Background: Intestinal parasitic infections are still a serious public health problem in developing countries, and the diagnosis of parasitic infections requires the first step of parasite/egg detection of samples. Automated detection can eliminate the dependence on professionals, but the current detection algorithms require large computational resources, which increases the lower limit of automated detection. Therefore, we have designed a lightweight deep-learning model, YAC-Net, to achieve rapid and accurate detection of parasitic eggs and reduce the cost of automation.
Methods: This paper uses the ICIP 2022 Challenge dataset for experiments, and the experiments are conducted using fivefold cross-validation. The YOLOv5n model is used as the baseline model, and then two improvements are made to the baseline model based on the specificity of the egg data. First, the neck of the YOLOv5n is modified to from a feature pyramid network (FPN) to an asymptotic feature pyramid network (AFPN) structure. Different from the FPN structure, which mainly integrates semantic feature information at adjacent levels, the hierarchical and asymptotic aggregation structure of AFPN can fully fuse the spatial contextual information of egg images, and its adaptive spatial feature fusion mode can help the model select beneficial feature and ignore redundant information, thereby reducing computational complexity and improving detection performance. Second, the C3 module of the backbone of the YOLOv5n is modified to a C2f module, which can enrich gradient information, improving the feature extraction capability of the backbone. Moreover, ablation studies are designed by us to verify the effectiveness of the AFPN and C2f modules in the process of model lightweighting.
Results: The experimental results show that compared with YOLOv5n, YAC-Net improves precision by 1.1%, recall by 2.8%, the F1 score by 0.0195, and mAP_0.5 by 0.0271 and reduces the parameters by one-fifth. Compared with some state-of-the-art detection methods, YAC-Net achieves the best performance in precision, F1 score, mAP_0.5, and parameters. The precision, recall, F1 score, mAP_0.5, and parameters of our method on the test set are 97.8%, 97.7%, 0.9773, 0.9913, and 1,924,302, respectively.
Conclusions: Compared with the baseline model, YAC-Net optimizes the model structure and simplifies the parameters while ensuring the detection performance. It helps to reduce the equipment requirements for performing automated detection and can be used to realize the automatic detection of parasite eggs under microscope images.
{"title":"A lightweight deep-learning model for parasite egg detection in microscopy images.","authors":"Wenbin Xu, Qiang Zhai, Jizhong Liu, Xingyu Xu, Jing Hua","doi":"10.1186/s13071-024-06503-2","DOIUrl":"10.1186/s13071-024-06503-2","url":null,"abstract":"<p><strong>Background: </strong>Intestinal parasitic infections are still a serious public health problem in developing countries, and the diagnosis of parasitic infections requires the first step of parasite/egg detection of samples. Automated detection can eliminate the dependence on professionals, but the current detection algorithms require large computational resources, which increases the lower limit of automated detection. Therefore, we have designed a lightweight deep-learning model, YAC-Net, to achieve rapid and accurate detection of parasitic eggs and reduce the cost of automation.</p><p><strong>Methods: </strong>This paper uses the ICIP 2022 Challenge dataset for experiments, and the experiments are conducted using fivefold cross-validation. The YOLOv5n model is used as the baseline model, and then two improvements are made to the baseline model based on the specificity of the egg data. First, the neck of the YOLOv5n is modified to from a feature pyramid network (FPN) to an asymptotic feature pyramid network (AFPN) structure. Different from the FPN structure, which mainly integrates semantic feature information at adjacent levels, the hierarchical and asymptotic aggregation structure of AFPN can fully fuse the spatial contextual information of egg images, and its adaptive spatial feature fusion mode can help the model select beneficial feature and ignore redundant information, thereby reducing computational complexity and improving detection performance. Second, the C3 module of the backbone of the YOLOv5n is modified to a C2f module, which can enrich gradient information, improving the feature extraction capability of the backbone. Moreover, ablation studies are designed by us to verify the effectiveness of the AFPN and C2f modules in the process of model lightweighting.