Pub Date : 2025-01-01DOI: 10.1016/j.exppara.2024.108878
Ian David Woolsey , Tonje Opsal , Lucy Robertson , Sokratis Ptochos , Lisbeth Hektoen
Faecal samples were obtained from 77 first season grazers from 20 Norwegian dairy herds in autumn 2020 for analysis of Cooperia oncophora and Ostertagia ostertagi infection. Strongylid eggs per gram of faeces (EPG) were determined for each sample and the samples underwent larval culture. DNA was extracted from the faeces at different stages of the culture preparation: from faecal slurry (FS), direct extraction before culture (DBC), and direct extraction after culture (DAC). Extracted DNA was subject to molecular assay for both C. oncophora and O. ostertagi using both a published ddPCR assay and a modified qPCR duplex assay targeting the ITS-2 gene. For C. oncophora, DBC samples contained significantly less ITS-2 copies than DAC and FS samples (p ≤ 0.0001 for both) for qPCR, but not between DAC and FS samples (p = 0.339). Droplet digital PCR with DBC samples yielded significantly fewer ITS-2 copies than DAC and FS samples (p ≤ 0.0001). For O. ostertagi, the ITS-2 copies in DBC samples differed significantly from levels in DAC and FS samples on the qPCR platform (p=0.002 and 0.044, respectively) as was the case with ddPCR (p ≤ 0.0001 and 0.0137). Overall, across the various processing techniques, C. oncophora results obtained on both ddPCR and qPCR platforms correlated with EPG better than was found for equivalent results for O. ostertagi and results are strongly indicative of poor correlation when EPG counts are low. Results from this study suggest that DAC faecal processing was of limited practical use.
{"title":"Molecular detection of Cooperia oncophora and Ostertagia ostertagi in cattle: Comparison of sample processing and detection on ddPCR and qPCR platforms","authors":"Ian David Woolsey , Tonje Opsal , Lucy Robertson , Sokratis Ptochos , Lisbeth Hektoen","doi":"10.1016/j.exppara.2024.108878","DOIUrl":"10.1016/j.exppara.2024.108878","url":null,"abstract":"<div><div>Faecal samples were obtained from 77 first season grazers from 20 Norwegian dairy herds in autumn 2020 for analysis of <em>Cooperia oncophora</em> and <em>Ostertagia ostertagi</em> infection. Strongylid eggs per gram of faeces (EPG) were determined for each sample and the samples underwent larval culture. DNA was extracted from the faeces at different stages of the culture preparation: from faecal slurry (FS), direct extraction before culture (DBC), and direct extraction after culture (DAC). Extracted DNA was subject to molecular assay for both <em>C. oncophora</em> and <em>O. ostertagi</em> using both a published ddPCR assay and a modified qPCR duplex assay targeting the ITS-2 gene. For <em>C. oncophora,</em> DBC samples contained significantly less ITS-2 copies than DAC and FS samples (<em>p</em> ≤ 0.0001 for both) for qPCR, but not between DAC and FS samples (<em>p</em> = 0.339). Droplet digital PCR with DBC samples yielded significantly fewer ITS-2 copies than DAC and FS samples (<em>p</em> ≤ 0.0001). For <em>O. ostertagi,</em> the ITS-2 copies in DBC samples differed significantly from levels in DAC and FS samples on the qPCR platform (<em>p=</em>0.002 and 0.044, respectively) as was the case with ddPCR (<em>p</em> ≤ 0.0001 and 0.0137). Overall, across the various processing techniques, <em>C. oncophora</em> results obtained on both ddPCR and qPCR platforms correlated with EPG better than was found for equivalent results for <em>O. ostertagi</em> and results are strongly indicative of poor correlation when EPG counts are low. Results from this study suggest that DAC faecal processing was of limited practical use.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108878"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142821969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.exppara.2024.108877
Leandro Rodrigues , Rodrigo Giglioti , Luciana Morita Katiki , André Lucio Franceschini Sarria , Germano Scholze , Cecília José Veríssimo
The cattle tick Rhipicephalus microplus is prevalent in tropical and subtropical regions, causing substantial economic losses due to its resistance to conventional acaricides. There is an urgent need to identify safe and effective new acaricidal agents. Essential oils and their volatile compounds are promising alternatives. Ensuring the use of optimal solvents or surfactants that do not compromise the acaricidal activity of these compounds during testing is crucial. This study aims to evaluate how compounds thymol, carvacrol and eugenol interact with xylol, methanol, ethanol, acetone, isopropyl alcohol, glycerol, dimethyl sulfoxide, castor oil, propylene glycol, vaseline, and Tween 80® to enhance (or to worse) their acaricidal efficacy against R. microplus. Larval mortality time were compared against one negative control (soybean oil) and two positive controls (commercial pour-on products). The experiments were conducted in 48-well polyethylene plates, with around 100 larvae immersed in 200 μl of each solvent at 100, 50, 25, 12.5, 6.25, 3.125 and 1.56% and diluted in soybean oil or water, according to solubility. Each volatile compound (Thymol, carvacrol and eugenol) was diluted in the tested solvents to assess larval mortality time. Xylol demonstrated the shortest larval mortality time, even at a minimum concentration (p < 0.05). In contrast, liquid vaseline exhibited the longest larval mortality time. When thymol, carvacrol, and eugenol were combined with xylol, they achieved the shortest larval mortality time. Conversely, when diluted in liquid vaseline they exhibited synergistic effects decreasing the mortality time. Tween 80® worsen the efficacy of thymol, carvacrol, and eugenol, resulting in prolonged larval mortality times. These findings emphasize the critical role of solvent selection, indicating the choice of solvent profoundly affects the formulation's effectiveness, directly influencing the activity of the active compounds.
