Detection and Genotyping of Francisella tularensis in Animal Hosts and Vectors from Six Different Natural Landscape Areas, Gansu Province, China

Objective. Tularemia, also known as hare fever, is caused by the bacterium Francisella tularensis (F. tularensis) transmitted through diseased wild animals, blood sucking insects, or contaminated water or food, which is distributed worldwide. The purpose of this study was to investigate F. tularensis infection in animal hosts and vectors from six different natural landscape areas in Gansu Province and to identify the genotypes of the detected F. tularensis. Methods. Rodents were captured by snap traps, and ticks were collected by dragging a cloth over the vegetation or from domestic animals. After species identification, DNA was isolated from the captured animals and detected by nested PCR assays targeting the F. tularensis fopA gene. The positive samples were further amplified to discriminate the species, and another two short-sequence tandem repeat regions (SSTR) were amplified to identify their genotypes. All positive fragments were sequenced and analyzed by ClustalX (5.0) and DNAClub software. Results. A total of 407 rodents of 12 species were captured, among which six rodent species were positive for F. tularensis, with an overall prevalence of 3.93%. The geographical difference in infection rate was statistically significant. At the SSTR9 locus, there were 7 genotypes among positive rodent samples. A total of 1864 ticks were tested for evidence of tularemia by nested PCR assays, 69 of which were positive, with an average positive rate of 3.70% for F. tularensis in ticks. The positive rates were significantly different among different regions. Seven genotypes were identified at the SSTR9 locus, one of which seemed dominant in positive tick samples. All positive samples had the same genotype at the SSTR16 locus. Conclusion. There is natural infection of F. tularensis among animal vectors and hosts in Gansu Province, with diverse genotypes.

Usefulness of Nested PCR Assay for the Molecular Diagnosis of Human Rickettsial Infection: A Study in Bangladesh

Background: Rickettsial infections are re-emerging arthropods born worldwide zoonotic disease caused by Rickettsia, which is responsible for spotted fever and typhus fever. The diagnosis of a rickettsial illness is important for appropriate antibiotic treatment. Aims: The study aimed to determine the diagnostic accuracy and clinical usefulness of using nested polymerase chain reaction (PCR) by comparing nested PCR, ELISA, and Weil-Felix (WF) tests. Methodology: This was a prospective type of cross-sectional study. A total of 135 clinically suspected rickettsial infection cases were enrolled. Peripheral blood was taken to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA, and ompB gene of Rickettsia by nested PCR. ELISA and Weil-Felix tests were done to compare with nested PCR. Results: Out of 135 cases, we detected Rickettsia in 70(51.85%) cases by nested PCR assay (p<0.01), 33((24.4%) by Weil- Felix test, 34 (25.18%) by ELISA. Only 26.66% of cases were PCR positive, which were negative by both ELISA and Weil-Felix test. Fifteen (11.11%) cases were positive by all three tests. Among 70 PCR positive rickettsia cases most frequently detected gene was ompB 42(60%), followed by 17kDa 34(48.58%); gltA 21(30%), and ompA 3(4.28%). Multiple gene combinations (ompB, 17kDa and gltA) detected in 98.57 % cases. Conclusion: Nested PCR assays showed the highest rate of detection of rickettsia cases than ELISA and Weil-Felix test. Multiple gene combinations (ompB, 17kDa, and gltA) showed the highest positivity. Therefore, diagnosis of rickettsial infection can be confirmed by PCR assay, and clinicians can plan appropriate treatment for these patients.

First report on clover proliferation phytoplasma related strain associated with Matthiola incana floral virescence in Uttar Pradesh, India