</p><p><strong>Results: </strong>The experimental results show that compared with YOLOv5n, YAC-Net improves precision by 1.1%, recall by 2.8%, the F1 score by 0.0195, and mAP_0.5 by 0.0271 and reduces the parameters by one-fifth. Compared with some state-of-the-art detection methods, YAC-Net achieves the best performance in precision, F1 score, mAP_0.5, and parameters. The precision, recall, F1 score, mAP_0.5, and parameters of our method on the test set are 97.8%, 97.7%, 0.9773, 0.9913, and 1,924,302, respectively.</p><p><strong>Conclusions: </strong>Compared with the baseline model, YAC-Net optimizes the model structure and simplifies the parameters while ensuring the detection performance. It helps to reduce the equipment requirements for performing automated detection and can be used to realize the automatic detection of parasite eggs under microscope images.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539555/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142583517","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-06DOI: 10.1186/s13071-024-06526-9
Ilaria Bernardini, Cristiana Poggi, Daniele Porretta, Jan Máca, Eleonora Perugini, Sara Manzi, Simona Gabrielli, Verena Pichler, Maria Stefania Latrofa, Josephus Fourie, Riccardo Paolo Lia, Frédéric Beugnet, Domenico Otranto, Marco Pombi
Background: Five species of the Phortica genus (Diptera: Drosophilidae) are known in Europe and the Middle East. Among these, Phortica variegata and Phortica okadai are better known for their role as vectors of the zoonotic eyeworm Thelazia callipaeda. Other species, such as Phortica semivirgo and Phortica oldenbergi, have been studied less. Given the paucity of data about these Phortica spp. vectors, we explored the population dynamics and ecology of Phortica spp. in an area highly endemic for T. callipeada (Manziana, Rome, Central Italy).
Methods: Phortica spp. flies were collected over a 3-year period (2018-2020) during their active season (April-October) with a sweep net while hovering around fermenting fruits or a human operator acting as baits. Collected flies were morphologically identified and tested for a T. callipeada infection and for the presence of Wolbachia, by polymerase chain reaction (PCR). Population dynamics of species collected was associated to environmental drivers through generalized additive models.
Results: Of the 5564 flies collected, 90.8% were P. variegata, 9.1% were P. oldenbergi, 0.05% were P. semivirgo, and one specimen was P. okadai. Only P. variegata scored molecularly infected with T. callipeada throughout the 3-year sampling period (1.8%). Phortica oldenbergi, observed consistently during the entire sampling period, exhibited a marked preference for fruit traps, contrasting with the lachryphagous activity of P. variegata. Analysis of environmental drivers of P. oldenbergi and P. variegata population dynamics indicated temperature, wind speed, and pressure as significant factors. In addition, Wolbachia pipientis endosymbiont was detected in P. oldenbergi and P. okadai.
Conclusions: For the first time, this study analysed several ecological aspects of Phortica species coexisting in a T. callipeada endemic area, highlighting different behaviors in the same environment and their vectorial role. Notably, this is also the first report of the presence of P. oldenbergi in Italy and P. okadai in Europe, underscoring the importance of extensive sampling for detecting potential vectors and alien species with direct implications for vector-borne disease epidemiology.