{"title":"Assessment of synergistic and antagonistic interactions between volatile compounds thymol, carvacrol, and eugenol diluted in solvents against Rhipicephalus microplus in in vitro tests","authors":"Leandro Rodrigues , Rodrigo Giglioti , Luciana Morita Katiki , André Lucio Franceschini Sarria , Germano Scholze , Cecília José Veríssimo","doi":"10.1016/j.exppara.2024.108877","DOIUrl":"10.1016/j.exppara.2024.108877","url":null,"abstract":"<div><div>The cattle tick <em>Rhipicephalus microplus</em> is prevalent in tropical and subtropical regions, causing substantial economic losses due to its resistance to conventional acaricides. There is an urgent need to identify safe and effective new acaricidal agents. Essential oils and their volatile compounds are promising alternatives. Ensuring the use of optimal solvents or surfactants that do not compromise the acaricidal activity of these compounds during testing is crucial. This study aims to evaluate how compounds thymol, carvacrol and eugenol interact with xylol, methanol, ethanol, acetone, isopropyl alcohol, glycerol, dimethyl sulfoxide, castor oil, propylene glycol, vaseline, and Tween 80® to enhance (or to worse) their acaricidal efficacy against <em>R. microplus</em>. Larval mortality time were compared against one negative control (soybean oil) and two positive controls (commercial <em>pour-on</em> products). The experiments were conducted in 48-well polyethylene plates, with around 100 larvae immersed in 200 μl of each solvent at 100, 50, 25, 12.5, 6.25, 3.125 and 1.56% and diluted in soybean oil or water, according to solubility. Each volatile compound (Thymol, carvacrol and eugenol) was diluted in the tested solvents to assess larval mortality time. Xylol demonstrated the shortest larval mortality time, even at a minimum concentration (p < 0.05). In contrast, liquid vaseline exhibited the longest larval mortality time. When thymol, carvacrol, and eugenol were combined with xylol, they achieved the shortest larval mortality time. Conversely, when diluted in liquid vaseline they exhibited synergistic effects decreasing the mortality time. Tween 80® worsen the efficacy of thymol, carvacrol, and eugenol, resulting in prolonged larval mortality times. These findings emphasize the critical role of solvent selection, indicating the choice of solvent profoundly affects the formulation's effectiveness, directly influencing the activity of the active compounds.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108877"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ticks vector a large number of significant pathogens, yet remain understudied due to the challenges in laboratory colonization. This study introduces innovative techniques for rearing Rhipicephalus sanguineus, focusing on in vivo tick feeding using ICR mice (Mus musculus) as a blood source. The research, conducted at the Walter Reed Army Institute of Research - Armed Forces Research Institute of Medical Sciences (WRAIR-AFRIMS), outlines the successful utilization of mice to support all stages of tick development. Ticks were retained on mice using Ethylene-Vinyl Acetate (EVA) foam capsules and cyclophosphamide was administered to the mice to prevent host immune response from interfering with tick feeding. These methods allowed the successful establishment and mass production of R. sanguineus tick colonies. The methods described herein hold promise for institutions seeking efficient tick production using a rodent model.
