.11%. This rapid, easy-to-perform, inexpensive, molecular detection assay could be included in the HPAIV surveillance toolbox.

An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

A study on the epidemiology of brucellosis in bovine population of peri-urban and rural areas of district Multan, southern Punjab, Pakistan.

BACKGROUND: Brucellosis is a zoonotic disease caused by a bacterial pathogen belonging to the genus Brucella. It is one of the most frequent bacterial zoonoses globally but unfortunately, it is still considered as a neglected disease in the developing world. Keeping in view, this study was conducted to determine the prevalence and risk determinants of brucellosis in large ruminants of peri-urban and rural areas of district Multan-Pakistan. For this purpose, blood samples (n = 490) were collected from the cattle (n = 245) and buffalo (n = 245) population of the study area and subjected to preliminary screening of brucellosis using local and imported RBPT reagents. All the samples were further analyzed using commercially available multi-specie indirect ELISA kit followed by their confirmation by PCR using genus and species-specific primers. Data obtained from lab analysis and questionnaires were subjected to statistical analysis for Pearson Chi-square, Odds Ratio and Confidence intervals (95%). RESULTS: The results showed that the maximum seropositivity was recorded with local RBPT reagent (VRI, Pakistan; 12.45%; 95%CI = 9.72-15.65%) followed by RBPT-IDEXX (12.24%; 95%CI = 9.52-15.45%) and RBPT-ID.vet (11.84%; 95%CI = 9.18-14.95%) however statistical difference was non-significant (P = 0.956). The ELISA results showed an overall seroprevalence rate of 11.22% (95%CI = 8.59-14.33%) with comparatively higher rate in cattle (12.65%; 95%CI = 8.82-17.44%) as compared to buffaloes (9.80%; 95%CI = 6.49-14.15%). The PCR analysis confirmed the presence of genus Brucella in all seropositive samples whereas frequency of B. abortus and B. melitensis in seropositive samples was 80% and 20%, respectively. The co-existence of both species was also observed in 5.45% samples. The statistical analysis showed a significant association of bovine brucellosis with herd size, breed, reproductive disorders, mode of insemination, educational status and farmers' awareness about brucellosis (P < 0.05). Conversely, locality, age, weight, gender, pregnancy status, parity and puberty status had no associations with brucellosis (P > 0.05). CONCLUSION: In conclusion, brucellosis is prevalent in large ruminants of district Multan, Pakistan. It is suggested to devise and implement stringent policies for the effective control and prevention of brucellosis in the region. Further, the current situation also warrants the need to strengthen interdisciplinary coordination among veterinarians and physicians in one health perspective to ensure and strengthen the human and animal health care systems in the region.

First report of Sarcocystis falcatula in naturally infected Razorbill auks (Alca torda) collected in Tunisian Mediterranean Sea shores.

Sarcocystis spp. are apicomplexan cyst-forming parasites that can infect numerous vertebrates, including birds. Sarcosporidiosis infection was investigated in three muscles (breast, right and left thigh muscle) and one organ (heart) of four Razorbill auks (Alca torda) stranded between November and December 2022 on the shores of the Mediterranean Sea in Nabeul and Bizerte governorates, Northern Tunisia. Two of the four tested A. torda were PCR positive for 18S rRNA Sarcocystis spp. gene. Among the examined 16 muscles/organs, only one breast and one right thigh were Sarcocystis spp. PCR-positive (12.5% ± 8.3, 2/16). Our results showed a relatively high molecular prevalence of Sarcocystis spp. in Razorbill auks (A. torda). Sarcocystis spp. sequence described in the present study (GenBank number: OR516818) showed 99.56-100% identity to Sarcocystis falcatula. In conclusion, our results confirmed the infection of Razorbill auks (A. torda) by S. falcatula. Further research is needed on different migratory seabirds' species in order to identify other Sarcocystis species.

Diversity of Sarcocystis parasites in southeastern Baltic Sea catchment ecosystems.