{"title":"Population dynamics of sympatric Phortica spp. and first record of stable presence of Phortica oldenbergi in a Thelazia callipaeda-endemic area of Italy.","authors":"Ilaria Bernardini, Cristiana Poggi, Daniele Porretta, Jan Máca, Eleonora Perugini, Sara Manzi, Simona Gabrielli, Verena Pichler, Maria Stefania Latrofa, Josephus Fourie, Riccardo Paolo Lia, Frédéric Beugnet, Domenico Otranto, Marco Pombi","doi":"10.1186/s13071-024-06526-9","DOIUrl":"10.1186/s13071-024-06526-9","url":null,"abstract":"<p><strong>Background: </strong>Five species of the Phortica genus (Diptera: Drosophilidae) are known in Europe and the Middle East. Among these, Phortica variegata and Phortica okadai are better known for their role as vectors of the zoonotic eyeworm Thelazia callipaeda. Other species, such as Phortica semivirgo and Phortica oldenbergi, have been studied less. Given the paucity of data about these Phortica spp. vectors, we explored the population dynamics and ecology of Phortica spp. in an area highly endemic for T. callipeada (Manziana, Rome, Central Italy).</p><p><strong>Methods: </strong>Phortica spp. flies were collected over a 3-year period (2018-2020) during their active season (April-October) with a sweep net while hovering around fermenting fruits or a human operator acting as baits. Collected flies were morphologically identified and tested for a T. callipeada infection and for the presence of Wolbachia, by polymerase chain reaction (PCR). Population dynamics of species collected was associated to environmental drivers through generalized additive models.</p><p><strong>Results: </strong>Of the 5564 flies collected, 90.8% were P. variegata, 9.1% were P. oldenbergi, 0.05% were P. semivirgo, and one specimen was P. okadai. Only P. variegata scored molecularly infected with T. callipeada throughout the 3-year sampling period (1.8%). Phortica oldenbergi, observed consistently during the entire sampling period, exhibited a marked preference for fruit traps, contrasting with the lachryphagous activity of P. variegata. Analysis of environmental drivers of P. oldenbergi and P. variegata population dynamics indicated temperature, wind speed, and pressure as significant factors. In addition, Wolbachia pipientis endosymbiont was detected in P. oldenbergi and P. okadai.</p><p><strong>Conclusions: </strong>For the first time, this study analysed several ecological aspects of Phortica species coexisting in a T. callipeada endemic area, highlighting different behaviors in the same environment and their vectorial role. Notably, this is also the first report of the presence of P. oldenbergi in Italy and P. okadai in Europe, underscoring the importance of extensive sampling for detecting potential vectors and alien species with direct implications for vector-borne disease epidemiology.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11542218/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-05DOI: 10.1186/s13071-024-06545-6
Elvis Quansah, Ji Zhao, Kenneth Kofi Eduful, Enock Kofi Amoako, Lucas Amenga-Etego, Faustina Halm-Lai, Qingli Luo, Jilong Shen, Chao Zhang, Li Yu
Background: PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location.
Methods: 72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed.
Results: A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings.
Conclusions: Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.
{"title":"Low nucleotide diversity of the Plasmodium falciparum AP2-EXP2 gene among clinical samples from Ghana.","authors":"Elvis Quansah, Ji Zhao, Kenneth Kofi Eduful, Enock Kofi Amoako, Lucas Amenga-Etego, Faustina Halm-Lai, Qingli Luo, Jilong Shen, Chao Zhang, Li Yu","doi":"10.1186/s13071-024-06545-6","DOIUrl":"10.1186/s13071-024-06545-6","url":null,"abstract":"<p><strong>Background: </strong>PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location.</p><p><strong>Methods: </strong>72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed.</p><p><strong>Results: </strong>A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings.</p><p><strong>Conclusions: </strong>Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539609/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142583525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-05DOI: 10.1186/s13071-024-06527-8
Mattia Calzolari, Andrea Mosca, Fabrizio Montarsi, Annalisa Grisendi, Mara Scremin, Paolo Roberto, Carlotta Tessarolo, Francesco Defilippo, Federica Gobbo, Cristina Casalone, Davide Lelli, Alessandro Albieri
Background: Knowledge of the distribution and abundance of disease-causing mosquito vectors is fundamental for assessing the risk of disease circulation and introduction. Aedes caspius (Pallas, 1771) and Aedes vexans (Meigen, 1830) have been implicated, to different extents, in the circulation of several arthropod-borne viruses (arboviruses). These two mosquitoes are vectors of Tahyna virus in Europe and are considered potential vectors of Rift Valley fever virus, a virus not present but at risk of introduction on the continent.
Methods: In this work, we analysed abundance data collected during West Nile virus (WNV) surveillance in northern Italy (Po Plain) via 292 CO2-baited traps to evaluate the distribution and density of these two non-target mosquitoes. We modelled the distribution and abundance of these two mosquito species in the surveyed area using two distinct spatial analysis approaches (geostatistical and machine learning), which yielded congruent results.