{"title":"Enhancing brown dog tick rearing (Rhipicephalus sanguineus): In vivo feeding using mice (Mus musculus) as blood sources","authors":"Siriporn Phasomkusolsil , Ratree Takhampunya , Rawiwan Im-erbsin , Phakorn Wilaisri , Jaruwan Tawong , Thanin Jitbantrengphan , Tanaporn Kornkan , Nantaporn Monkanna , Alyssa N. Mann , Erica J. Lindroth","doi":"10.1016/j.exppara.2024.108879","DOIUrl":"10.1016/j.exppara.2024.108879","url":null,"abstract":"<div><div>Ticks vector a large number of significant pathogens, yet remain understudied due to the challenges in laboratory colonization. This study introduces innovative techniques for rearing <em>Rhipicephalus sanguineus</em>, focusing on in vivo tick feeding using ICR mice (<em>Mus musculus</em>) as a blood source. The research, conducted at the Walter Reed Army Institute of Research - Armed Forces Research Institute of Medical Sciences (WRAIR-AFRIMS), outlines the successful utilization of mice to support all stages of tick development. Ticks were retained on mice using Ethylene-Vinyl Acetate (EVA) foam capsules and cyclophosphamide was administered to the mice to prevent host immune response from interfering with tick feeding. These methods allowed the successful establishment and mass production of <em>R. sanguineus</em> tick colonies. The methods described herein hold promise for institutions seeking efficient tick production using a rodent model.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108879"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142821952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Toxoplasmosis which is caused by T. gondii, is common among humans and animals. T. gondii is a threat to the fetus and individuals with immune disorders, especially patients with acquired immunodeficiency syndrome (AIDS) and individuals who undergo organ transplants. Therefore, quick diagnosis and accurate differentiation of acute and chronic stages are essential. One of the important serological methods in differentiating stages of the disease and the time of acquiring the infection is evaluating the IgG avidity. In this systematic review and meta-analysis, keywords were searched in databases including PubMed, Science Direct, ProQuest, Scopus, and Google Scholar. Included studies were collected after checking the inclusion and exclusion criteria, and according to the PRISMA flow chart. Finally, the data were analyzed by StatsDirect statistical software and random-effects model. A total of 10 studies (26 datasets) were eligible for analysis. The random effects model estimated the prevalence of low IgG avidity in acute toxoplasmosis using in-house IgG avidity tests of 84% and chronic toxoplasmosis infection using in-house IgG avidity of 91%. The IgG avidity test can be a helpful diagnostic tool in differentiating between acute and chronic stages. Also, this review emphasizes that the use of recombinant or chimeric proteins is preferable to TLA in differentiating acute and chronic stages. It can be concluded that choosing more effective antigens (multi-epitope and multi-stage) and performing more detailed studies on the design of an avidity kit to differentiate the stage of infection is required.
{"title":"Design and optimization of IgG avidity test for differentiating acute from chronic human toxoplasmosis: A systematic review and meta-analysis","authors":"Mostafa Tork , Shahabeddin Sarvi , Hossein Asgarian-Omran , Mitra Sadeghi , Bahareh Basirpour , Maryam Hatami Nejad , Shirzad Gholami , Seyed Abdollah Hosseini , Ahmad Daryani , Sargis A. Aghayan","doi":"10.1016/j.exppara.2024.108883","DOIUrl":"10.1016/j.exppara.2024.108883","url":null,"abstract":"<div><div>Toxoplasmosis which is caused by <em>T. gondii</em>, is common among humans and animals. <em>T. gondii</em> is a threat to the fetus and individuals with immune disorders, especially patients with acquired immunodeficiency syndrome (AIDS) and individuals who undergo organ transplants. Therefore, quick diagnosis and accurate differentiation of acute and chronic stages are essential. One of the important serological methods in differentiating stages of the disease and the time of acquiring the infection is evaluating the IgG avidity. In this systematic review and meta-analysis, keywords were searched in databases including PubMed, Science Direct, ProQuest, Scopus, and Google Scholar. Included studies were collected after checking the inclusion and exclusion criteria, and according to the PRISMA flow chart. Finally, the data were analyzed by StatsDirect statistical software and random-effects model. A total of 10 studies (26 datasets) were eligible for analysis. The random effects model estimated the prevalence of low IgG avidity in acute toxoplasmosis using in-house IgG avidity tests of 84% and chronic toxoplasmosis infection using in-house IgG avidity of 91%. The IgG avidity test can be a helpful diagnostic tool in differentiating between acute and chronic stages. Also, this review emphasizes that the use of recombinant or chimeric proteins is preferable to TLA in differentiating acute and chronic stages. It can be concluded that choosing more effective antigens (multi-epitope and multi-stage) and performing more detailed studies on the design of an avidity kit to differentiate the stage of infection is required.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108883"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142893058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.exppara.2024.108865
Abeer M.A. Mahgoub , Mona Ibrahim Ali , Enas Yahia Abu-Sarea , Sara Ahmed Rady , Ibrahim Rabea Bayoumi Ali , Doaa Reda Sayed
Human trichinosis is a serious foodborne parasitic zoonosis. Diagnosing human trichinosis is usually difficult due to the nonspecific clinical picture and the limited effectiveness of serological tests in acute infections. While ELISA can detect circulating Trichinella antigens, aiding in early diagnosis, its sensitivity may be low. The application of nanoparticles can improve the sensitivity of ELISA and allow a specific early diagnosis of the disease. This work compares the nano chitosan beads-based ELISA (NCSB-ELISA) and traditional sandwich ELISA for the detection of circulating Trichinella spiralis (T. spiralis) crude extract-antigen (CEA) in serum samples of experimentally infected mice.