Currently, research on apicomplexan Sarcocystis parasites is mainly carried out by analyzing animal carcasses. However, environmental studies would not only allow faster detection of possible sources of infection but also avoid the use of animals for investigations. Therefore, in the current study, we aimed to identify tested Sarcocystis species in sediment collected from water bodies located in the southeastern Baltic countries. A total of 99 sediment samples were collected during the summer from different types of water bodies in Estonia, Latvia, Lithuania, and Poland. Species-specific nested PCR targeting cox1 gene was used for the detection of selected Sarcocystis species (S. cruzi, S. bovifelis, S. hirsuta, S. arieticanis, S. tenella, S. capracanis, S. miescheriana, and S. bertrami) infecting livestock. The results showed a statistically lower (p < 0.05) occurrence of Sarcocystis parasites in Estonia (50%) compared to three countries, where the detection rate of Sarcocystis spp. DNA was remarkably higher, ranging from 88 to 100%. Among Sarcocystis species tested, S. cruzi (83.8%) and S. arieticanis (55.6%) using cattle and sheep as their intermediate hosts were most commonly identified. The detection rates of some of the analyzed Sarcocystis species were significantly different in southeastern Baltic countries. It is discussed that the detection rates of certain Sarcocystis species depend not only on the number of animals per 1 km2 but also on various ecological factors and farming practices that differ in the amount of contact domestic animals have with predators and the potential for animals to become infected through natural water or food sources.

Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.

Molecular Identification and Characterization of Novel Taxonomic Subgroups and New Host Plants in 16SrI and 16SrII Group Phytoplasmas and Their Evolutionary Diversity on Hainan Island, China.

Phytoplasmas are a group of plant prokaryotic pathogens distributed worldwide. To comprehensively reveal the diversity of the pathogens and the diseases they cause on Hainan, a tropical island with abundant biodiversity in China, a survey of phytoplasmal diseases was performed from 2009 to 2022. Herein, molecular identification and genetic analysis were conducted based on the conserved genes of phytoplasmas. The results indicated that phytoplasmas could be detected in 138 samples from 18 host plants among 215 samples suspected to be infected by the pathogens. The phytoplasma strains from 27 diseased samples of 4 host plants belonged to the 16SrI group and the strains from 111 samples of 14 hosts belonged to the 16SrII group. Among them, 12 plants, including important tropical cash crops such as Phoenix dactylifera, cassava, sugarcane, and Piper nigrum, were first identified as hosts of phytoplasmas on Hainan Island. Based on BLAST and iPhyClassifier analyses, seven novel 16Sr subgroups were proposed to describe the relevant phytoplasma strains, comprising the 16SrI-AP, 16SrI-AQ, and 16SrI-AR subgroups within the 16SrI group and the 16SrII-Y, 16SrII-Z, 16SrII-AB, and 16SrII-AC subgroups within the 16SrII group. Genetic variation and phylogenetic analysis indicated that the phytoplasma strains identified in this study and those reported previously on Hainan Island mainly belong to four 16Sr groups (including I, II, V, and XXXII) and could infect 44 host plants, among which the 16SrI and 16SrII groups were the prevalent 16Sr groups associated with 43 host plant species. The diversity of host plants infected by the phytoplasmas made it difficult to monitor and control their related diseases. Therefore, strengthening inspection and quarantine during the introduction and transit of the related phytoplasmal host crops would effectively curb the spread and prevalence of the phytoplasmas and their related lethal diseases.

First Report of 'Candidatus Phytoplasma ziziphi'-Related Strains Infecting Peony (Paeonia suffruticosa Andr.) Showing Leaf Yellows Symptoms in China.