Results: Both species are more abundant close to the Po River than elsewhere, but Ae. caspius is present in the eastern and western parts of the plain, linked with the occurrence of rice fields and wetlands, while Ae. vexans is observed in the middle area of the plain.
Conclusions: Presence and abundance data at the municipality level were obtained and made available through this work. This work demonstrates the importance of maintaining and improving entomological surveillance programs with an adequate sampling effort.
{"title":"Distribution and abundance of Aedes caspius (Pallas, 1771) and Aedes vexans (Meigen, 1830) in the Po Plain (northern Italy).","authors":"Mattia Calzolari, Andrea Mosca, Fabrizio Montarsi, Annalisa Grisendi, Mara Scremin, Paolo Roberto, Carlotta Tessarolo, Francesco Defilippo, Federica Gobbo, Cristina Casalone, Davide Lelli, Alessandro Albieri","doi":"10.1186/s13071-024-06527-8","DOIUrl":"10.1186/s13071-024-06527-8","url":null,"abstract":"<p><strong>Background: </strong>Knowledge of the distribution and abundance of disease-causing mosquito vectors is fundamental for assessing the risk of disease circulation and introduction. Aedes caspius (Pallas, 1771) and Aedes vexans (Meigen, 1830) have been implicated, to different extents, in the circulation of several arthropod-borne viruses (arboviruses). These two mosquitoes are vectors of Tahyna virus in Europe and are considered potential vectors of Rift Valley fever virus, a virus not present but at risk of introduction on the continent.</p><p><strong>Methods: </strong>In this work, we analysed abundance data collected during West Nile virus (WNV) surveillance in northern Italy (Po Plain) via 292 CO<sub>2</sub>-baited traps to evaluate the distribution and density of these two non-target mosquitoes. We modelled the distribution and abundance of these two mosquito species in the surveyed area using two distinct spatial analysis approaches (geostatistical and machine learning), which yielded congruent results.</p><p><strong>Results: </strong>Both species are more abundant close to the Po River than elsewhere, but Ae. caspius is present in the eastern and western parts of the plain, linked with the occurrence of rice fields and wetlands, while Ae. vexans is observed in the middle area of the plain.</p><p><strong>Conclusions: </strong>Presence and abundance data at the municipality level were obtained and made available through this work. This work demonstrates the importance of maintaining and improving entomological surveillance programs with an adequate sampling effort.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11539340/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142583522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1186/s13071-024-06512-1
Chunfu Li, Rui Ma, Ai Gao, Na Jiang, Chunli Sang, Yanli Zhang, Haoqiang Tian, Jian Li, Wei Hu, Xinyu Feng
Background: Ticks are vectors of numerous pathogens, with their bacterial composition, abundance, diversity, and interaction influencing both their growth and disease transmission efficiency. Despite the abundance of ticks in Inner Mongolia, China, comprehensive data on their microbial communities are lacking. This study aims to analyze the microbial communities within ticks from Inner Mongolia to inform innovative control strategies for interrupting pathogen transmission.
Methods: Tick samples were collected from animals and vegetation in multiple locations across Inner Mongolia and stored at - 80 °C. Ticks were identified using morphological keys and molecular biology methods. Full-length 16S rRNA gene sequencing was performed on collected samples. Bacterial community composition and diversity were mainly analyzed using bioinformatic tools such as QIIME, phyloseq, and DESeq2. Alpha diversity was assessed using Chao1, ACE, and Shannon indices, while beta diversity was evaluated using Bray-Curtis dissimilarity matrices. LEfSe analysis was applied to identify taxa associated with ecological and biological variables.
