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AbstractPurposeMaterials and MethodsResultsConclusionThe study focused on developing a rapid PCR-based detection method and employing gamma irradiation techniques to manage Ralstonia solanacearum, aiming to produce brown rot-free export-quality potatoes. This initiative seeks to enhance potato exports from Bangladesh.Samples of potato tubers and soil were collected from various commercially significant potato-growing areas, resulting in a total of 168 Ralstonia solanacearum isolates from potato tubers and soil across 12 regions. The detection of R. solanacearum in the enriched tuber extract and soil were conducted using the primer pairs (PS-1, PS-2) and (759, 760). For the gamma irradiation experiment, petri dishes containing R. solanacearum cultures were subjected to different doses of gamma rays at the Bangladesh Institute of Nuclear Agriculture using a 60Co source. The irradiation doses applied to the samples were 0–6.0KGy.Morphological identification based on pink/light red colonies on TTC medium was confirmed R. solanacearum in 148 isolates. PCR using species-specific primers (PS-1/PS-2) and (759, 760) verified 26 isolates (14 tubers, 12 soil), producing 553 bp and 281 bp fragments in latently infected tubers and soil samples respectively. Gamma irradiation at 2.5 kGy damaged R. solanacearum’s DNA and cells, preventing brown rot, while higher doses eliminated it entirely. This offers a promising strategy to enhance safety of stored potatoes, potentially mitigating economic losses from this quarantine pathogen.The study developed a PCR detection method and gamma irradiation techniques to manage R. solanacearum, enhancing the export quality of potatoes. [ABSTRACT FROM AUTHOR]

The ash dieback disease caused by Hymenoscyphus fraxineus is widespread in Germany and is the subject of intensive research efforts. The fungus identification is based on the genomic internal transcribed spacer (ITS) region, which can also be the site of intragenomic variability. In Germany, despite intense research efforts, only a few numbers of H. fraxineus sequence data are recorded. Therefore, this study aims to characterize H. fraxineus isolates obtained from diseased ash leaves in Brandenburg (Germany). Fungal isolates from infected ash leaf tissue were analyzed molecularly using species-specific primers and based on sequencing the ITS region of rDNA. The analysis of the two identified sequences revealed two base substitutionscompared to the reference sequences. Thus, they show an identity of 98.8%–100% to the reference sequences and support the assumption that H. fraxineus has multiple copies of the ITS region. The phylogenetic grouping with reference sequences did not show a distinct cluster for the European, particularly the German, sequences. This indicates that the evolvement of the genetic variability of the ITS region is still an ongoing process. [ABSTRACT FROM AUTHOR]

ABSTRACT An S‐RNase‐based gametophytic self‐incompatibility (GSI) system, a mechanism that forces outbreeding by preventing self‐fertilization, characterizes the genus Malus. Knowledge of the self‐incompatibility (S) genotypes of apple cultivars is crucial for choosing pollen donors for fruit production and breeding. Even though the S‐allele of most commercial apple cultivars has already been identified, limited information is available about the S‐allele diversity within local germplasm collections. In this study, 67 S‐allele combinations of local apple accessions were identified. The allele S3 was the most common among local apple accessions followed by S1 and S7. The main aim of this study is to provide new information on cultivar compatibility, and these results will be used to set up new parent selection in apple breeding programmes as well as pollinator selection for the commercial orchard. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In order to identify the major virus species and infection types affecting cigar tobacco (Nicotiana tabacum) in Hainan Province, tobacco leaf samples suspected of being infected with viral diseases were collected from 7 cigar tobacco growing regions in Hainan. Specific primers of 6 viruses and universal primers of geminivirus were used for PCR detection, and the primers were designed according to the amplified sequences of TYLCV, SiLCV and AYVCNV to obtain their complete genome sequence. The results showed that 59 out of 64 samples were detected with 5 viruses (TMV, CMV, TYLCV, SiLCV and AYVCNV), and the detection rate was 92.19%. The detection rate of TMV was the highest (73.45%), followed by CMV (39.07%). The single infection rate was 60.94%, with TMV (45.31%), accounting for the highest proportion. The mixed infection rate was 31.25%, and the proportion of TMV+CMV was the highest (26.57%). Therefore, TMV was the dominant virus affecting cigar tobacco in Hainan, and the main type of mixed infection was TMV+CMV. In addition, the complete DNA-A sequences of TYLCV, SiLCV and AYVCNV were obtained. [ABSTRACT FROM AUTHOR]

Brown rot of potato is caused by a soil borne bacterium, Ralstonia solanacearum(Smith), a major limiting factor in the production of many crop plants around the world. An experiment was conducted to assess the status of brown rot disease in potatoes with the development of a rapid detection technique and approaches for production of brown rot free export quality potatoes to boost up potato export from Bangladesh. Survey results revealed a variation in latent infection (%) in tuber samples collected from six major growing areas viz. Dinajpur, Rangpur, Bogra, Joypurhat, Munshigonj and Comilla. The lowest (30%) latent infection was detected in tuber samples collected from Joypurhat as compared to Rangpur and Munshigonj in case of Asterix. However, in Diamant, the lowest (40%) latent infection was observed in Rangpur while 80% latent infection was recorded in Dinajpur and 88% was recorded in Munshigonj. Intriguingly, no latent infection was detected in Cardinal collected from Dinajpur while 40% latent infection was detected in samples from Joypurhat. However, 60% latent infection was detected in Granola which was collected from Dinajpur. The detection of R. solanacearum was reported in latently infected tubers by PCR using primers corresponding to 16SrDNA. In this study, PCR with primers PS-1 and PS-2 (16S rDNA) was used to detect R. solanacearum in latently infected tubers. An amplicon size corresponding to 553bp were obtained using enriched tuber extract as template. [ABSTRACT FROM AUTHOR]

Abstract Background Pneumocystis jirovecii pneumonia (PJP) is an interstitial pneumonia caused by pneumocystis jirovecii (PJ). The diagnosis of PJP primarily relies on the detection of the pathogen from lower respiratory tract specimens. However, it faces challenges such as difficulty in obtaining specimens and low detection rates. In the clinical diagnosis process, it is necessary to combine clinical symptoms, serological test results, chest Computed tomography (CT) images, molecular biology techniques, and metagenomics next-generation sequencing (mNGS) for comprehensive analysis. Purpose This study aims to overcome the limitations of traditional PJP diagnosis methods and develop a non-invasive, efficient, and accurate diagnostic approach for PJP. By using this method, patients can receive early diagnosis and treatment, effectively improving their prognosis. Methods We constructed an intelligent diagnostic model for PJP based on the different Convolutional Neural Networks. Firstly, we used the Convolutional Neural Network to extract CT image features from patients. Then, we fused the CT image features with clinical information features using a feature fusion function. Finally, the fused features were input into the classification network to obtain the patient's diagnosis result. Results In this study, for the diagnosis of PJP, the accuracy of the traditional PCR diagnostic method is 77.58%, while the mean accuracy of the optimal diagnostic model based on convolutional neural networks is 88.90%. Conclusion The accuracy of the diagnostic method proposed in this paper is 11.32% higher than that of the traditional PCR diagnostic method. The method proposed in this paper is an efficient, accurate, and non-invasive early diagnosis approach for PJP.