Peony (Paeonia suffruticosa Andr.), belonging to family Paeoniaceae, is an important medicinal and ornamental plant. During August of each year from 2016 to 2023, peony plants at Heze city were found to exhibit leaf yellows symptoms. The incidence rate of the symptomatic plant was recorded from 10% to 30% in four peony gardens with about 200 acres. Total DNA was extracted from 0.10 g fresh plant leaf tissues from 24 symptomatic and 8 asymptomatic samples using rapid plant genomic DNA isolation kit (Aidlab Biotechnology, Beijing, China). The extracted DNA was amplified by nested polymerase chain reaction using universal primers R16mF2/R16mR1 followed by R16F2/R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) specific for the 16S rRNA gene and new designed tuf gene specific primers JWB-tuforfF1 (5'-ATGGCTGAAATATTTTCAAGAG-3') and JWB-tuforfR1 (5'-TTATTCTATGATTTTAATAACAG-3') followed by JWB-tuforfF2 (5'-ATGTAAACGTAGGAACTATTGG-3') and JWB-tuforfR2 (5'- TCCGATAGTTCTTCCACCTTCAC-3'). Amplicons of about 1.25 kb and 1.02 kb (16S rRNA gene and tuf gene, respectively) were obtained in 8 symptomatic samples from four peony gardens. However, no amplification was obtained in any of the asymptomatic samples. The representative amplicons of 16S rRNA and tuf genes of three positive samples (Heze-9, -18 and -27) were cloned into a zero background pLB-simple vector (Tiangen Biotechnology, Beijing, China) and sequenced by Taihe Biotechnology, Beijing, China. Sequences obtained in the study were deposited in NCBI GenBank with accession numbers PP504882, PP504883 and PP504884 for the 16S rRNA gene as well as PP530237, PP530238 and PP530239 for the tuf gene. The phytoplasma strain under the study was described as peony yellows (PeY) phytoplasma, PeY-Heze strain. Alignment analysis by DNAMAN software showed that three 16S rRNA gene sequences obtained in the study shared 99.36% to 99.60% sequence identity and three tuf gene sequences obtained in the study were identical. BLAST analysis of the 16S rRNA gene sequences of the PeY-Heze phytoplasma strains showed 99.60%-99.84% sequence identity with 'Candidatus Phytoplasma ziziphi' (GenBank accession: CP025121). And tuf sequences of the strains showed 100% similarity with 'Ca. P. ziziphi' (CP025121). Interestingly, the virtual RFLP patterns derived from three 16Sr RNA gene sequences obtained in the study by iPhyClassifier (Zhao et al., 2009) were different from the reference patterns of all previously established 16Sr groups/subgroups. The most similar are the reference pattern of the 16Sr group VII, subgroup E (AY741531), with a similarity coefficient of 0.72, which is less than 0.85. These phytoplasma strains may represent a new 16Sr group. Phylogenetic analysis based on 16S rRNA genes using MEGA 7.0 by neighbor-joining (NJ) method with 1000 bootstrap value indicated that PeY-Heze strains clustered into one clade with the phytoplasma strains of 'Ca. P. ziziphi' with 68% bootstrap value. Although there are several reports available on 'Ca. P. solani' infecting peony in Shandong Province, China (Gao et al., 2013). To our knowledge, this is the first report of 'Ca. P. ziziphi'-related strains infecting peony in China. The findings in this study will be beneficial to the detection, quarantine, and prevention of peony yellows phytoplasmas in China.

Occurrence of Phytoplasmas Belonging to 16SrII Group Associated with Cyanthillium cinereum Witches'-Broom Diseases in China.