Results: A total of 5,048,137 high-quality read counts were obtained, forming an average of 789.3 OTUs per sample. Proteobacteria, Firmicutes, and Bacteroidetes were the most dominant phyla. Bacterial community composition varied significantly with geography, with Dermacentor nuttalli showing a higher abundance of Rickettsia in Xilingol League, while other regions had different dominant genera. The microbial community also differed based on the feeding status of ticks. Additionally, the microbiota of engorged ticks showed organ specificity. Pathogen detection efforts revealed the presence of nine pathogens across all three tick species. D. nuttalli was found to carry a significantly higher burden of pathogenic bacteria, making it the most potentially threatening tick species in Inner Mongolia.
Conclusions: The study highlights significant variations in tick microbiomes influenced by geographic location, feeding status, and tick species. It underscores the importance of enhancing tick and tick-borne disease surveillance in Inner Mongolia for early detection and control of emerging pathogens.
{"title":"Deciphering the microbial communities in ticks of Inner Mongolia: ecological determinants and pathogen profiles.","authors":"Chunfu Li, Rui Ma, Ai Gao, Na Jiang, Chunli Sang, Yanli Zhang, Haoqiang Tian, Jian Li, Wei Hu, Xinyu Feng","doi":"10.1186/s13071-024-06512-1","DOIUrl":"10.1186/s13071-024-06512-1","url":null,"abstract":"<p><strong>Background: </strong>Ticks are vectors of numerous pathogens, with their bacterial composition, abundance, diversity, and interaction influencing both their growth and disease transmission efficiency. Despite the abundance of ticks in Inner Mongolia, China, comprehensive data on their microbial communities are lacking. This study aims to analyze the microbial communities within ticks from Inner Mongolia to inform innovative control strategies for interrupting pathogen transmission.</p><p><strong>Methods: </strong>Tick samples were collected from animals and vegetation in multiple locations across Inner Mongolia and stored at - 80 °C. Ticks were identified using morphological keys and molecular biology methods. Full-length 16S rRNA gene sequencing was performed on collected samples. Bacterial community composition and diversity were mainly analyzed using bioinformatic tools such as QIIME, phyloseq, and DESeq2. Alpha diversity was assessed using Chao1, ACE, and Shannon indices, while beta diversity was evaluated using Bray-Curtis dissimilarity matrices. LEfSe analysis was applied to identify taxa associated with ecological and biological variables.</p><p><strong>Results: </strong>A total of 5,048,137 high-quality read counts were obtained, forming an average of 789.3 OTUs per sample. Proteobacteria, Firmicutes, and Bacteroidetes were the most dominant phyla. Bacterial community composition varied significantly with geography, with Dermacentor nuttalli showing a higher abundance of Rickettsia in Xilingol League, while other regions had different dominant genera. The microbial community also differed based on the feeding status of ticks. Additionally, the microbiota of engorged ticks showed organ specificity. Pathogen detection efforts revealed the presence of nine pathogens across all three tick species. D. nuttalli was found to carry a significantly higher burden of pathogenic bacteria, making it the most potentially threatening tick species in Inner Mongolia.</p><p><strong>Conclusions: </strong>The study highlights significant variations in tick microbiomes influenced by geographic location, feeding status, and tick species. It underscores the importance of enhancing tick and tick-borne disease surveillance in Inner Mongolia for early detection and control of emerging pathogens.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11533347/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142576758","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1186/s13071-024-06515-y
Zoë Tess Lara Lindhorst, Sebastian Brandstetter, Maria Sophia Unterköfler, Barbara Eigner, Joachim Spergser, Marc Colyn, Peter Steinbach, Duško Ćirović, Nikica Šprem, Tomislav Dumić, Vincenzo Veneziano, Franz Müller, Josef Harl, Georgiana Deak, Angela Monica Ionică, Mike Heddergott, Hans-Peter Fuehrer
Background: Vector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans.
Methods: Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp.
Results: The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples.
Conclusions: The large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health.