Background: Pneumocystis jirovecii pneumonia (PJP) is an interstitial pneumonia caused by pneumocystis jirovecii (PJ). The diagnosis of PJP primarily relies on the detection of the pathogen from lower respiratory tract specimens. However, it faces challenges such as difficulty in obtaining specimens and low detection rates. In the clinical diagnosis process, it is necessary to combine clinical symptoms, serological test results, chest Computed tomography (CT) images, molecular biology techniques, and metagenomics next-generation sequencing (mNGS) for comprehensive analysis. Purpose: This study aims to overcome the limitations of traditional PJP diagnosis methods and develop a non-invasive, efficient, and accurate diagnostic approach for PJP. By using this method, patients can receive early diagnosis and treatment, effectively improving their prognosis. Methods: We constructed an intelligent diagnostic model for PJP based on the different Convolutional Neural Networks. Firstly, we used the Convolutional Neural Network to extract CT image features from patients. Then, we fused the CT image features with clinical information features using a feature fusion function. Finally, the fused features were input into the classification network to obtain the patient's diagnosis result. Results: In this study, for the diagnosis of PJP, the accuracy of the traditional PCR diagnostic method is 77.58%, while the mean accuracy of the optimal diagnostic model based on convolutional neural networks is 88.90%. Conclusion: The accuracy of the diagnostic method proposed in this paper is 11.32% higher than that of the traditional PCR diagnostic method. The method proposed in this paper is an efficient, accurate, and non-invasive early diagnosis approach for PJP. [ABSTRACT FROM AUTHOR]

Introduction: Enterovirus D68 (EV-D68) belongs to the Picornaviridae family, genus Enterovirus. It is mostly known as a respiratory virus causing upper and lower respiratory tract infections, but it is also rarely associated with a variety of central nervous system complications, with acute flaccid myelitis being reported most frequently. This study assesses the incidence, seasonality, clinical presentation, and molecular epidemiology of the EV-D68 strain in EV-positive children hospitalized between 2014 and 2022 at the largest pediatric medical center in Slovenia. Methods: EV-D68 was detected using specific qRT-PCR, whereas partial VP1 sequences were obtained with Sanger sequencing, and further analyzed using the software CLC Main Workbench version 7 and MEGA version X. Results: EV-D68 was detected in 154 out of 1,145 (13.4%) EV-positive children. In the two epidemic years, 2014 and 2016, EV-D68 was most frequently detected in the summer and early autumn, peaking in September. The median age of EVD68-infected children was 3 years (IQR 1-3 years), with a female: male ratio of 1:1.17. Rhinorrhea was present in 74.0% of children, respiratory distress in 82.5%, and hypoxemia requiring supplemental oxygen in 44.1%. Out of 154 patients, 80.0% were hospitalized, with a median stay of 2 days (IQR 1-3 days). Lower respiratory tract infection was observed in 89.0% of EV-D68-positive patients, with bronchitis and bronchiolitis being most frequently diagnosed. No central nervous system manifestations of EV-D68 infection were observed in the study cohort. Phylogenetic analysis of partial VP1 sequences of EV-D68 revealed close similarity to the EV-D68 variants that were circulating in other European countries in these years. Discussion: Slovenia faced two EV-D68 epidemics in 2014 and 2016; however, after 2016 only nine more cases were detected until the end of the study period. Based on the results of this study, EV-D68 was a frequent cause of lower respiratory tract infection among EV-positive patients. However, none of the patients we studied needed ICU treatment, and none developed acute flaccid paralysis. Our results indicate that EV-D68 is not present constantly, so additional monitoring studies should be conducted in the future to better understand the implications of this EV type in human disease. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cacopsylla mali, C. melanoneura, C. picta, C. pruni, C. pyri, and C. pyrisuga belong among important pests of fruit trees. They harm trees by sucking and transmitting pathogenic bacteria of the ‘Candidatus Phytoplasma’, 16SrX group, which leads to declining and death of fruit trees. Protection against these pests depends on their early reliable identification and monitoring in fruit nurseries and orchards. In general, the determination of adult psyllids is based on morphological criteria, however, it is difficult or even impossible to perform determination using eggs and nymphs. In this study, a recently developed real-time PCR method validated for the determination of adult psyllids, was applied for the identification of other developmental stages. A total, 46 samples of eggs and nymph instars N1 to N5 were successfully determined, indicating the PCR identification system can be reliably used also for these types of specimens. Specific PCRs detected three species – C. melanoneura, C. pyri a C. pruni which were also confirmed by sequencing of the cytochrome c oxidase subunit I (COI) gene. Thus, this innovative molecular method can be used for a quick and routine determination of selected Cacopsylla species infecting fruit trees in all developmental stages.

Abstract Background Q fever and toxoplasmosis are economically important zoonoses as they cause considerable losses in livestock (cattle, sheep and goats) and wildlife (antelopes, giraffes, lions, and cheetahs) through reproductive disorders such as abortions and stillbirths. Q fever and toxoplasmosis testing in South Africa is conducted by the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR). However, both zoonoses are understudied and not monitored in South Africa as they are not considered controlled or notifiable diseases in the Animal Disease Act 35 of 1984. A retrospective study was conducted on Q fever (2007–2009) and toxoplasmosis (2007–2017) using diagnostic laboratory data at the ARC-OVR. Also, we report on sporadic abortion and stillbirth cases in livestock from diagnostic tissue samples submitted for Coxiella burnetii polymerase chain reaction (PCR) detection at the ARC-OVR. Results During 2007 to 2009, 766 animal samples were tested for C. burnetii antibodies and seropositivity was 0.9% (95%CI: 0.3–1.7) with sheep (1.9%; 95%CI: 0.6–4.4) having the highest seropositivity followed by cattle (0.7%; 95%CI: 0.09–2.6), while all goats (0.0%; 95%CI: 0.0–4.2) and wildlife (0.0%; 95%CI: 0.0–2.5) tested were negative. From 2007 to 2017, 567 sera were tested for T. gondii antibodies; overall seropositivity was 12.2% (95%CI: 9.6–15). Wildlife had highest seropositivity to T. gondii antibodies (13.9%; 95%CI: 9.0–19.7) followed by goats (12.9%; 95%CI: 9.2–17.4) and sheep (12.3%; 95%CI: 5.1–23.8) while seropositivity in cattle was 2.4% (95%CI: 0.06–12.9). Of 11 animals tested by C. burnetii PCR detection (2021–2022), 10 (91.0%) were positive. The amplicon sequences showed similarity to Coxiella burnetii strain 54T1 transposase gene partial coding sequence. Conclusions We have confirmed the occurrence of the causative agents of Q fever and toxoplasmosis in livestock and wildlife in South Africa, with data limitations. These zoonoses remain of importance with limited information about them in South Africa. This study provides baseline information for future studies on Q fever and toxoplasmosis in South African livestock and wildlife, as well other African countries. Due to limited data collection experienced in this study, it is recommended that improvements in data collection samples tested should include associated factors such as sex, age, and breed of the animals.