Fifty-seven mice included in this study were classified into 3 groups: T. spiralis infected group (Group I) (36 mice), which was equally subdivided into six subgroups according to the time of sacrifice (6, 8, 10, 12, 14 and 16) days post-infection (dpi), cross-reactivity group (Group II) (9 mice) and negative control group (Group III) (12 mice). T. spiralis AW-CEA prepared from the adult worms were used to produce anti- T. spiralis IgG-polyclonal antibodies in rabbits; these antibodies were utilized to detect AW-CEA in serum samples by traditional sandwich ELISA and NCSB-ELISA. Using NCSB-ELISA, T. spiralis AW-CEA was detected in sera collected at 8 dpi, with a sensitivity of 50% and a specificity of 100%. Meanwhile, traditional sandwich ELISA could not detect the antigen at the same time interval. Both ELISA were able to detect the antigen in samples collected at 10, 12, 14 and 16 dpi with a sensitivity of 16.67%, 50%, 67.67% and 83.67%, respectively, for traditional sandwich-ELISA and a specificity of 100% at 10, 12 and 14 dpi while at 16 dpi specificity was decreased to 90.91%. In contrast, the sensitivity of NCSB-sandwich ELISA on the same days was 66.67%, 83.34%, 100% and 100%, respectively, with a specificity of 100% at all days. False positive detection of T. spiralis AW-CEA in the serum of mice in GII was recorded on day 16 pi by only traditional sandwich ELISA.
This study concluded that NCSB-ELISA is a promising and sensitive technique for the early and specific diagnosis of acute trichinosis in an animal model.
{"title":"In vivo study of nano chitosan beads-based ELISA versus traditional sandwich ELISA for the early diagnosis of trichinosis","authors":"Abeer M.A. Mahgoub , Mona Ibrahim Ali , Enas Yahia Abu-Sarea , Sara Ahmed Rady , Ibrahim Rabea Bayoumi Ali , Doaa Reda Sayed","doi":"10.1016/j.exppara.2024.108865","DOIUrl":"10.1016/j.exppara.2024.108865","url":null,"abstract":"<div><div>Human trichinosis is a serious foodborne parasitic zoonosis. Diagnosing human trichinosis is usually difficult due to the nonspecific clinical picture and the limited effectiveness of serological tests in acute infections. While ELISA can detect circulating Trichinella antigens, aiding in early diagnosis, its sensitivity may be low. The application of nanoparticles can improve the sensitivity of ELISA and allow a specific early diagnosis of the disease. This work compares the nano chitosan beads-based ELISA (NCSB-ELISA) and traditional sandwich ELISA for the detection of circulating <em>Trichinella spiralis</em> (<em>T</em>. <em>spiralis</em>) crude extract-antigen (CEA) in serum samples of experimentally infected mice.</div><div>Fifty-seven mice included in this study were classified into 3 groups: <em>T. spiralis</em> infected group (Group I) (36 mice), which was equally subdivided into six subgroups according to the time of sacrifice (6, 8, 10, 12, 14 and 16) days post-infection (dpi), cross-reactivity group (Group II) (9 mice) and negative control group (Group III) (12 mice). <em>T. spiralis</em> AW-CEA prepared from the adult worms were used to produce anti- <em>T. spiralis</em> IgG-polyclonal antibodies in rabbits; these antibodies were utilized to detect AW-CEA in serum samples by traditional sandwich ELISA and NCSB-ELISA. Using NCSB-ELISA, <em>T. spiralis</em> AW-CEA was detected in sera collected at 8 dpi, with a sensitivity of 50% and a specificity of 100%. Meanwhile, traditional sandwich ELISA could not detect the antigen at the same time interval. Both ELISA were able to detect the antigen in samples collected at 10, 12, 14 and 16 dpi with a sensitivity of 16.67%, 50%, 67.67% and 83.67%, respectively, for traditional sandwich-ELISA and a specificity of 100% at 10, 12 and 14 dpi while at 16 dpi specificity was decreased to 90.91%. In contrast, the sensitivity of NCSB-sandwich ELISA on the same days was 66.67%, 83.34%, 100% and 100%, respectively, with a specificity of 100% at all days. False positive detection of <em>T. spiralis</em> AW-CEA in the serum of mice in GII was recorded on day 16 pi by only traditional sandwich ELISA.</div><div>This study concluded that NCSB-ELISA is a promising and sensitive technique for the early and specific diagnosis of acute trichinosis in an animal model.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108865"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142754845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.exppara.2024.108884
Desiree V. Pontarolo, Marcelo B. Molento