{"title":"Molecular analysis of vector-borne pathogens in Eurasian badgers (Meles meles) from continental Europe.","authors":"Zoë Tess Lara Lindhorst, Sebastian Brandstetter, Maria Sophia Unterköfler, Barbara Eigner, Joachim Spergser, Marc Colyn, Peter Steinbach, Duško Ćirović, Nikica Šprem, Tomislav Dumić, Vincenzo Veneziano, Franz Müller, Josef Harl, Georgiana Deak, Angela Monica Ionică, Mike Heddergott, Hans-Peter Fuehrer","doi":"10.1186/s13071-024-06515-y","DOIUrl":"10.1186/s13071-024-06515-y","url":null,"abstract":"<p><strong>Background: </strong>Vector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans.</p><p><strong>Methods: </strong>Between 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp.</p><p><strong>Results: </strong>The pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples.</p><p><strong>Conclusions: </strong>The large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536541/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142576760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1186/s13071-024-06542-9
Kamal Eddine Benallal, Mohammed Mefissel, Yassine Dib, Jérôme Depaquit, Daniel Kavan, Zoubir Harrat, Vít Dvořák, Petr Volf, Petr Halada
Background: Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus, which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis.
Methods: In the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS.
Results: In total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia (Sergentomyia) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri, suggesting that these vector species are opportunistic feeders.
Conclusions: Integrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp.
背景:沙蝇(双翅目:Psychodidae)是各种病原体(主要是利什曼原虫)的重要传播媒介。在旧大陆,传播病原体最重要的属是 Phlebotomus 属,其中包括许多已被证实或疑似传播利什曼原虫的物种,而 Sergentomyia 属作为人类病原体的传播媒介至今仍未得到证实。阿尔及利亚是受人类利什曼病影响最严重的国家之一:在本研究中,我们分别在撒哈拉北部和中部有人类利什曼病病例记录的两个省份加尔达伊亚和伊利济进行了昆虫学调查。我们的目标是了解当地沙蝇物种在利什曼病寄生虫传播中的作用,并分析它们对血餐的偏好。采集到的沙蝇通过形态学和分子方法进行鉴定,包括 DNA 条形码和基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)蛋白质分析。此外,还利用 MALDI-TOF MS 对雌性血餐进行了肽质量图谱分析:结果:两省共采集了 640 份沙蝇标本,分别属于 Phlebotomus 和 Sergentomyia 属。Sergentomyia antennata 和 Se. fallax 是 Ghardaïa 省最多的沙蝇物种,Ph. papatasi 和 Ph. alexandri 是伊利济省最多的沙蝇物种。此外,在伊利济还发现了一个新的沙蝇物种,名为 Sergentomyia (Sergentomyia) imihra n. sp:结合形态分析、DNA 标记测序和蛋白质分析的综合方法识别并描述了一种新的 Sergentomyia 物种,使阿尔及利亚沙蝇动物群的种类增至 27 种。建议对撒哈拉沙漠中部的沙蝇进行进一步监测,以确定迄今未知的 Se. imihra n. sp.
{"title":"Phlebotomine sand fly survey, blood meal source identification, and description of Sergentomyia imihra n. sp. in the central Sahara of Algeria.","authors":"Kamal Eddine Benallal, Mohammed Mefissel, Yassine Dib, Jérôme Depaquit, Daniel Kavan, Zoubir Harrat, Vít Dvořák, Petr Volf, Petr Halada","doi":"10.1186/s13071-024-06542-9","DOIUrl":"10.1186/s13071-024-06542-9","url":null,"abstract":"<p><strong>Background: </strong>Phlebotomine sand flies (Diptera: Psychodidae) are important vectors of various pathogens, mainly Leishmania parasites. In the Old World, the most important genus in term of pathogens transmission is the genus Phlebotomus, which includes many proven or suspected vectors of several Leishmania species, while the genus Sergentomyia remains so far unproven as a vector of human pathogens. Algeria is one of the most affected countries by human leishmaniasis.</p><p><strong>Methods: </strong>In the present study, an entomological survey was carried out in two provinces, Ghardaïa and Illizi, located in the north and central Sahara, respectively, where cases of human leishmaniasis are recorded. Our goal was to understand the role of the local sand fly species in the transmission of Leishmania parasites and to analyze their blood meal preferences. Collected sand flies were identified by a combination of morphological and molecular approaches that included DNA-barcoding and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling. In addition, female blood meals were analyzed by peptide mass mapping using MALDI-TOF MS.