This research investigated the incidence of two foodborne pathogens, Salmonella spp. and Escherichia coli in water used during live transportation of climbing perch (Anabas testudineus). Experiments were conducted in three commercially important fish supply channels of Bangladesh, starting from an important production area, Muktagacha, Mymensingh to Dhaka (supply channel 1), Sylhet (supply channel 2), and Rajshahi (supply channel 3). Water samples were collected from 0 hours (at the time of loading) to reaching at the final destination (unloading points/ retail markets) at every 2 hours interval during transportation. To assess the prevalence of Salmonella spp. and E. coli, Salmonella-Shigella (SS) agar and Eosin methylene blue (EMB) agar plate counts were done. For confirmation and PCR detection, a total of 52 isolates were obtained from those cultured plates based on the colony characteristics. The finding showed a gradual increase in SS- and EMB agar plate counts in all the supply channels. Out of the 52 isolates, 10 (19%) were detected as positive for Salmonella spp., while 38 (73%) as positive for E. coli and the rest were unidentified. It was assessed that the pond waters of the cultured areas might be the primary sources of contamination of these two foodborne pathogens. [ABSTRACT FROM AUTHOR]

There have been high expectations in recent years of using xenotransplantation and regenerative medicine to treat humans, and pigs have been utilized as the donor model. Pigs used for these clinical applications must be microbiologically safe, that is, free of infectious pathogens, to prevent infections not only in livestock, but also in humans. Currently, however, the full spectrum of pathogens that can infect to the human host or cause disease in transplanted porcine organs/cells has not been fully defined. In the present study, we thus aimed to develop a larger panel for the detection of pathogens that could potentially infect xenotransplantation donor pigs. Our newly developed panel, which consisted of 76 highly sensitive PCR detection assays, was able to detect 41 viruses, 1 protozoa, and a broad range of bacteria (by use of universal 16S rRNA primers). The applicability of this panel was validated using blood samples from uterectomy‐born piglets, and pathogens suspected to be vertically transmitted from sows to piglets were successfully detected. We estimate that, at least for viruses and bacteria, the number of target pathogens detected by the developed screening panel should suffice to meet the microbiological safety levels required worldwide for xenotransplantation and/or regenerative therapy. This panel provides greater diagnosis options to produce donor pigs so that it would render unnecessary to screen for all pathogens listed. Instead, the new panel could be utilized to detect only required pathogens within a given geographic range where the donor pigs for xenotransplantation have been and/or are being developed. [ABSTRACT FROM AUTHOR]

IntroductionEnterovirus D68 (EV-D68) belongs to the Picornaviridae family, genus Enterovirus. It is mostly known as a respiratory virus causing upper and lower respiratory tract infections, but it is also rarely associated with a variety of central nervous system complications, with acute flaccid myelitis being reported most frequently. This study assesses the incidence, seasonality, clinical presentation, and molecular epidemiology of the EV-D68 strain in EV-positive children hospitalized between 2014 and 2022 at the largest pediatric medical center in Slovenia.MethodsEV-D68 was detected using specific qRT-PCR, whereas partial VP1 sequences were obtained with Sanger sequencing, and further analyzed using the software CLC Main Workbench version 7 and MEGA version X.ResultsEV-D68 was detected in 154 out of 1,145 (13.4%) EV-positive children. In the two epidemic years, 2014 and 2016, EV-D68 was most frequently detected in the summer and early autumn, peaking in September. The median age of EV-D68–infected children was 3 years (IQR 1–3 years), with a female: male ratio of 1:1.17. Rhinorrhea was present in 74.0% of children, respiratory distress in 82.5%, and hypoxemia requiring supplemental oxygen in 44.1%. Out of 154 patients, 80.0% were hospitalized, with a median stay of 2 days (IQR 1–3 days). Lower respiratory tract infection was observed in 89.0% of EV-D68–positive patients, with bronchitis and bronchiolitis being most frequently diagnosed. No central nervous system manifestations of EV-D68 infection were observed in the study cohort. Phylogenetic analysis of partial VP1 sequences of EV-D68 revealed close similarity to the EV-D68 variants that were circulating in other European countries in these years.DiscussionSlovenia faced two EV-D68 epidemics in 2014 and 2016; however, after 2016 only nine more cases were detected until the end of the study period. Based on the results of this study, EV-D68 was a frequent cause of lower respiratory tract infection among EV-positive patients. However, none of the patients we studied needed ICU treatment, and none developed acute flaccid paralysis. Our results indicate that EV-D68 is not present constantly, so additional monitoring studies should be conducted in the future to better understand the implications of this EV type in human disease.

Background: Q fever and toxoplasmosis are economically important zoonoses as they cause considerable losses in livestock (cattle, sheep and goats) and wildlife (antelopes, giraffes, lions, and cheetahs) through reproductive disorders such as abortions and stillbirths. Q fever and toxoplasmosis testing in South Africa is conducted by the Agricultural Research Council-Onderstepoort Veterinary Research (ARC-OVR). However, both zoonoses are understudied and not monitored in South Africa as they are not considered controlled or notifiable diseases in the Animal Disease Act 35 of 1984. A retrospective study was conducted on Q fever (2007–2009) and toxoplasmosis (2007–2017) using diagnostic laboratory data at the ARC-OVR. Also, we report on sporadic abortion and stillbirth cases in livestock from diagnostic tissue samples submitted for Coxiella burnetii polymerase chain reaction (PCR) detection at the ARC-OVR. Results: During 2007 to 2009, 766 animal samples were tested for C. burnetii antibodies and seropositivity was 0.9% (95%CI: 0.3–1.7) with sheep (1.9%; 95%CI: 0.6–4.4) having the highest seropositivity followed by cattle (0.7%; 95%CI: 0.09–2.6), while all goats (0.0%; 95%CI: 0.0–4.2) and wildlife (0.0%; 95%CI: 0.0–2.5) tested were negative. From 2007 to 2017, 567 sera were tested for T. gondii antibodies; overall seropositivity was 12.2% (95%CI: 9.6–15). Wildlife had highest seropositivity to T. gondii antibodies (13.9%; 95%CI: 9.0–19.7) followed by goats (12.9%; 95%CI: 9.2–17.4) and sheep (12.3%; 95%CI: 5.1–23.8) while seropositivity in cattle was 2.4% (95%CI: 0.06–12.9). Of 11 animals tested by C. burnetii PCR detection (2021–2022), 10 (91.0%) were positive. The amplicon sequences showed similarity to Coxiella burnetii strain 54T1 transposase gene partial coding sequence. Conclusions: We have confirmed the occurrence of the causative agents of Q fever and toxoplasmosis in livestock and wildlife in South Africa, with data limitations. These zoonoses remain of importance with limited information about them in South Africa. This study provides baseline information for future studies on Q fever and toxoplasmosis in South African livestock and wildlife, as well other African countries. Due to limited data collection experienced in this study, it is recommended that improvements in data collection samples tested should include associated factors such as sex, age, and breed of the animals. [ABSTRACT FROM AUTHOR]