Cyanthillium cinereum, which belongs to the family of Asteraceae, is an annual or perennial herbaceous plant with significant medicinal uses for treating colds and fever. During September to November of 2020, C. cinereum showing symptoms of witches'-broom were found in economic forests distributed in Ding'an, Hainan Province of China, with 20% incidence. The symptoms of the plant were consistent with infections by 'Candidatus Phytoplasma' species. To identify the pathogen, five symptomatic and three asymptomatic C. cinereum samples were collected. Total DNAs were extracted using 0.10 g fresh leaf tissues of symptomatic and asymptomatic C. cinereum through a CTAB DNA extraction method according to Doyle and Doyle (1990). PCR amplification were performed employing the primer pairs of R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) specific for the conserved gene fragments of 16S rRNA and secA from phytoplasma. The PCR products were purified and sequenced through Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China), and the obtained sequences were deposited in GenBank. The phytoplasmal 16S rRNA and secA gene fragments obtained in the study were all identical with the length of 1325 bp (GenBank accession: PP098738) and 741 bp (PP072217), respectively. The phytoplasma strain was described as CcWB-hnda. A BLAST search based on 16S rRNA genes indicated that CcWB-hnda strain was identical to phytoplasmas belonging to 16SrII group like peanut witches'-broom phytoplasma strain T48 (OR239773) and 'Ca. Phytoplasma aurantifolia' strain TB2022 (CP120449). Virtual RFLP profiles based on 16S rRNA gene fragments obtained by iPhyClassifier (Zhao et al., 2009) showed that CcWB-hnda strain was a member of 16SrII-A subgroup with 1.00 similarity coefficient to the reference phytoplasma strain (L33765). A BLAST search based on secA genes indicated that CcWB-hnda had 100% sequence identity with phytoplasmas belonging to 16SrII group such as 'Ca. Phytoplasma aurantifolia' isolate TB2022 (CP120449), Vigna unguiculata witches'-broom phytoplasma (OR661282) and Emilia sonchifolia witches'-broom phytoplasma (MW353710). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing Neighbor-Joining method with 1000 bootstrap value (Kumar et al., 2016; Felsenstein, 1985) demonstrated that CcWB-hnda was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value respectively. To our knowledge, this is the first report of C. cinereum infected by phytoplasmas belonging to 16SrII-A subgroup in China. Identification of the vector insects of the pathogens is necessary in future, revealing the epidemiology of the related diseases. Phytoplasmas belonging to same 16Sr group or subgroup can infect different plants and spread through them in nature. The finding in this study will be beneficial to epidemic monitoring and early warning of C. cinereum witches'-broom disease and the related plant diseases caused by the phytoplasmas belonging to 16SrII group.

First report of Lasiodiplodia pseudotheobromae causing soft rot of plum in China.

