scholarly journals Real-time PCR on skin biopsies for super-spreaders’ detection in bovine besnoitiosis.

2020 ◽  
Author(s):  
Christelle Grisez ◽  
Leslie Bottari ◽  
Françoise Prévot ◽  
Jean-Pierre Alzieu ◽  
Emmanuel Liénard ◽  
...  
Keyword(s):  
Real Time ◽  
Control Strategy ◽  
Real Time Pcr ◽  
Treatment Success ◽  
Acute Stage ◽  
Besnoitia Besnoiti ◽  
Skin Sample ◽  
Production Type ◽  
Bovine Besnoitiosis ◽  
Skin Biopsies

Abstract Background: Bovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the Apicomplexa Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. Methods: Previous real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (N = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time.Results: Among the 518 seropositive animals, a low proportion of individuals (14.5%) showed Ct values below 36, 17.8% had doubtful results (36 < Ct ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographic location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. Conclusions: Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

scholarly journals Real-time PCR on skin biopsies for super-spreaders’ detection in bovine besnoitiosis

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Christelle Grisez ◽  
Leslie Bottari ◽  
Françoise Prévot ◽  
Jean-Pierre Alzieu ◽  
Emmanuel Liénard ◽  
...  
Keyword(s):  
Real Time ◽  
Control Strategy ◽  
Real Time Pcr ◽  
Treatment Success ◽  
Acute Stage ◽  
Besnoitia Besnoiti ◽  
Skin Sample ◽  
Production Type ◽  
Bovine Besnoitiosis ◽  
Skin Biopsies

Abstract Background Bovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the apicomplexan Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. Methods Previous real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (n = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time. Results Among the 518 seropositive animals, a low proportion of individuals (14.5%) showed Cq values below 36, 17.8% had doubtful results (36 < Cq ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographical location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. Conclusions Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

scholarly journals Real-time PCR on skin biopsies for super-spreaders’ detection in bovine besnoitiosis

2020 ◽  
Author(s):  
Christelle Grisez ◽  
Leslie Bottari ◽  
Françoise Prévot ◽  
Jean-Pierre Alzieu ◽  
Emmanuel Liénard ◽  
...  
Keyword(s):  
Real Time ◽  
Control Strategy ◽  
Real Time Pcr ◽  
Treatment Success ◽  
Acute Stage ◽  
Besnoitia Besnoiti ◽  
Skin Sample ◽  
Production Type ◽  
Bovine Besnoitiosis ◽  
Skin Biopsies

Abstract Background: Bovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the apicomplexan Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. Methods: Previous real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (n = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time.Results: Among the 518 seropositive animals, a low proportion of individuals (14.5%) showed Cq values below 36, 17.8% had doubtful results (36 < Cq ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographical location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. Conclusions: Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

scholarly journals Real-time PCR on skin biopsies for super-spreaders’ detection in bovine besnoitiosis.

2020 ◽  
Author(s):  
Christelle Grisez ◽  
Leslie Bottari ◽  
Françoise Prévot ◽  
Jean-Pierre Alzieu ◽  
Emmanuel Liénard ◽  
...  
Keyword(s):  
Real Time ◽  
Control Strategy ◽  
Real Time Pcr ◽  
Treatment Success ◽  
Acute Stage ◽  
Besnoitia Besnoiti ◽  
Skin Sample ◽  
Production Type ◽  
Bovine Besnoitiosis ◽  
Skin Biopsies

Abstract Background: Bovine besnoitiosis, an emerging disease in Europe that can be transmitted by vectors, is caused by the apicomplexan Besnoitia besnoiti. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. Methods: Previous real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (n = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled at the tail base and their skin sample analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time.Results: Among the 518 seropositive animals, a low proportion of individuals (14.5%) showed Cq values below 36, 17.8% had doubtful results (36 < Cq ≤ 40) and 67.8% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographical location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. Conclusions: Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

scholarly journals Super-spreaders: detection and culling in the control of bovine besnoitiosis.