Matthiola incana R. Br. (Fam: Brassicaceae) is an ornamental, commonly known as hoary stock has an extremely fragrant flowers, which blooms in dense clusters in a large variety of colors. During a survey of flower nurseries in March 2019 at Indian Institute of Sugarcane Research campus, Lucknow, floral virescence (MiV) symptoms (Fig. 1 A, B) were observed in M. incana pots with an incidence of over 40%. Leaf yellows symptoms were also observed on a weed Acalypha indica (AiLY) in Matthiola nursery (Fig. 1 C). Nested PCR assays were carried out to detect and identify the possible association of phytoplasmas with MiV and AiLY symptoms. Three each of symptomatic MiV and AiLY samples and two non-symptomatic samples were collected and processed for DNA extraction from the leaf midrib by CTAB method. Hishimonus phycitis (HP) (Hemiptera: Cicadellidae) leafhopper feeding on MiV symptomatic plants was also collected and DNA was extracted. The DNA of 8 symptomatic and 4 non-symptomatic plants and from the 10 leafhopper was used as a template for PCR assays. Phytoplasma specific 16Sr RNA gene specific primers (P1/P7 and 3Far/3Rev; Schneider et al. 1995; Manimekalai et al. 2010) and multilocus genes’ specific primer pairs for secA (SecAfor1/SecArev3;SecAfo5r/SecARev2; Bekele et al. 2011), secY (SecYF1(VI)/SecYR1(VI);SecYF2(VI)/SecYR1(VI); Lee et al. 2010) and rp genes (rpFIC/rp(I)R1A; rp(VI)F2/ rp(VI)R2; Martini et al. 2007) were employed as previously described. Amplified products of ~1.3kb, ~600bp, ~1.7kb and ~1.0kb of 16S rRNA, secA, secY and rp genes of phytoplasma were consistently amplified in all the MiV and AiLY samples and in the HP leafhopper. No amplifications were achieved in any of the asymptomatic plant samples. Amplified products of all the four genes of MiV, AiLY and HP isolates were purified, sequenced and submitted in GenBank. Sequence comparison and phylogeny analysis of the sequences of the four genes of MiV, AiLY and HP isolates revealed 99% - 100% sequence identity and clustering with clover proliferation phytoplasma related strains (16SrVI group)(Fig.2 A,B,C and D). The virtual RFLP analysis of 17 restriction endonucleases corresponding to the 16S rDNA sequence of MiV, AiLY and HP phytoplasma strains by pDraw program, assigned them into a novel phytoplasma subgroup strain under 16SrVI group, since its HpaII restriction profile was different to earlier classified 16SrVI subgroups but was very close to16SrVI-E subgroup (GenBank acc. no. AY270156) (Fig 3). Earlier, peanut witches’ broom (16SrII-A) phytoplasma was identified associated with M. incana from Italy (Davino et al. 2007). However, the association of clover proliferation phytoplasma (16SrVI) related strain associated with virescence symptom of M. incana is the first report in world. The weed (A. indica) and HP leafhopper were also reported as additional hosts of 16SrVI subgroup related new strain in India, which needs further investigation. The report of a new host and new subgroup of clover proliferation phytoplasma related strain in India is having an epidemiological significance and warrants attention.

Molecular Detection and Identification of Babesia spp., and Trypanosoma spp. in One-humped Camel (Camelus dromedarius) in Halayeb and Shalateen, Egypt

Phylogenetic analysis of blood parasite infections including Babesia (B.) bovis, Babesia microti and Trypanosoma (T.) spp. in one-humped camel (Camelus dromedarius) (n= 142) breeds in Halayeb and Shalateen, in Upper Egypt were performed in the current study. Polymerase chain reaction (PCR) assays targeting the Rhoptry Associated Protein-1 (RAP-1), Babesia microti small subunit rRNA (ss-rRNA) and internal transcribed spacer 1 (ITS1) genes were used to detect the prevalence of B. bovis, B. microti and Trypanosoma spp. in camels, respectively. Nested PCR assays were used for the detection of Babesia spp. (B. bovis and B. microti). While, KIN-multi species PCR reaction was employed to detect and identify trypanosome DNA in camels. B. microti was detected in (17/142) with infection rate (11.97 %). Sequencing and phylogenetic analyses revealed that B. microti detected in camel was closely related to the German strain in rats and voles in France. B. bovis was also detected in (4/142) with infection rate (2.81%). The sequence and phylogenetic analyses revealed that the isolated B. bovis was closely related to strains isolated from Argentine, USA and Brazil. Moreover, T. evansi was detected in (8/142) with infection rate (5.63%). Sequence and phylogenetic analyses revealed that isolated T. evansi was closely related to T. theileri that was detected from cattle in Brazil. This study provides the first evidence of B. microti in camel in Egypt and highlights the possible role of one-humped camels in maintaining the enzootic cycle of Babesia transmission in Egypt.

Malaria parasites in macaques in Thailand: stump-tailed macaques (Macaca arctoides) are new natural hosts for Plasmodium knowlesi, Plasmodium inui, Plasmodium coatneyi and Plasmodium fieldi

Background Certain species of macaques are natural hosts of Plasmodium knowlesi and Plasmodium cynomolgi, which can both cause malaria in humans, and Plasmodium inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. cynomolgi, P. inui, Plasmodium coatneyi and Plasmodium fieldi in non-human primates from 4 new locations in Thailand. Methods A total of 93 blood samples from Macaca fascicularis, Macaca leonina and Macaca arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Results Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Conclusions The prevalence and species of Plasmodium varied among the wild and captive macaques, and between macaques at 4 sampling sites in Thailand. Macaca arctoides is a new natural host for P. knowlesi, P. inui, P. coatneyi and P. fieldi.