</p><p><strong>Results: </strong>In total, 640 sand fly specimens belonging to Phlebotomus and Sergentomyia genera were collected in the two provinces. Sergentomyia antennata and Se. fallax were most abundant species in Ghardaïa, and Ph. papatasi and Ph. alexandri in Illizi. In addition, a new sand fly species was described in Illizi named Sergentomyia (Sergentomyia) imihra n. sp. Blood meal analysis of the engorged females revealed various mammalian hosts, especially goats, but also humans for Phlebotomus papatasi and Ph. alexandri, suggesting that these vector species are opportunistic feeders.</p><p><strong>Conclusions: </strong>Integrative approach that combined morphological analysis, sequencing of DNA markers, and protein profiling enabled the recognition and description of a new Sergentomyia species, raising the number of the Algerian sand fly fauna to 27 species. Further sand fly surveillance in the central Sahara is recommended to identify the thus-far unknown males of Se. imihra n. sp.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536750/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142576761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-04DOI: 10.1186/s13071-024-06482-4
Matthew Lukenge, Rickard Ignell, Sharon Rose Hill
Background: The decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult.
Methods: The feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined.
Results: Aedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae-but not that of Ae. aegypti-demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged.
Conclusions: Taken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies.
背景:吸食血食的决定主要取决于食血节肢动物对源自血液的腺嘌呤核苷酸(尤其是三磷酸腺苷(ATP))的敏感性和特异性。尽管以前曾努力确定和描述 ATP 和其他腺嘌呤核苷酸的特异性和敏感性,以及其他来自血液的吞噬刺激剂在秃蝇科中的作用,但仍很难对不同物种进行比较:方法:在碳酸盐缓冲液存在的情况下,使用膜进食试验评估了黄热病蚊子埃及伊蚊和非洲疟疾病媒冈比亚按蚊对腺嘌呤核苷酸的进食反应。用肉眼和分光光度计对所有蚊子的吞食比例和吞食量进行评分。此外,还检测了预诱杀冈比亚伊蚊的比例以及吞食量和预诱杀量:结果:埃及伊蚊比冈比亚伊蚊对腺嘌呤核苷酸更敏感,但两种伊蚊都对这些促噬剂保持特异性,显示出剂量依赖性的双峰摄食模式,从而扩大了我们对全血或无血摄食假说的理解。冈比亚疟蚊对碳酸氢盐缓冲液的摄食--而埃及疟蚊对碳酸氢盐缓冲液的摄食--证明了血液吞噬刺激剂的编码方式存在物种特异性差异。据观察,腺嘌呤核苷酸与牛血清白蛋白或不与牛血清白蛋白一起使用时,可剂量依赖性地调节冈比亚疟原虫的诱食比例和诱食量,但不能调节充血量:总之,这项研究的结果拓展了我们对蚊子如何对血餐成分做出不同评估和反应的理解,并为进一步的生理和分子研究提供了基础。
{"title":"Differential sensitivity and specificity of Aedes aegypti and Anopheles gambiae to adenine nucleotide phagostimulants-an all-or-none response?","authors":"Matthew Lukenge, Rickard Ignell, Sharon Rose Hill","doi":"10.1186/s13071-024-06482-4","DOIUrl":"10.1186/s13071-024-06482-4","url":null,"abstract":"<p><strong>Background: </strong>The decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult.</p><p><strong>Methods: </strong>The feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined.</p><p><strong>Results: </strong>Aedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae-but not that of Ae. aegypti-demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged.</p><p><strong>Conclusions: </strong>Taken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11536708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142576759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Background: Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established.
Methods: In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay.
Results: A SGLV-specific LAMP assay was established with a detection limit of 1 × 10-2 copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples.
Conclusions: We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV.