2020 ◽  
Author(s):  
Christelle Grisez ◽  
Leslie Bottari ◽  
Françoise Prévot ◽  
Jean-Pierre Alzieu ◽  
Emmanuel Liénard ◽  
...  
Keyword(s):  
Real Time ◽  
Control Strategy ◽  
Real Time Pcr ◽  
Treatment Success ◽  
Geographic Location ◽  
Acute Stage ◽  
Mechanical Transmission ◽  
Besnoitia Besnoiti ◽  
Production Type ◽  
Bovine Besnoitiosis

Abstract Background Bovine besnoitiosis is an emerging vector-borne disease in Europe caused by the Apicomplexa Besnoitia besnoiti. The mechanical transmission from infected to naïve hosts is permitted by horse flies and stable flies. Bovine besnoitiosis is difficult to control due to the complexity of its diagnosis in the acute stage of the disease, poor treatment success and chronically asymptomatic cattle acting as parasite reservoirs. When serological prevalence is low, detection and specific culling of seropositive cattle is feasible; however, economic considerations preclude this approach when serological prevalence is high. The aims of this study were to evaluate the accuracy of detection of super-spreaders in highly infected herds and to test their selective elimination as a new control strategy for bovine besnoitiosis. Methods Real-time PCR analyses performed on skin tissues from 160 asymptomatic animals sampled at slaughterhouses showed that the tail base was the best location to evaluate the dermal parasite DNA load. All seropositive animals (N = 518) from eight dairy or beef cattle farms facing a high serological prevalence of besnoitiosis were sampled and their skin analysed by real-time PCR. A recommendation of rapid and selective culling of super-spreaders was formulated and provided to the cattle breeders. Subsequent serological monitoring of naïve animals was used to evaluate the interest of this control strategy over time. Results Among the 518 seropositive animals, a low proportion of individuals (13%) showed Ct values below 36, 17% had doubtful results (36 < Ct ≤ 40) and 70% had negative PCR results. These proportions were grossly similar on the eight farms, regardless of their production type (beef or dairy cattle), size, geographic location or history of besnoitiosis. Within two weeks of the biopsy, the rapid culling of super-spreaders was implemented on only three farms. The numbers of newly infected animals were lower on these farms compared to those where super-spreaders were maintained in the herd. Conclusions Real-time PCR analyses performed on skin biopsies of seropositive cattle showed huge individual variabilities in parasite DNA load. The rapid culling of individuals considered as super-spreaders seems to be a new and encouraging strategy for bovine besnoitiosis control.

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis

2009 ◽  
Vol 129 (1-2) ◽  
pp. 115-118 ◽  
Author(s):  
Yvette M. Schlotter ◽  
Eveline Z. Veenhof ◽  
Bas Brinkhof ◽  
Victor P.M.G. Rutten ◽  
Bart Spee ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Quantitative Real Time Pcr ◽  
Skin Biopsies ◽  
Healthy Dogs ◽  
Selection Of

scholarly journals Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine skin biopsies

2007 ◽  
Vol 146 (3-4) ◽  
pp. 352-356 ◽  
Author(s):  
Helder C.E. Cortes ◽  
Yara Reis ◽  
Bruno Gottstein ◽  
Andrew Hemphill ◽  
Alexandre Leitão ◽  
...  
Keyword(s):  
Real Time ◽  
Besnoitia Besnoiti ◽  
Its1 Rdna ◽  
Skin Biopsies

scholarly journals In vitro effects of arylimidamides against Besnoitia besnoiti infection in Vero cells

Parasitology ◽  
2011 ◽  
Vol 138 (5) ◽  
pp. 583-592 ◽  
Author(s):  
H. C. E. CORTES ◽  
N. MULLER ◽  
D. BOYKIN ◽  
C. E. STEPHENS ◽  
A. HEMPHILL
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Neospora Caninum ◽  
Vero Cells ◽  
Besnoitia Besnoiti ◽  
Drug Treatments ◽  
Important Addition ◽  
In Vitro Effects ◽  
Intracellular Proliferation