Plum (Prunus salicina) is one of the most important fruit tree species worldwide (Valderrama-Soto et al. 2021). In June 2023, the postharvest soft rot symptoms were observed on plum fruits in several fruit markets of Guiyang city, Guizhou province, China. The disease incidence in these markets ranged from 20 to 25% with 70% disease severity. Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. In severe conditions, whole fruits become rotted and were covered with white fungal mycelia. Small sections (5 × 3 mm) from 6 diseased plum fruits were surface sterilized by using 75% ethanol for 30 s followed by 0.1% mercuric chloride solution for 5 min, rinsed three times with ddH2O, and then transferred onto potato dextrose agar (PDA) and incubated at 25 ± 2°C for three days. Three pure cultures (GUCC23-0001 to GUCC23-0003) were obtained by transferring a single hyphal tip to new PDA plates. Colonies of these isolates were grayish-white initially, gradually turning to whitish brown with fluffy aerial mycelia and uneven edges and finally turned to a dark gray colony after five days of inoculation. The pseudoparaphyses were hyaline, cylindrical, aseptate, and rounded at apex. Conidia were ellipsoidal, hyaline, unicellular, and 24.2 to 28.6 × 12.3 to 15.5 µm in size (n = 30) (Fig. S1), which were similar to the morphology of Lasiodiplodia pseudotheobromae (Alves et al. 2008). Furthermore, fungal DNA was extracted from fresh mycelia of PDA after seven days by using fungus genomic DNA extraction kit (Biomiga, USA). Partial DNA sequences from four loci including internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), and polymerase II second largest subunit (rpb2) were amplified with ITS1 and ITS4 (White et al. 1990), EF1-688F and EF1-1251R (Alves et al. 2008), Bt2a and Bt2b (Glass and Donaldson 1995), and RPB2-LasF and RPB2-LasR, respectively (Cruywagen et al. 2017). GenBank accession numbers are OR361680, OR361681, OR361682 for ITS, OR423394, OR423395, OR423396 for tef1, OR423397, OR423398, OR423399 for tub2, and OR423391, OR423392, OR423393 for rpb2, and gene sequencing showed 99.6 to 100% identity with ex-type strain of L. pseudotheobromae (CBS 116459). Phylogenetic analysis also placed our isolates in a highly supported clade with the reference isolate of L. pseudotheobromae (Fig. S2). Another experiment was designed to confirm the pathogenicity test for additional confirmation. Five mm mycelial plugs of L. pseudotheobromae from a three day old culture on PDA were placed on five surface-sterilized and non-wounded plum fruits for 12 hours and incubated at 25°C ± 2°C for four days. Sterilized fungus free PDA plugs were used as a negative control. Mycelial plugs were removed after 12 hours following which whole fruits were incubated in plastic boxes at 25°C ± 2°C. The experiment was repeated twice. The pathogenicity was evaluated under control conditions in laboratory (relative humidity, 70 ± 5% and temperature 25 ± 5˚C). Plum fruits showed rotting, which was characterized by water soaked fruit tissue, softening and presence of whitish mycelia four days post inoculation. These symptoms and signs were similar to the initially observed symptoms on plums in the markets. No disease symptoms were observed on the control fruits. The re-isolated fungus obtained from inoculated plum fruits was very similar to those isolated from diseased samples in morphology, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of L. pseudotheobromae causing postharvest fruit rot of plum in China. In 2022, the total planting area of plum was 1946.5 thousand hectares, which produces approximately 6626300 tons of plum (Food and Agriculture Organization of the United Nations, 2022). Based on the disease incidence and severity reported in the current study, soft rot of plum may be responsible for nearly 35% of yield losses under severe. Therefore, our study laid a theoretical foundation for the prevention and control of this post-harvest disease of plum.

Serological and Molecular Detection of Toxoplasma gondii in Domestic Pigs Intended for Human Consumption and Potential Occupational Hazard to Pig Farmers in India.

Toxoplasma gondii, an important food-borne zoonotic parasite, poses a worldwide public health hazard. Domestic pigs are considered one of the main intermediate hosts in the zoonotic transmission of T. gondii. To date, seroepidemiological information on T. gondii in domestic pigs in India is very scarce, and there are no reports of occupational hazards to pig farmers in this country. Here, we aimed at estimating the occurrence of T. gondii (antibodies and parasite DNA) in slaughtered pigs and pig farmers in Central India. Seroprevalence was determined in 410 serum samples from slaughtered pigs and 103 sera from pig farmers using an in-house prepared antigen-based modified agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and indirect-fluorescent antibody test (IFAT). Anti-T. gondii IgG antibodies were detected in 200 pigs (up to 48.8%, confidence interval [95% CI]: 40.4-52.2) and 44 pig farmers (up to 42.7%, 95% CI: 35.6-47.3) using MAT, ELISA, and IFAT. Inter-rater agreement showed an excellent agreement (kappa κ = 0.9) among the different serological tests suggesting similar detection potential of these tests. Recently acquired infections in all seropositive subjects were determined using IgG avidity testing and polymerase chain reaction (PCR). IgG avidity showed that 20 (10.3%) of slaughtered pigs and 8 (19.5%) pig farmers had a recently acquired infection. PCR for B1 and 529 repeats was performed in the heart tissues of slaughtered pigs and the blood cells of pig farmers. T. gondii DNA was detected in 14 (7.2%) slaughtered pigs and 5 (12.2%) pig farmers. Univariate analysis revealed that adult animals (>1 year), cats and rodents on the farm, and outdoor access are common factors (p ≤ 0.05) associated with T. gondii infection in pigs. Our results indicate that T. gondii is widely distributed in slaughtered pigs and pig farmers at risk of infection, highlighting a potential zoonotic transmission and health risk to consumers.