Malaria parasites in macaques in Thailand: stump-tailed macaques (Macaca arctoides) are new natural hosts for Plasmodium knowlesi, Plasmodium inui, Plasmodium coatneyi and Plasmodium fieldi

Background: Certain species of macaques are natural hosts of Plasmodium knowlesi and Plasmodium cynomolgi, which can both cause malaria in humans, and Plasmodium inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. cynomolgi, P. inui, Plasmodium coatneyi and Plasmodium fieldi in non-human primates from 4 new locations in Thailand.Methods: A total of 93 blood samples from Macaca fascicularis, Macaca leonina and Macaca arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Results: Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Conclusions: The prevalence and species of Plasmodium varied among the wild and captive macaques, and between macaques at 4 sampling sites in Thailand. Macaca arctoides is a new natural host for P. knowlesi, P. inui, P. coatneyi and P. fieldi.

Malaria parasites in macaques in Thailand: Stump-tailed macaques (Macaca arctoides) are new natural hosts for Plasmodium knowlesi, P. inui, P. coatneyi and P. fieldi.

Background: Certain species of macaques are natural hosts of Plasmodium knowlesi and P. cynomolgi, which can both cause malaria in humans, and P. inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi in non-human primates from 4 new locations in Thailand. Methods: A total of 93 blood samples from Macaca fascicularis, M. leonina and M. arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Results: Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Conclusions: The prevalence and species of Plasmodium varied among the wild and captive macaques, and between macaques at 4 sampling sites in Thailand. Macaca arctoides is a new natural host for P. knowlesi, P. inui, P. coatneyi and P. fieldi.

Detection of SARS-CoV-2 in wastewater in Japan by multiple molecular assays-implication for wastewater-based epidemiology (WBE)

ABSTRACTPresence of SARS-coronavirus-2 (SARS-CoV-2) in wastewater sample has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of COVID-19 outbreak. The purpose of this study was to verify the detection limit of WBE for COVID-19. In total, 27 influent wastewater samples were collected from four wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, numbers of the confirmed COVID-19 cases in these prefectures increased from almost 0 to around 20 per 100,000 peoples. SARS-CoV-2 RNA in the samples were identified by several PCR-based assays. Among the 27 samples, 7 were positive for SARS-CoV-2 by at least one out of the three quantitative RT-PCR assays. These samples were also positive by RT-nested PCR assays. The detection frequency became higher when the number of total confirmed SARS-CoV-2 cases in 100,000 peoples became above 10 in each prefecture. However, SARS-CoV-2 could also be detected with a low frequency when the number was below 1.0. Considering that the number of the confirmed cases does not necessarily reflect the actual prevalence of the infection at the time point, data on the relationship between the number of infection cases and concentration in wastewater needs to be accumulated further.

Malaria Parasites in Macaques in Thailand: Stump-Tailed Macaques (Macaca Arctoides) are New Natural Hosts for Plasmodium Knowlesi, P. Inui, P. Coatneyi and P. Fieldi.

Background:Certain species of macaques are natural hosts ofPlasmodium knowlesi and P. cynomolgi, which can both cause malaria in humans, and P. inui, which can be experimentally transmitted to humans. A significant number of zoonotic malaria cases have been reported in humans throughout Southeast Asia, including Thailand. There have been only two studies undertaken in Thailand to identify malaria parasites in non-human primates in 6 provinces. The objective of this study was to determine the prevalence of P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldiin non-human primates from 4 new locations in Thailand. Methods:A total of 93 blood samples from Macaca fascicularis, M. leonina and M. arctoides were collected from four locations in Thailand: 32 were captive M. fascicularisfrom Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wildM. arctoidesfromPrachuap Kiri Khan Province (PRA), and 25 were wild M. leoninafrom Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detectP. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.Results:Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularisat CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9had double and 2 had triple infections.The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi. Conclusions:The prevalence and species of Plasmodiumvaried among the wild and captive macaques, and betweenmacaques at 4 sampling sites in Thailand. Macaca arctoidesis a new natural host for P. knowlesi, P. inui,P. coatneyi and P. fieldi.

Metagenomic datasets of cerebrospinal fluid from a small cohort of MS/non-MS patients do not show DNA from the fungal genus Trichosporon

In [2] Alonso et al used nested PCR assays together with next-generation sequencing to find Trichosporon species in the nervous tissue of 10 patients with MS. We deemed it possible that the fungus would be present in cerebrospinal fluid (CSF) samples. Whole metagenomic shotgun (WMGS) sequencing allows detection of any organism in a sample. With Trichosporon any detection would be a true positive because these fungi are not known to be on the skin, or as typical lab contamination. We screened public WMGS datasets of CSF from a cohort of 43 Canadian patients (28 MS, 13 non-MS)[1], using Kraken2[7], the ultrafast kmer-based classifier, using a fungal database augmented with all cleaned available Trichosporon genome assemblies from the NCBI. Blasting the marked reads against an equally augmented blastn database revealed no alignments with an evalue &lt;= 1e-50. In general Kraken2 marked not more than 5 consecutive kmers in any read, which is a clear negative.