SUMMARYThe in vitro effects of 4 arylimidamides (DB811, DB786, DB750 and DB766) against the proliferative tachyzoite stage of the apicomplexan parasite Besnoitia besnoiti were investigated. These four compounds had been shown earlier to exhibit in vitro activities in the nanomolar range against the related apicomplexans Neospora caninum and Toxoplasma gondii. Real-time-PCR was used to assess B. besnoiti intracellular proliferation in vitro. Preliminary assessment by light microscopy identified DB811 and DB750 as the most promising compounds, while DB786 and DB766 were much less effective. Three-day-growth assays and quantitative real-time PCR was used for IC50 determination of DB811 (0·079 μM) and DB750 (0·56 μM). Complete growth inhibition was observed at 1·6 μM for DB 811 and 1·7 μM for DB750. However, when infected cultures were treated for 14 days, proliferation of parasites occurred again in cultures treated with DB750 from day 4 onwards, while the proliferation of DB811-treated tachyzoites remained inhibited. Electron microscopy of B. besnoiti-infected fibroblast cultures fixed and processed at different time-points following the initiation of drug treatments revealed that DB811 exerted a much higher degree of ultrastructural alterations compared to DB750. These results show that arylimidamides such as DB811 could potentially become an important addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Application of Real-time PCR to Recognize Atypical Mycobacteria in Archival Skin Biopsies

2007 ◽  
Vol 16 (2) ◽  
pp. 81-86 ◽  
Author(s):  
Lesla S. Bruijnesteijn van Coppenraet ◽  
Vincent T. H. B. M. Smit ◽  
Kate E. Templeton ◽  
Eric C. J. Claas ◽  
Edward J. Kuijper
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Atypical Mycobacteria ◽  
Skin Biopsies

scholarly journals Co Expression of GMFβ, IL33, CCL2 and SDF1 Genes in the Acute Stage of Toxoplasmosis in Mice Model and Relation for Neuronal Impairment

2021 ◽  
Author(s):  
Mehri Najafi ◽  
Razieh Amini ◽  
Amir Hossein Maghsood ◽  
Mohammad Fallah ◽  
Faeze Foroughi-Parvar
Keyword(s):  
Gene Expression ◽  
Neurodegenerative Diseases ◽  
Real Time ◽  
Real Time Pcr ◽  
Early Stage ◽  
Rna Extraction ◽  
Acute Stage ◽  
Mice Model ◽  
Toxoplasma Infection ◽  
Inflammatory Protein

Background: Toxoplasma gondii is an obligate intracellular parasite that migrates through macrophages or dendritic cells to neurons and nerve cells. Glia Maturation Factor (GMF) is a pre-inflammatory protein that is expressed in the central nervous system (CNS). GMFβ expression is related to IL33 and CCL2 and SDF1 in some neurodegenerative diseases. According to the importance of GMFβ in neurodegenerative diseases and its association with IL33, CCL2 and SDF1 genes, this study was designed to determine the level of expression of these genes in the brains of mice with acute toxoplasmosis. Methods: Tachyzoites of T. gondii RH strains were injected to 5 Swiss Albino mice. At the same time, healthy mice were inoculated with the Phosphate-buffered saline (PBS). Their brains were removed and kept at -70 oC in order to RNA extraction, cDNA syntheses and Real Time PCR performance. The level of gene expression was investigated with SYBR Green Quantitative Real-Time PCR. Results: GMFβ gene expression increased significantly (P=0.003) 3.26 fold in Toxoplasma infected mice in comparison to the control. GMFβ gene expression was associated with increased expression level of IL33, CCL2, and SDF1 genes. Conclusion: Considering the prominent role of GMFβ in CNS as well as the immune system, the elevation of GMFβ, IL33, CCL2 and SDF1 genes expression in the early stage of toxoplasmosis is associated with the occurrence of neuropathological alterations. Detection of these genes as an indication of brain damage in the early stages of Toxoplasma infection can prevent neurodegenerative disorders following acquired toxoplasmosis.

scholarly journals A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings

2021 ◽  
Vol 9 (3) ◽  
pp. 588
Author(s):  
Prakash Ghosh ◽  
Abhijit Sharma ◽  
Narayan Bhattarai ◽  
Kumar Abhishek ◽  
Thilini Nisansala ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Real Time ◽  
Real Time Pcr ◽  
Evaluation Study ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Skin Sample ◽  
Diagnostic Efficacy ◽  
Kala Azar

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45–98.42) and 88.85% (95% CI: 85.08–91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

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