Fasciolosis is a food-borne anthropozoonotic disease caused by Fasciola spp. that affects multiple hosts, including ruminants and humans. In vitro testing of anthelmintics is of interest to establish the drug's activity without the need for time-consuming and expensive in vivo assays. This study was set to establish a discriminatory dose (DD) by running a dose-titration in vitro experiment (egg hatch test, EHT) of albendazole sulfoxide (ABZ.SO) and nitroxynil (NTX) on eggs of a field strain of Fasciola hepatica. Eggs were recovered from adult parasites isolated from intact bovine livers obtained from a single farm in Paraná, Brazil (FhPar2022 strain) with no ABZ or NTX treatment history. Two hundred eggs were exposed to 18 and 14 concentrations of ABZ.SO and NTX, respectively, for 12h and incubated for 16 days. Egg development and integrity were determined every other day, establishing an index of morphological modification of the different phases. A concentration-dependent effect was observed for egg development in both compounds. ABZ.SO solutions prevent egg hatch, except for the two lowest concentrations. We observed no egg hatch at 6.250–100.0 μmol L−1 for NTX. NTX had an inhibition concentration of 50% (IC50) of 0.043 μmol L−1 with a correlation coefficient of (R2) 0.961. ABZ.SO had an IC50 of 0.00099 μmol L−1 with a low R2 of 0.417. Morphological damage was also associated with the increasing concentration of both drugs. Moreover, it was noted that most eggs that reached the eye spot type could hatch, except at 0.39 and 3.12 μmol L−1 of NTX. In ABZ.SO, hatching occurred only at 0.00038, 0.0007, and 0.0015 μmol L-1 concentrations. The obtained DDs of 0.043 μmol L−1 for NTX and 0.00099 μmol L−1 for ABZ.SO can be used to monitor efficacy in field isolates.
{"title":"Discriminatory dose of nitroxynil and albendazole sulfoxide using a modified egg hatch test of Fasciola hepatica","authors":"Desiree V. Pontarolo, Marcelo B. Molento","doi":"10.1016/j.exppara.2024.108884","DOIUrl":"10.1016/j.exppara.2024.108884","url":null,"abstract":"<div><div>Fasciolosis is a food-borne anthropozoonotic disease caused by <em>Fasciola</em> spp. that affects multiple hosts, including ruminants and humans. <em>In vitro</em> testing of anthelmintics is of interest to establish the drug's activity without the need for time-consuming and expensive <em>in vivo</em> assays. This study was set to establish a discriminatory dose (DD) by running a dose-titration <em>in vitro</em> experiment (egg hatch test, EHT) of albendazole sulfoxide (ABZ.SO) and nitroxynil (NTX) on eggs of a field strain of <em>Fasciola hepatica</em>. Eggs were recovered from adult parasites isolated from intact bovine livers obtained from a single farm in Paraná, Brazil (<em>Fh</em>Par2022 strain) with no ABZ or NTX treatment history. Two hundred eggs were exposed to 18 and 14 concentrations of ABZ.SO and NTX, respectively, for 12h and incubated for 16 days. Egg development and integrity were determined every other day, establishing an index of morphological modification of the different phases. A concentration-dependent effect was observed for egg development in both compounds. ABZ.SO solutions prevent egg hatch, except for the two lowest concentrations. We observed no egg hatch at 6.250–100.0 μmol L<sup>−1</sup> for NTX. NTX had an inhibition concentration of 50% (IC<sub>50</sub>) of 0.043 μmol L<sup>−1</sup> with a correlation coefficient of (R<sup>2</sup>) 0.961. ABZ.SO had an IC<sub>50</sub> of 0.00099 μmol L<sup>−1</sup> with a low R<sup>2</sup> of 0.417. Morphological damage was also associated with the increasing concentration of both drugs. Moreover, it was noted that most eggs that reached the eye spot type could hatch, except at 0.39 and 3.12 μmol L<sup>−1</sup> of NTX. In ABZ.SO, hatching occurred only at 0.00038, 0.0007, and 0.0015 μmol L-1 concentrations. The obtained DDs of 0.043 μmol L<sup>−1</sup> for NTX and 0.00099 μmol L<sup>−1</sup> for ABZ.SO can be used to monitor efficacy in field isolates.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108884"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-01-01DOI: 10.1016/j.exppara.2024.108880
Sophia Bigot , Amin Ahmed Ouameur , Gaétan Roy , Raouia Fakhfakh, Jean-François Ritt, Danielle Légaré, Marc Ouellette
The protozoan parasite Leishmania has a large family of major facilitator membrane proteins part of the Folate Biopterin Transporter (FBT) family. The chromosome 10 of Leishmania has a cluster of 7 FBT genes including the S-Adenosyl methionine (AdoMet) transporter and the functionally characterized folate transporters FT1 and FT5. Six of the 7 FBT proteins coded by this locus are located at the plasma membrane as determined by gene fusions with the green fluorescent protein. We deleted the whole locus of 7 genes (>30 kb) using CRISPR-Cas9 genome editing as a first step in studying the potential function of the four uncharacterized FBT genes from the locus. This knock out strain was viable, highly resistant to sinefungin (an AdoMet analogue) and to methotrexate (a folate analogue) but not to allopurinol, pentamidine or 5-fluorouracil. We similarly studied another FBT family member whose gene is encoded on chromosome 19. The protein was also located at the plasma membrane and its gene was dispensable for growth and not associated to any of the drug tested. Our work has indicated that large diploid deletion is achievable in Leishmania and the cell lines produced here will serve to better understand the function and putative substrates of these FBT proteins yet to be characterized.