The darkling beetle, Alphitobius diaperinus , and the poultry red mite, Dermanysuss gallinae are among the most common pests of poultry farms. Both pests can be carriers and reservoirs of various pathogens including zoonotic ones like Salmonella. Salmonellosis is one of the most common foodborne diseases reported in the EU. We developed a semi-nested PCR method for the direct detection of Salmonella enterica. When testing the specificity of the novel PCR, we successfully detected various S. enterica strains, whereas Escherichia coli and Citrobacter strains gave negative results. The authenticity of the PCR products was confirmed by DNA sequencing. The sensitivity of the semi-nested PCR was tested on serial dilution of bacterial cultures and extracted DNA. We found our new method more sensitive than the previous PCRs. We also screened ectoparasite samples, collected from a poultry farm in Hungary, and three out of the eight samples were positive for S. Enteritidis. This novel PCR seems suitable for the detection of S. enterica strains in poultry ectoparasites without the need of sample pre-enrichment. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Medical Equipment Journal is the property of Chinese Medical Equipment Journal Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The south of Western Siberia traditionally belongs to the regions with unfavorable epidemiological situation for tick-transmitted infections. In Novosibirsk and its suburbs, approximately 20 thousand people annually seek for medical care after tick bites. The purpose of this work was to determine human-attacking tick species composition in the territory of Novosibirsk and its suburbs by identifying nucleotide sequence of tick mitochondrial gene cytochrome oxidase COI fragment during the 2018 spring-summer season, determination of infections of individual ticks with viral and bacterial tick-borne infections, followed by genotyping identified pathogens by PCR methods, by determining nucleotide sequences and phylogenetic analysis. It is established that ticks of five species attack the humans: Ixodes persulcatus, Ixodes pavlovskyi, Dermacentor reticulatus, Dermacentor marginatus, Dermacentor nuttali in the Novosibirsk metropolis. The majority of tick attacks on humans are associated with I. pavlovskyi (43.6%) and D. reticulatus (41.2%) ticks. The genetic material of the tick-borne encephalitis virus was detected in 3.6%, borrelia 13.8% and Rickettsia 23.1% individual ticks collected from humans. Genotyping of the tick-borne encephalitis virus (TBEV) in the collected ticks showed the presence of TBEV Siberian and Far Eastern genotype in 83% and 17% cases, respectively. The Kemerovo virus could not be detected. Genetic material from Borrelia spp. and Rickettsia spp. has been detected in all five tick species attacking the local human population. The bulk of the identified Borrelia spp. isolates were genotyped as Borrelia garinii (86%) and 13% of the isolates as Borrelia afzelii species. The genetic material of Borrelia miyamotoi was found in one individual tick. Ticks of the genus Dermacentor have been found to be infected by Rickettsia raoultii, and ticks of the genus Ixodes were largely infected with Rickettsia tarasevichiae, and I. persulcatus tick was infected by Rickettsia helvetica in a single case. Thus, the human population of Novosibirsk and its suburbs is attacked by five species of ixodid ticks dominated by I. pavlovskyi and D. reticulatus ticks. The genetic material of the following tick-borne pathogens was found: Siberian and Far Eastern TBEV genotype, B. garinii, B. afzelii, B. miyamotoi, R. raoulti, R. tarasevichiae and R. helvetica in the individual ticks collected from humans.

Abstract Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig’s immunity may be a reason those sites are undergoing highly positive selection.

Lisianthus (Eustoma grandiflorum) has become a major flowering plant in Okinawa, the southernmost prefecture of Japan. Its cultivation area has increased steadily with each passing year for two decades. Simultaneously, many types of lisianthus diseases related to damping-off symptoms have also increased dramatically. To create a strategy for preventing the disease, disease symptoms and pathogenic organisms of primary problematic disease with seasonal variation in the emergence were investigated. The symptoms were diagnosed as Fusarium stem rot (Kukigusare-byo) and the pathogen of the disease was identified as Fusarium avenaceum based on multigene sequences analyses. Indeed, the PCR result of the isolated strain in this study was the same as that isolated from lisianthus plants with Fusarium stem rot in Hokkaido Prefecture. Furthermore, the pathogen is clustered separately from the other F. avenaceum strains isolated from lisianthus in the USA. Diseased lisianthus plants spread throughout greenhouses even though several fungicides were applied. Additionally, they appeared from November to January and increased to 0.3% of the total number. Fusarium stem rot was found in 43.8% of the total number of farms from 2020-2021 in Okinawa Main Island. [ABSTRACT FROM AUTHOR]

The study was designed to investigate the effects of Sparassis latifolia polysaccharides(SLPs) on skeletal calcium metabolism in lead-contaminated mice. The mice were randomly divided into control group, lead group, low-dose SLPs group, medium-dose SLPs group, and high-dose SLPs group. After 8 weeks of feeding, the bones were collected to determine the contents of divalent metal elements by inductively coupled plasma emission spectrometer, the contents of parathyroid hormone (PTH), bone glaprotein(BGP), calcitonin(CT), and the activity of alkaline phosphatase(AKP) by enzyme-linked immunosorbent assay, observe morphology of mouse femur tissue by HE staining, and detect the expression levels of calcium metabolism related genes in bones by real-time fluorescence quantitative polymerase chain reaction. The results showed that compared with the control group, the contents of Pb2+, PTH and the expression levels of PMCA1b mRNA in bones in the lead group were significantly increased, while the contents of Ca2+, Fe2+, Cu2+, Mn2+, BGP, and CT, the AKP activity, and the expression levels of TRPV5, TRPV6, and NCX1 mRNA were significantly decreased. The distribution of bone trabecula was disordered and lacunae appeared at the margin of bone trabecula in lead group. Compared with the lead group, the Fe2+ content was significantly increased in the medium-dose SLPs group. The contents of Pb2+, PTH and the expression levels of PMCA1b mRNA in high-dose SLPs group were significantly decreased, while the contents of Ca2+ and CT, AKP activity, and the expression levels of TRPV5 and TRPV6 mRNA were significantly increased. In the high-dose SLPs group, the bone trabecular was more evenly distributed and the margin lacunae of bone trabeculae was less. It indicated that SLPs could improve skeletal imbalance of calcium and iron elements induced by lead in mice by promoting the excretion of lead, reducing the PTH level in bones, increasing the CT content and the AKP activity in bones, alleviate the damage of lead on morphological structures in bone tissues, and regulate the expression of calcium metabolism related genes to further cause a regulatory effect on skeletal calcium metabolism disorder induced by lead in mice. [ABSTRACT FROM AUTHOR]

Cordyceps farinosa, an entomopathogenic fungus, infects and leads to high mortality of Thitarodes armoricanus larvae, which die soon after the infection of C. farinose, usually before the colonization of Ophiocordyceps sinensis owing to competitive inhibition and fruiting body formation. Therefore, monitoring C. farinosa in the O. sinensis cultivation environment is critical for minimizing the C. farinosa infection-induced losses. In this study, we initially designed a PCR primer pair (Tar-1F/Tar-1R) through open reading frame prediction and homology comparison of the C. farinosa genome sequence. This primer pair can detect both C. farinosa and Samsoniella hepiali. To further distinguish, primers (ITS5-172/ITS4-95) were then designed to selectively amplify the large ribosomal subunit sequences in the C. farinosa genome. All these primers were applied in combination for detection of C. farinosa in soil samples. The sensitivity reached a detection limit of 1 × 106 spores/g soil. In addition, these primers can detect the presence of C. farinosa in dead T. armoricanus larval samples. This newly established rapid detection method provides important information for C. farinosa control during O. sinensis cultivation. [ABSTRACT FROM AUTHOR]