{"title":"Development of a LAMP assay for the rapid visual detection of the emerging tick-borne Songling virus.","authors":"Zheng Gui, Yuanning Ren, Qiqi Guo, Weiying Yang, Ziyan Liu, Ning Liu, Yunzhi Peng, Yu Liu, Jingfeng Yu, Lichao Sun, Zedong Wang","doi":"10.1186/s13071-024-06552-7","DOIUrl":"10.1186/s13071-024-06552-7","url":null,"abstract":"<p><strong>Background: </strong>Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established.</p><p><strong>Methods: </strong>In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay.</p><p><strong>Results: </strong>A SGLV-specific LAMP assay was established with a detection limit of 1 × 10<sup>-2</sup> copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples.</p><p><strong>Conclusions: </strong>We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11529016/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-31DOI: 10.1186/s13071-024-06458-4
Bo Chen, Qi Zhang, Sen Wang, Xing-Ai Guan, Wan-Xin Luo, Dong-Fang Li, Yue He, Shu-Jing Huang, Ya-Ting Zhou, Jun-Long Zhao, Lan He
Background: Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.
Methods: The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).
Results: In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.
Conclusions: This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.
背景:巴贝西亚原虫(Babesia duncani)是引起人类巴贝西亚原虫病的一种病原体。它不仅可通过蜱虫叮咬传播,还可通过输血传播,对公共卫生构成重大威胁。因此,要开发药物和疫苗,就必须了解这种病原体的基因功能。然而,条件基因敲除工具的缺乏阻碍了对这种病原体的研究。在基因功能研究中,广泛使用的是一种快速、可逆的条件性基因敲除系统--辅助素诱导降解子(AID)系统。因此,迫切需要在 B. duncani 中建立 AID 系统来研究重要的基因功能:方法:通过多序列比对和保守结构域分析,确定并确认了B. duncani中Skp1-Cullin-F-box(SCF)复合体的内源基因。通过同源重组策略构建转基因寄生虫株,实现了Oryza sativa的F-box蛋白TIR1(OsTIR1)的表达。聚合酶链反应(PCR)、Western 印迹和间接免疫荧光测定(IFA)被用来确认正确的单克隆寄生虫菌株。在吲哚-3-乙酸(IAA)处理后,通过 Western 印迹和活细胞荧光显微镜检测了用 AID 降解子标记的增强型绿色荧光蛋白(eGFP)的降解情况:结果:本研究通过序列比对和结构域分析鉴定了B.duncani中SCF复合体的Skp1、Cul1和Rbx1。通过同源重组构建并确认了表达 OsTIR1 基因的纯 BdTIR1 菌株。该菌株在生长速度和不同寄生形式的比例方面与野生型(WT)无明显差异。使用 500 μM IAA 成功诱导了标记有 AID 降解子的 eGFP 降解。Western 印迹灰度分析表明 eGFP 表达水平降低了 61.3%,而荧光强度分析表明荧光强度降低了 77.5%。将 IAA 浓度提高到 2 mM 会加速 eGFP 的降解,并增强降解的程度:本研究通过使用 IAA 诱导 eGFP 的快速降解,证明了 AID 系统在调节蛋白质水平方面的功能,为研究与 B. duncani 的入侵、排出和毒力相关的重要基因功能提供了重要的研究工具。此外,它还为尚未开发出 AID 系统的类囊体寄生虫提供了一种构建策略。
{"title":"Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.","authors":"Bo Chen, Qi Zhang, Sen Wang, Xing-Ai Guan, Wan-Xin Luo, Dong-Fang Li, Yue He, Shu-Jing Huang, Ya-Ting Zhou, Jun-Long Zhao, Lan He","doi":"10.1186/s13071-024-06458-4","DOIUrl":"10.1186/s13071-024-06458-4","url":null,"abstract":"<p><strong>Background: </strong>Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.</p><p><strong>Methods: </strong>The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).</p><p><strong>Results: </strong>In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.</p><p><strong>Conclusions: </strong>This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.</p>","PeriodicalId":19793,"journal":{"name":"Parasites & Vectors","volume":null,"pages":null},"PeriodicalIF":3.0,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11526643/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}