{"title":"Studies of the FBT family transporters in Leishmania infantum by gene deletion and protein localization","authors":"Sophia Bigot , Amin Ahmed Ouameur , Gaétan Roy , Raouia Fakhfakh, Jean-François Ritt, Danielle Légaré, Marc Ouellette","doi":"10.1016/j.exppara.2024.108880","DOIUrl":"10.1016/j.exppara.2024.108880","url":null,"abstract":"<div><div>The protozoan parasite <em>Leishmania</em> has a large family of major facilitator membrane proteins part of the Folate Biopterin Transporter (FBT) family. The chromosome 10 of <em>Leishmania</em> has a cluster of 7 FBT genes including the S-Adenosyl methionine (AdoMet) transporter and the functionally characterized folate transporters FT1 and FT5. Six of the 7 FBT proteins coded by this locus are located at the plasma membrane as determined by gene fusions with the green fluorescent protein. We deleted the whole locus of 7 genes (>30 kb) using CRISPR-Cas9 genome editing as a first step in studying the potential function of the four uncharacterized FBT genes from the locus. This knock out strain was viable, highly resistant to sinefungin (an AdoMet analogue) and to methotrexate (a folate analogue) but not to allopurinol, pentamidine or 5-fluorouracil. We similarly studied another FBT family member whose gene is encoded on chromosome 19. The protein was also located at the plasma membrane and its gene was dispensable for growth and not associated to any of the drug tested. Our work has indicated that large diploid deletion is achievable in <em>Leishmania</em> and the cell lines produced here will serve to better understand the function and putative substrates of these FBT proteins yet to be characterized.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"268 ","pages":"Article 108880"},"PeriodicalIF":1.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142871894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-01DOI: 10.1016/j.exppara.2024.108863
Maryam Shirazian , Niloofar Taghipour , Amir Ahmad Akhavan , Seyyed Javad Seyyed Tabaei , Mohammad Reza Abaei , Fahimeh Firouzjaie , Mahboubeh Fatemi , Nariman Mosaffa , Vahideh Moin Vaziri
Early interactions between Leishmania-macrophages (MQ) of host and effect of sand fly saliva are central to leishmaniasis outcome. Macrophages are able to kill or act as long-term hosts of parasite depending on host immunity. It proved that immunogenic proteins in sand fly saliva mostly have an exacerbating effect on leishmaniasis by up-regulating cytokines. We have explored expression of Arginase 1 (ARG1) and inducible Nitric Oxide Synthase (iNOS) genes in macrophages of three different rodents, BALB/c (susceptible), C57BL/6 (resistant) and Rhombomys opimus (R. opimus, natural reservoir) in presence of Leishmania major (L. major), salivary gland homogenate (SGH) of Phlebotomus papatasi (Ph. papatasi) and finally L. major + SGH. Stationary phase of promastigotes was used; salivary glands were extracted from female of Ph. papatasi (3–5 day-old/non-blood fed). SGH prepared by sonication. Macrophages harvested from the peritoneal cavity of each rodent and grouped as follow; 1) macrophage (control group), 2) MQ + L.major 3) MQ + SGH 4) MQ + L.major + SGH. After 6 h of incubation, culture medium supernatant collected, RNA extraction and cDNA synthesis performed, expression level of desired genes checked by Real-time PCR. ARG1 expression pattern in MQ + SGH and MQ + L.major group showed the highest and lowest expressions level respectively in BALB/c and C57BL/6. But, in MQ + L.major + SGH, although the highest ARG1 expression happened in BALB/c again, the lowest one observed in R. opimus. On the other hand, iNOS expression showed significant increase in all treated group of C57BL/6 macrophages. Interestingly, iNOS expression showed significant differences in MQ + L.major group of C57BL/6 in comparison to other rodents. Expression of ARG1 and iNOS in macrophages of BALB/c and C57BL/6 and R. opimus are different and can justify their clinical outcome of disease. The difference in gene expression pattern is related to the genetics of host and shows that genetic differences between hosts can affect the immune responses caused by saliva proteins even of the same species of sand fly.