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Simple Summary: Transmission of Mycobacterium avium subspecies paratuberculosis (MAP) from infected adult cattle to susceptible calves occurs mainly through contamination of the calf environment, i.e., the calving pen and the pre-weaned calf rearing area, with adult faeces. Control programmes for Johne's disease utilise a risk assessment and management plan (RAMP) to evaluate the risk of transmission of infection on a specific farm, however the assessment is limited by its subjective, point-in-time nature. The objective of this study was to evaluate a novel environmental sampling protocol for measurement of MAP contamination of the calf environment on infected farms. Across 28 infected farms, 46% had detectable MAP in the calf environment, with 21% of farms having detectable MAP in the pre-weaned calf area. There was no significant association found between the RAMP scores for each farm and the result of environmental testing for MAP, and there was a moderate correlation found between RAMP scores and the level of MAP contamination as quantified by PCR. We conclude that environmental sampling is a potentially useful tool to objectively measure transmission risk in the calf environment on farms, as a complement to the annual RAMP. One of the pillars of Johne's disease control is to break the cycle of transmission from infectious adult animals to young susceptible animals. Many control programmes utilise a risk assessment and management plan (RAMP) to identify specific risks for transmission of infection on individual farms and then recommend tailored biosecurity measures to mitigate the risk. It is important that infected farms in particular, practice effective biocontainment in the calving area and pre-weaned calf area. The objectives of this study were: (1) to determine what proportion of MAP-infected farms (PCR-positive) enrolled in a control programme had detectable MAP in their calf environment; (2) to compare RAMP scores and apparent within-herd prevalence (aWHP) of PCR-positive and PCR-negative farms; (3) to evaluate the correlation between RAMP scores, aWHP and levels of contamination based on PCR test Ct value. A novel environmental sampling protocol combining manure samples and boot swab samples was conducted in the calving area and pre-weaned calf area on 28 dairy farms with PCR-confirmed MAP infection. All samples were tested with PCR. Logistic regression modelling was used to evaluate the association between RAMP score and aWHP and PCR outcome. Overall, 46% of farms had positive PCR samples in either the calving area, pre-weaned calf area or both. The calving area was positive in 36% of farms and the pre-weaned calf area was positive in 21% of farms. There was a moderate, negative correlation (r = −0.45; p = 0.08) observed between RAMP scores and Ct value at the sample-level that slightly missed the required significance level. No significant association was identified between RAMP scores or aWHP and PCR test outcome (positive or negative). It was concluded that environmental sampling of the calving area and pre-weaned calf area has potential applications in the assessment of transmission risk on infected farms and could be used to monitor the efficacy of biosecurity measures over time. Further research conducted on a larger scale is required to support these results. [ABSTRACT FROM AUTHOR]

Almond witches'-broom (AlmWB) is a serious phytoplasma disease of stone fruits; thus far reported from Iran and Lebanon. In the present study the host range of two Iranian AlmWB phytoplasma strains, known as PEAB and AlKI, were examined through graft-inoculation assay. Two-year-old stone fruit seedlings of almond (three cultivars), peach, plum, Myrobalan plum, apricot and sweet cherry were separately graft-inoculated by scions obtained from symptomatic almond seedlings infected by PEAB or AlKI phytoplasma strains. The presence of phytoplasma in the inoculated seedlings, and the type and development of symptoms were evaluated and recorded monthly over one year. Results showed that both phytoplasma strains could be transmitted to almond ('Pooya', 'Mamaei' and 'Talkhe' cultivars), peach and plum seedlings. The disease symptoms were more obvious in almond cultivars. AlmWB phytoplasma strains could not be detected in the inoculated sweet cherry and the Myrobalan plum seedlings. On the other hand, infection of apricot seedlings could only be detected in seedlings inoculated with the PEAB strain. The homologous of the AYWB SAP11 effector protein, known to play a crucial role in phytoplasma infection and symptom expression, was sequence-analyzed in both PEAB and AlKI strains. The SAP11 nucleotide sequence of AlKI strain was identical to that of the SA213 AlmWB phytoplasma strain from Lebanon. Analysis showed high SAP11 sequence diversity between AlmWB phytoplasmas and phytoplasma strains of other 16Sr groups. Despite poor sequence conservation, the structural features and the motifs of SAP11 protein were conserved between AlmWB and distantly related phytoplasma strains; speculating a similar function for this gene in diverse phytoplasmas. [ABSTRACT FROM AUTHOR]

In April 2023, a substantial mortality event struck the primary Nile tilapia (Oreochromis niloticus) breeding areas in Maoming City, Guangdong Province, resulting in considerable economic losses. To unravel the etiology of this disease outbreak, thorough farm field necropsies were conducted, encompassing detailed descriptions of gross clinical signs and diagnostic sample collection. Clinically, affected tilapia primarily manifested multifocal superficial skin ulcers, mild focal congestion, and hemorrhage in the jaws, eyes' irises, and other integumentary regions. Tissue specimens from diseased fish were screened for Francisella spp. , using a polymerase chain reaction (PCR) assay. The PCR product targeting polymerase gene showed 99.64 % nucleotide sequence identity to the published sequence of Francisella noatunensis subsp. orientalis (F. noatunensis subsp. orientalis). Histopathological examination of selected tissues revealed acute to subacute, severe necrotizing bacterial splenitis accompanied by diffuse vasculitis, marked necrotizing nephritis, mild necrotizing hepatitis, and prominent proliferative vasculitis. Transmission electron microscopy (TEM) further demonstrated that F. noatunensis subsp. orientalis was predominantly found in macrophage cytoplasm, appearing as round, spheroidal bacteria. Koch's postulates were successfully confirmed through challenge testing. Moreover, F. noatunensis subsp. orientalis exhibited high sensitivity to Gentamicin, Vancomycin, Minocycline, Imipenem, and Ceftazidime. In conclusion, F. noatunensis subsp. orientalis has emerged as a significant pathogen affecting Nile tilapia in China, characterized by systemic multi-organ necrotizing vasculitis with the spleen and head kidney being the primary targets. • The isolation and characterization of a new pathogen of Nile tilapia in Guangdong, China. • F. noatunensis subsp. orientalis is the pathogen causing disease and mortality in Nile tilapia. • The spleen and head kidney are the main target organs of F. noatunensis subsp. orientalis infection. • It may be a serious infection disease in this region of China. [ABSTRACT FROM AUTHOR]

Background: The accurate occurrence rate of C. auris infections is still not clear, mainly due to the defects in detection and identification tools routinely used. In this study, we used conventional PCR and real‐time PCR assays for sensitive and specific detection/identification of C. auris from either yeast isolates or clinical specimens collected from various patients in different parts of Iran. Our survey is the first large‐scale study rating the incidence of C. auris infections in Iran. Methods: A total of 439 yeast isolates and 590 clinical specimens were screened by specific C. auris‐PCR, targeting the ITS region. The validity of positive samples was assessed by sequencing. Results: Four out of 590 clinical specimens (0.68%) were positive by conventional PCR, while in real‐time PCR performed on 100 clinical samples, including those four samples positive in conventional samples, 6 samples were positive. A complete agreement of the identification of positive cases with sequencing results was documented. Among 439 culture isolates, none was positive for C. auris. After following up and resampling of the patients with positive PCR, only one specimen showed positive culture for C. auris, which was confirmed by sequencing. Conclusion: C. auris is not a common cause of systemic or superficial fungal infections in Iran, and a few detected positive cases can be considered as a commensal, coloniser or infecting yeast which may potentially emerge in some clinical and therapeutical conditions. Mycological and phenotypical assays are not sensitive approaches for isolation/identification of C. auris, unless a specific and sensitive molecular‐based method is applied. [ABSTRACT FROM AUTHOR]

Campylobacter, a major cause of food‐borne gastroenteritis worldwide, colonize the gastrointestinal tract of a wide range of animals, being birds the main reservoir. The mechanisms involved in the interaction of Campylobacter with the different hosts are poorly understood. The cytolethal distending toxin, encoded in the cdtABC operon, is considered a pivotal virulence factor during human infection. Differences in the prevalence of cdtABC genes in Campylobacter isolates from three distinct origins (wild birds, broiler chickens and humans) prompted us to further characterize their allelic variability. The sequence of cdtABC is highly conserved among broiler and human isolates. A high diversity of cdtABC alleles was found among wild bird isolates, including several alleles that do not produce any functional CDT. These results suggest that specific variants of the cdtABC operon might define the host range of specific Campylobacter jejuni isolates. Moreover, our data indicate that PCR methodology is inaccurate to characterize the prevalence of the cdt genes, since negative PCR detection can be the result of divergences in the sequence used for primer design rather than indicating the absence of a specific gene. [ABSTRACT FROM AUTHOR]

The ongoing circulation of SARS‐CoV‐2 variants of concern (VOCs) has caused global concerns, because VOCs could escape current vaccines, antiviral drugs, and diagnosis. Analyzing mutations and intra‐host diversities in different and widespread VOCs can provide important insights to virus adaptive evolution and validity of vaccines, antiviral drugs, and diagnosis. In this study, by analyzing 1744 high‐throughput sequencing data for intra‐host single‐nucleotide variations (iSNVs) and 3,668,205 genome sequences for mutations in different VOCs, it was found that Omicron variant is still evolving at high speed, especially having high iSNVs frequency in its S and N genes. The efficacies of antibodies or detection primers targeting these two genes are at high risks to be invalid. Instead, highly conserved regions such as NSP8 gene could be better therapeutic and detection targets. Furthermore, mutations in later VOCs could be traced to the minor alleles in the previous variant samples such as Alpha and Delta in different countries. Finally, it was found that mutations C14408T in RdRp and A18163G in NSP14 gene might be associated with the higher genetic diversity in Omicron. Our findings not only contribute to understanding the adaptive evolution of SARS‐CoV‐2 VOCs, but also provide useful information for both drugs and diagnostic kits development. [ABSTRACT FROM AUTHOR]

Pyrenophora tritici-repentis (Ptr), family Pleosporaceae, is a common wheat pathogen in all wheat-growing regions around the globe. It is widely studied in North America, South America, and North Africa, while data about the fungus genetic diversity in Europe is still insufficient. This study aimed to describe the variation of morphological traits and toxin production of strains collected in Latvia and Belarus. Twenty-one isolates from Latvia, and 12 from Belarus were sampled in 2019 for morphological evaluation in culture and necrotrophic effector gene determination by PCR. All isolates were grouped into nine different morphotypes. Five of these morphotypes were unique for isolates from Latvia, one for Belarus, and three morphotypes were occurring in both countries. No association between the host and the pathogen morphotype was observed. ToxA gene was detected in 44% of the analysed isolates. For 52% of the isolates, PCR did not confirm the presence of any known effector genes of Pyrenophora tritici-repentis. ToxB and toxb were found only in one isolate from Latvia. The studies need to be continued to evaluate the diversity of the pathogen depending on the host species. [ABSTRACT FROM AUTHOR]

Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection. [ABSTRACT FROM AUTHOR]

Abstract The ongoing circulation of SARS‐CoV‐2 variants of concern (VOCs) has caused global concerns, because VOCs could escape current vaccines, antiviral drugs, and diagnosis. Analyzing mutations and intra‐host diversities in different and widespread VOCs can provide important insights to virus adaptive evolution and validity of vaccines, antiviral drugs, and diagnosis. In this study, by analyzing 1744 high‐throughput sequencing data for intra‐host single‐nucleotide variations (iSNVs) and 3,668,205 genome sequences for mutations in different VOCs, it was found that Omicron variant is still evolving at high speed, especially having high iSNVs frequency in its S and N genes. The efficacies of antibodies or detection primers targeting these two genes are at high risks to be invalid. Instead, highly conserved regions such as NSP8 gene could be better therapeutic and detection targets. Furthermore, mutations in later VOCs could be traced to the minor alleles in the previous variant samples such as Alpha and Delta in different countries. Finally, it was found that mutations C14408T in RdRp and A18163G in NSP14 gene might be associated with the higher genetic diversity in Omicron. Our findings not only contribute to understanding the adaptive evolution of SARS‐CoV‐2 VOCs, but also provide useful information for both drugs and diagnostic kits development.

The highly degraded DNA content in processed food samples results in limited efficiency in detecting GMOs. Generally, conventional DNA isolation techniques from transgenic plant seeds or raw materials were available in the literature, whereas studies on DNA isolation techniques from processed food samples were more limited. Also, many processed food products contain genomic DNA from numerous complex plants or animal sources. In the present study, we proposed some beneficial modifications for high-quality DNA isolation of processed foods such as biscuits, cakes, crackers, corn chips, and flours. For this purpose, isolation protocols were investigated to obtain high molecular weight and quality DNA from food samples, the first step of GMO analysis in processed foods. To control the gene region of the target organism from the obtained DNA samples, PCR detection was performed with soybean and maize-specific primers. According to the statistical analysis, the A260/A280 ratios were the lowest in cake (1.58) and and highest in biscuit (1.83). The highest values of the total DNA presence belong to soy flour samples (211.80 ug/ul), and the lowest amount belongs to corn flour, cake, and corn chips samples Among the four isolation methods tested, the modified Wizard-CTAB method showed better results in most of the tested food products. Results showed that the modified Wizard- CTAB could be used in different food products for studies on corn and soybean specific genes and GMO detection. [ABSTRACT FROM AUTHOR]