{"title":"Expression pattern of ARG1 and iNOS genes in macrophages of Rhombomys opimus, BALB/c and C57BL/6 mice exposed to Leishmania major and salivary gland homogenates of Phlebotomus papatasi","authors":"Maryam Shirazian , Niloofar Taghipour , Amir Ahmad Akhavan , Seyyed Javad Seyyed Tabaei , Mohammad Reza Abaei , Fahimeh Firouzjaie , Mahboubeh Fatemi , Nariman Mosaffa , Vahideh Moin Vaziri","doi":"10.1016/j.exppara.2024.108863","DOIUrl":"10.1016/j.exppara.2024.108863","url":null,"abstract":"<div><div>Early interactions between <em>Leishmania</em>-macrophages (MQ) of host and effect of sand fly saliva are central to leishmaniasis outcome. Macrophages are able to kill or act as long-term hosts of parasite depending on host immunity. It proved that immunogenic proteins in sand fly saliva mostly have an exacerbating effect on leishmaniasis by up-regulating cytokines. We have explored expression of Arginase 1 (ARG1) and inducible Nitric Oxide Synthase (iNOS) genes in macrophages of three different rodents, BALB/c (susceptible), C57BL/6 (resistant) and <em>Rhombomys opimus</em> (<em>R. opimus</em>, natural reservoir) in presence of <em>Leishmania major</em> (<em>L. major</em>), salivary gland homogenate (SGH) of <em>Phlebotomus papatasi</em> (<em>Ph. papatasi</em>) and finally <em>L. major</em> + SGH. Stationary phase of promastigotes was used; salivary glands were extracted from female of <em>Ph. papatasi</em> (3–5 day-old/non-blood fed). SGH prepared by sonication. Macrophages harvested from the peritoneal cavity of each rodent and grouped as follow; 1) macrophage (control group), 2) MQ + <em>L.major</em> 3) MQ + SGH 4) MQ + <em>L.major</em> + SGH. After 6 h of incubation, culture medium supernatant collected, RNA extraction and cDNA synthesis performed, expression level of desired genes checked by Real-time PCR. ARG1 expression pattern in MQ + SGH and MQ + <em>L.major</em> group showed the highest and lowest expressions level respectively in BALB/c and C57BL/6. But, in MQ + <em>L.major</em> + SGH, although the highest ARG1 expression happened in BALB/c again, the lowest one observed in <em>R. opimus</em>. On the other hand, iNOS expression showed significant increase in all treated group of C57BL/6 macrophages. Interestingly, iNOS expression showed significant differences in MQ + <em>L.major</em> group of C57BL/6 in comparison to other rodents. Expression of ARG1 and iNOS in macrophages of BALB/c and C57BL/6 and <em>R. opimus</em> are different and can justify their clinical outcome of disease. The difference in gene expression pattern is related to the genetics of host and shows that genetic differences between hosts can affect the immune responses caused by saliva proteins even of the same species of sand fly.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"267 ","pages":"Article 108863"},"PeriodicalIF":1.4,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142709684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-20DOI: 10.1016/j.exppara.2024.108864
Reem Hoblos , Karl Khalil , Marc Karam , Samer Bazzi
Cutaneous leishmaniasis caused mainly by Leishmania major (L. major) is one of the trending models used to investigate induced hyperalgesia and the involved cytokines. Previous studies approached the role of several cytokines in the observed hyperalgesia, but the molecular mechanisms orchestrating such a response still needed to be addressed. In this study, we inspect the role of the NF-κB in the modulation of L. major-prompted hyperalgesia and cytokine expression in BALB/c mice by administering celastrol, a potent blocker of this transcription factor. Intraperitoneal injection of 0.5 mg/kg and 1 mg/kg of celastrol attenuated the L. major-induced thermal hyperalgesia in BALB/c mice for 15 days and 21 days, respectively, as detected by hot plate and tail flick behavioral assessments. Cytokine levels were quantified in the infected paws of BALB/c mice using Sandwich ELISA. The administration of 1 mg/kg celastrol decreased TNF-α levels in L. major infected mice for 23 days, and IL-1β expression declined significantly for 23 days using both celastrol dosages. However, no significant change was observed in the levels of IL-10 in our experimental groups. The activation of NF-κB was detected by observing the phosphorylation levels of the p65 subunit using PathScan phospho-ELISA. The level of NF-κB phosphorylation was elevated in L. major infected BALB/c mice. Only administering 1 mg/kg celastrol suppressed the phosphorylation of p65, thus inactivating NF-kB. In conclusion, our results provide new insights into the correlation between the activation of NF-kB, the induction of thermal hyperalgesia, and the expression of TNF-α and IL-1β in the L. major-induced hyperalgesia model.