Fusarium wilt of cotton, caused by the soilborne fungus Fusarium oxysporum f.sp. vasinfectum race 4 (Fov4, VCG0114), is a serious disease that has adversely affected cotton production in California since 2001 and in the Upper Rio Grande Region near El Paso in the border areas between Texas and New Mexico since 2017. Fov4 was isolated from cotton stem samples collected from wilted or asymptomatic plants adjacent to the wilting plants from the El Paso area. The MT genotype was the only Fov4 genotype found in two fields in the Lower Valley of El Paso and the T genotype was the only Fov4 genotype found in one field in the Upper Valley Region of El Paso. No N, MiT or mixed genotypes of Fov4 were detected in any of these fields. Furthermore, the F. oxysporum f.sp. vasinfectum isolates were exclusively Fov4. The results support the observation that the MT genotype of Fov4 is mainly distributed in the Lower Valley of El Paso and the T genotype of Fov4 is mainly distributed in the Upper Valley Region of El Paso. Virulence of the MT genotype of Fov4 was higher than that of the T genotype. PCR methods previously developed for Fov4 specific PCR detection and multiplex Fov4 genotyping were validated by VCG analyses of the newly obtained isolates. These PCR methods were adapted for the in planta detection of Fov4, but Fov4 genotyping was limited to the detection of T, MT and MiT genotypes. It is important to continue to monitor dynamic population structure of Fov4 genotypes to prevent the spread of Fov4 and optimize disease control. [ABSTRACT FROM AUTHOR]

Bovine respiratory disease (BRD) is a very important disease that contributes to economic losses in dairy and beef cattle breeding worldwide. The molecular testing of material from 296 calves showing BRD symptoms from 74 dairy herds located in south-western Poland was performed in 2019–2021. Molecular tests were performed using a commercial kit "VetMAXTM Ruminant Respiratory Screening Kit" (Thermo Fisher Scientific) for the simultaneous detection of genetic material of seven pathogens responsible for BRD. At least one pathogen was detected in 95.95% of herds. The overall prevalence was: Pasteurella multocida 87.84%, Mannheimia haemolytica 44.59%, bovine coronavirus (BcoV) 32.43%, Mycoplasma bovis 29.73%, Histophilus somni 28.38%, bovine parainfluenza virus type 3 (BPIV-3) 13.51%, and bovine respiratory syncytial virus (BRSV) 10.81%. Twenty-nine configurations of pathogen occurrences were found. Bacterial infections were the most frequently recorded as 56.7% of all results. Coinfections mainly consisted of two pathogens. Not a single purely viral coinfection was detected. The most frequent result was a single P. multocida infection accounting for 18.31% of all results. The statistically significant correlation (p = 0.001) with the highest strength of effect (ϕ 0.38) was between M. bovis and H. somni. [ABSTRACT FROM AUTHOR]

In radiation-resistant bacteria belonging to the genus Deinococcus , transposition events of insertion sequences (IS elements) leading to phenotypic changes from a reddish color to white were detected following exposure to gamma irradiation and hydrogen peroxide treatment. This change resulted from the integration of IS elements into the phytoene desaturase gene, a key enzyme in the carotenoid biosynthesis pathway. To facilitate species identification and distinguish among Deinococcus strains, the gyr B gene encoding the B subunit of DNA gyrase was utilized. The s gnificance of the gyr B gene is well recognized not only in genome replication through the regulation of supercoiling but also in phylogenetic analysis providing support for 16S rRNA-based identification. Its mutation rate surpasses that of the 16S rRNA gene, offering greater resolution between closely related species, particularly those exhibiting >99% similarity. In this study, phylogenetic analysis was conducted comparing the 16S rRNA and gyr B gene sequences of Deinococcus species. Species-specific and genus-specific primers targeting Deinococcus species were designed and experimentally validated for selective amplification and rapid identification of the targeted species. This approach allows for the omission of 16S rRNA sequencing in the targeted Deinococcus species. Therefore, the gyr B gene is useful for identifying bacterial species and genus-level detection from individual microbes or microbial consortia using specialized primer sets for PCR amplification. [Display omitted] • This manuscript has three important aspects following: firstly, the gyr B primer sets proved invaluable for the rapid and straightforward genus- and species-specific identification of Deinococcus strains, distinguishing them from bacteria of other genera. • Secondly, the phylogenetic tree exhibits higher resolution within gyr B gene than 16S rRNA of over 99% sequence identical Deinococcus genus. • Thirdly, exploring evolutionary aspects within the bacterial gyr B gene of the Deinococcus genus and other bacteria based on sequence variation and available structure models. [ABSTRACT FROM AUTHOR]

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method. [ABSTRACT FROM AUTHOR]

The present study was designed with the aim of isolation and identification of Salmonella by conventional culture method and their confirmation by polymerase chain reaction (PCR). Antibacterial sensitivity study of isolated Salmonella from cattle faeces was also performed. During the study period of July 2017 to June 2018, a total of 200 faecal samples were collected from different government and private farms in Sylhet district of Bangladesh. Out of 200 samples, 24 (12%) were found to be positive for Salmonella by conventional culture methods. Among the twenty four suspected colonies of Salmonella, seventeen were confirmed by biochemical test and same number was detected by PCR estimating a prevalence of 8.5% (17/200). The prevalence was higher in calves under 1 year of age (16%) compared with older animals (11.25% of 1–2 years; 10% of above 2 years of age) but without statistically significant differences (χ2=4.835, P=0.089). Moreover, in diarrhoeic animals the preva-lence was significantly higher (32.14%, χ2=49.414, P

Abstract Microbial degradation of recalcitrant alkanes under anaerobic conditions results in the accumulation of heavy oil fraction in oil reservoirs. Hydroxylation of alkanes is an important activation mechanism under anaerobic conditions, but the diversity and distribution of the responsible microorganisms in the subsurface environment are still unclear. The lack of functional gene polymerase chain reaction (PCR) primers and commercially available intermediate degradation chemical compounds are the major obstacles for this research. In this investigation, PCR primers for the ahyA gene (encoding alkane hydroxylase) were designed, evaluated, and improved based on the nucleotide sequences available. Using microbial genomic DNA extracted from oil-contaminated soil and production water samples of oil reservoirs, ahyA gene nucleotide sequences were amplified and retrieved successfully from production water sample Z3-25 of Shengli oilfield. Additionally, the signature biomarker of 2-acetylalkanoic acid was detected in both Shengli and Jiangsu oilfields. These results demonstrate that anaerobic hydroxylation is an active mechanism used by microorganisms to degrade alkanes in oxygen-depleted oil reservoirs. This finding expands the current knowledge of biochemical reactions about alkane degradation in subsurface ecosystems. In addition, the PCR primers designed and tested in this study serve as an effective molecular tool for detecting the microorganisms responsible for anaerobic hydroxylation of alkanes in this and other ecosystems.