{"title":"The role of NF-κB transcription factor in the regulation of cytokine induced thermal hyperalgesia in a Leishmania major model in BALB/c mice","authors":"Reem Hoblos , Karl Khalil , Marc Karam , Samer Bazzi","doi":"10.1016/j.exppara.2024.108864","DOIUrl":"10.1016/j.exppara.2024.108864","url":null,"abstract":"<div><div>Cutaneous leishmaniasis caused mainly by <em>Leishmania major</em> (<em>L. major</em>) is one of the trending models used to investigate induced hyperalgesia and the involved cytokines. Previous studies approached the role of several cytokines in the observed hyperalgesia, but the molecular mechanisms orchestrating such a response still needed to be addressed. In this study, we inspect the role of the NF-κB in the modulation of <em>L. major-prompted</em> hyperalgesia and cytokine expression in BALB/c mice by administering celastrol, a potent blocker of this transcription factor. Intraperitoneal injection of 0.5 mg/kg and 1 mg/kg of celastrol attenuated the <em>L. major</em>-induced thermal hyperalgesia in BALB/c mice for 15 days and 21 days, respectively, as detected by hot plate and tail flick behavioral assessments. Cytokine levels were quantified in the infected paws of BALB/c mice using Sandwich ELISA. The administration of 1 mg/kg celastrol decreased TNF-α levels in <em>L. major</em> infected mice for 23 days, and IL-1β expression declined significantly for 23 days using both celastrol dosages. However, no significant change was observed in the levels of IL-10 in our experimental groups. The activation of NF-κB was detected by observing the phosphorylation levels of the p65 subunit using PathScan phospho-ELISA. The level of NF-κB phosphorylation was elevated in <em>L. major</em> infected BALB/c mice. Only administering 1 mg/kg celastrol suppressed the phosphorylation of p65, thus inactivating NF-kB. In conclusion, our results provide new insights into the correlation between the activation of NF-kB, the induction of thermal hyperalgesia, and the expression of TNF-α and IL-1β in <em>the L. major-</em>induced hyperalgesia model.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"267 ","pages":"Article 108864"},"PeriodicalIF":1.4,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142692833","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-11-13DOI: 10.1016/j.exppara.2024.108862
Mikkel C.E. Ward, Ann M. Fallon
Oxidative stress generated as a normal byproduct of aerobic metabolism is minimized by the enzyme catalase (CAT; EC 1.11.1.6), which reduces hydrogen peroxide to molecular oxygen and water. In various mosquitoes, hydrogen peroxide and/or CAT activity have been implicated in oxidative responses to viral and protozoal pathogens as well as in ovarian maturation and insecticide resistance. We combined features of various CAT assays to develop a simple micro-assay that enables comparison of enzyme activities in individual mosquito tissues on a microscope slide. Activity recovered in the supernatant of mosquito whole body homogenates was inhibited by the CAT-specific inhibitor 3-amino-1,2,4-triazole. Activity was higher in blood-fed mosquitoes, consistent with exogenous enzyme in vertebrate blood. Triton X-100 improved evaluation of dissected organs, and accurate comparisons required careful removal of extraneous tissues. In unfed mosquitoes baseline CAT activity was lower in ovaries than in midgut or fatbody, but increased as oocytes matured after a blood meal, and was detectable in a single mature egg. CAT has unusual kinetics and can be difficult to assay directly. Our observations provide a simple approach for direct evaluation of CAT activity independent of changes in transcript levels and results of RNAi-based interference.
{"title":"A rapid and simple micro-assay to assess catalase activity in individual mosquito tissues","authors":"Mikkel C.E. Ward, Ann M. Fallon","doi":"10.1016/j.exppara.2024.108862","DOIUrl":"10.1016/j.exppara.2024.108862","url":null,"abstract":"<div><div>Oxidative stress generated as a normal byproduct of aerobic metabolism is minimized by the enzyme catalase (CAT; EC 1.11.1.6), which reduces hydrogen peroxide to molecular oxygen and water. In various mosquitoes, hydrogen peroxide and/or CAT activity have been implicated in oxidative responses to viral and protozoal pathogens as well as in ovarian maturation and insecticide resistance. We combined features of various CAT assays to develop a simple micro-assay that enables comparison of enzyme activities in individual mosquito tissues on a microscope slide. Activity recovered in the supernatant of mosquito whole body homogenates was inhibited by the CAT-specific inhibitor 3-amino-1,2,4-triazole. Activity was higher in blood-fed mosquitoes, consistent with exogenous enzyme in vertebrate blood. Triton X-100 improved evaluation of dissected organs, and accurate comparisons required careful removal of extraneous tissues. In unfed mosquitoes baseline CAT activity was lower in ovaries than in midgut or fatbody, but increased as oocytes matured after a blood meal, and was detectable in a single mature egg. CAT has unusual kinetics and can be difficult to assay directly. Our observations provide a simple approach for direct evaluation of CAT activity independent of changes in transcript levels and results of RNAi-based interference.</div></div>","PeriodicalId":12117,"journal":{"name":"Experimental parasitology","volume":"267 ","pages":"Article 108862"},"PeriodicalIF":1.4,"publicationDate":"2024-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142617226","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号