Human tissue samples soaked in formaldehyde solution are rare resources in many cases, and the extraction of their DNA is a challenging issue. In this paper, genomic DNA was extracted from four formalin soaked human embryo skin and muscle samples by using an improved method for extracting genomic DNA from samples soaked in formaldehyde solution and a generic genomic DNA extraction kit. The purity and concentration of extracted DNA were determined by UV spectrophotometry, and the DNA solution was subjected to agarose gel electrophoresis and imaging. The results showed that the concentration and purity of genomic DNA obtained by the improved extraction method were better than those obtained by the general method. PCR amplification was carried out with the extracted genomic DNA template. Electrophoresis results showed that the improved extraction method had better PCR amplification effect. Our study provides methodological guidance and a reference for the direction of improvement for the extraction of genomic DNA from precious samples soaked in formaldehyde solution [ABSTRACT FROM AUTHOR]

Powdery mildew is a serious disease of economically important hazelnut crop in Turkey. Hazelnut production has been extremely affected by the disease in terms of quality and quantity. The disease is caused by two different fungi, namely Erysiphe corylacearum and Phyllactinia guttata. E. corylacearum has been shown to be the responsible one predominantly for the recent economic damage. The fungi produce a mycelium network on hazelnut plants before they sporulate and visually detected. Early detection of these pathogens is important for management as well as understanding their spread and epidemics. In this study, a PCR assay was developed for the detection of both pathogens from hazelnut plant leaves by targeting their ribosomal DNA genes in their internal transcribed spacer (ITS) regions. Two sets of specific primers were designed for the detection of E. corylacearum and P. guttata at an early stage of infection. As a result of PCR, a specific band of 578 bp was observed. The amplicon sequencing confirmed the presence of only E. corylacearum, but not P. guttata. Therefore, this PCR-based test can identify plants that are infected with powdery mildew before they show any visual signs. From there, the infected plants can be treated or removed before the fungus has a chance to produce spores that infect neighboring plants. These results would help tackle the eradication of powdery mildew.

Abstract Background Most lactobacilli found in animal intestines are generally non-motile, but there are few exceptions. Our previous work showed that Lactobacillus agilis BKN88, which is a highly motile strain originating from a chicken, takes advantage of motility in gut colonization in murine models, and thus motile lactobacilli likely have unique ecological characteristics conferred by motility. However, the ecology and habitat of gut-derived motile lactobacilli are still rarely understood. In addition, the limited availability of motile Lactobacillus isolates is one of the major obstacles for further studies. To gain insight into the ecology and habitat of the motile lactobacilli, we established a routinely applicable detection method for motile lactobacilli using PCR and subsequent selective isolation in semi-solid MRS medium for the collection of additional motile lactobacilli from animal feces. Results We applied the PCR detection using motile lactobacilli-specific primers, based on the motor switch protein gene (fliG) of flagella, to 120 animal feces, followed by selective isolation performed using 45 animal feces. As a result, motile lactobacilli were detected in 44 animal feces. In the selective isolation, 29 isolates of L. agilis and 2 isolates of L. ruminis were obtained from 8 animal species. Conclusions These results indicated that motile lactobacilli are distributed in different animal species. Moreover, phylogenetic analysis of the L. agilis isolates suggests co-evolution with the host, and adaptation to a particular environmental niche.

Over the last years, an increasing rate of ixodes tick bites has been registered in the northern regions of the European Russia. In addition, the number of subjects request medical assistance due to tick bites has been dramatically increased in the Komi Republic. In addition, incidence of tick-borne encephalitis was also increased particularly starting since 2009. However, highly limited data on pathogen genetic diversity related to viral tick-borne infections in this region are currently available. Taiga ticks (Ixodespersulcatus) collected from the Komi Republic southern and central part vegetation were examined to identify and genotype tick-borne viruses. Individual ticks were used to identify by RT-PCR viral RNA coupled to tick-borne encephalitis and Kemerovo viruses. Viral genome fragment sequencing allowed to unambiguously identify these viruses. It was found that viral RNA tick-borne encephalitis was detected in 6.8±1.2% individual ticks. Moreover, tick-linked isolate genotyping based on analyzing E protein gene fragment nucleotide sequence derived from tick-borne encephalitis discovered that 35% and 65% isolates belonged to the Far Eastern and Siberian subtype, respectively. In addition, subsequent phylogenetic analysis demonstrated that at least four variants of the Siberian and Far Eastern subtypes of tick-borne encephalitis virus were detected, which were close to the viruses circulating in the Urals and Siberia. In contrast, prevalence of Kemerovo virus in taiga ticks was 0.8±0.2%. Sequencing of Kemerovo virus RNA-dependent RNA polymerase gene fragment showed around 94% homology with the remainder of the Kemerovo virus strains. Phylogenetic analysis of the Kemerovo virus genome fragments demonstrated at least two subtypes circulating in the Komi Republic. Thus, it was suggested that tick-borne encephalitis virus was introduced relatively recently from the Urals and Siberian region into the natural foci of the Komi Republic. Moreover, genetic differences found in Kemerovo virus strains presume for them a longer lasting evolution throughout the natural foci of this region. In addition, a potential role for birds and their ticks in rapid spreading of viral tick-borne infections in the Komi Republic is also discussed. Thus, the data on genetic diversity of the viral agents related to tick-born encephalitis and Kemerovo fever may be useful for improving their diagnostics, prevention and treatment in the Komi Republic.

The mangrove forest is located in the coastal intertidal zone and plays a vital role in buffering against erosion from storm surges, currents, waves, and tides. This ecosystem also serves for ecotourism due to its heterogeneous habitats, which supported a high diversity of plants and animals. Small mammals are among the abundant but poorly recognised inhabitants in mangrove forests. Apart from their role as primary consumer and prey, they are host to various ectoparasites, which potentially become the primary vector for tick-borne diseases among visitors. Therefore, this study aims to determine the small mammal assemblages and their ectoparasite prevalence in mangroves forests of Peninsular Malaysia. The capture-release method was applied for small mammal sampling, and each captured individual was screened and collected for ectoparasites before released. DNA barcoding using CO1 genes was performed for small mammal species verification, whereby ectoparasite identification was based on morphological identification and molecular verification using 16S rDNA genes. A total of 94 small mammals from 6 species were captured across sites, dominated by Rattus tiomanicus (n=74). From this number, 15 individuals (16% prevalence) were infested by ticks, 22 individuals (23%) were infested by mites, whereas 4 individuals (4%) were infested by both ticks and mites. Five species of ticks and one mite species were identified; Amblyomma cordiferum, Ixodes granulatus, Haemaphysalis hystricis, Dermacentor auratus, Dermacentor atrosignatus, and Laelaps echidninus. This study reveals a relatively poor diversity of small mammals in the mangroves forest, of which highly infested with a diversity of ectoparasites, elucidating the relationship of hostectoparasite associations in the riparian zone. This information is crucial to inform visitors to these areas, ultimately safeguard against ectoparasite-borne disease.

[Objectives] This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana. [Methods] With sterilized tube seedling leaves as the recipient material and GBP as the reporter gene, optimization experiments were carried out in terms of infection time and method, co-cultivation time and method, and PCR detection technology. [Results] The transformation effect was better under the conditions of shaking Agrobacterium liquid, infection time of 1 -2 h, and co-cultivation on sterilized filter paper for 2 d. After co-cultivation, the recipient material was first subjected to recover}' culture, and then used for Hyg gradient screening, which was conducive to obtaining resistant transformants. The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants, and the proportion of transgenic plants was 16.7%. [Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants. [ABSTRACT FROM AUTHOR]