Application of conventional and real-time fluorescent ITS1 rDNA PCR for detection of Besnoitia besnoiti infections in bovine 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.

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

Parasites & Vectors ◽

10.1186/s13071-020-04405-7 ◽

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.

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

10.21203/rs.3.rs-37696/v4 ◽

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.

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

10.21203/rs.3.rs-37696/v3 ◽

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.

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

Veterinary Immunology and Immunopathology ◽

10.1016/j.vetimm.2008.12.004 ◽

2009 ◽

Vol 129 (1-2) ◽

pp. 115-118 ◽

Cited By ~ 41

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

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

Parasitology ◽

10.1017/s0031182011000114 ◽

2011 ◽

Vol 138 (5) ◽

pp. 583-592 ◽

Cited By ~ 18

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.

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

Diagnostic Molecular Pathology ◽

10.1097/pdm.0b013e318033625d ◽

2007 ◽

Vol 16 (2) ◽

pp. 81-86 ◽

Cited By ~ 15

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

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Quantitative real time polymerase chain reaction assays for the sensitive detection of Besnoitia besnoiti infection in cattle

Veterinary Parasitology ◽

10.1016/j.vetpar.2011.01.038 ◽

2011 ◽

Vol 178 (3-4) ◽

pp. 208-216 ◽

Cited By ~ 39

Author(s):

G. Schares ◽

A. Maksimov ◽

W. Basso ◽

G. Moré ◽

J.P. Dubey ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Sensitive Detection ◽

Chain Reaction ◽

Besnoitia Besnoiti ◽

Polymerase Chain

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A Prospective Observational Study of Mycophenolate Mofetil Treatment in Progressive Diffuse Cutaneous Systemic Sclerosis of Recent Onset

The Journal of Rheumatology ◽

10.3899/jrheum.111229 ◽

2012 ◽

Vol 39 (6) ◽

pp. 1241-1247 ◽

Cited By ~ 50

Author(s):

FABIAN A. MENDOZA ◽

SARAH J. NAGLE ◽

JASON B. LEE ◽

SERGIO A. JIMENEZ

Keyword(s):

Mycophenolate Mofetil ◽

Systemic Sclerosis ◽

Observational Study ◽

Pulmonary Function ◽

Real Time ◽

Real Time Pcr ◽

Prospective Observational Study ◽

Pulmonary Function Tests ◽

Recent Onset ◽

Skin Biopsies

Objective.A prospective observational study of mycophenolate mofetil (MMF) treatment in patients with diffuse progressive cutaneous systemic sclerosis (SSc) of recent onset.Methods.Twenty-five previously untreated consecutive patients with recent-onset (< 24 mo) diffuse progressive cutaneous SSc received MMF as the only disease-modifying therapy. Modified Rodnan skin score (mRSS) and affected body surface area (BSA) were compared from initiation of MMF to study end. Pulmonary function tests performed at the same institution before therapy and at study end were available in 15 patients. Histopathology and real-time PCR assessment of fibrosis-related gene expression were performed before and after treatment in skin biopsies from 3 patients.Results.At 18.2 ± 8.73 months of MMF therapy (median 2000 mg/day) the mRSS decreased from 24.56 ± 8.62 to 14.52 ± 10.9 (p = 0.0004) and the affected BSA from 36% ± 16% to 14% ± 13.3% (p = 0.00001). Pulmonary function tests remained stable from initiation of MMF to the end of the study. Skin histopathology showed a remarkable reduction in accumulation of fibrotic tissue. Real-time PCR of skin biopsies demonstrated a marked decrease in expression of fibrosis-related genes.Conclusion.Patients with diffuse progressive cutaneous SSc of recent onset treated with MMF experienced marked improvement in skin involvement and stabilization of pulmonary function. Skin biopsies from 3 patients demonstrated histopathological improvement and decreased expression of fibrosis-related genes.

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Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosis

Parasitology ◽

10.1017/s0031182019001707 ◽

2019 ◽

Vol 147 (4) ◽

pp. 401-409 ◽

Cited By ~ 2

Author(s):

Ershun Zhou ◽

Liliana M. R. Silva ◽

Iván Conejeros ◽

Zahady D. Velásquez ◽

Manuela Hirz ◽

...

Keyword(s):

Protozoan Parasite ◽

Polymorphonuclear Neutrophils ◽

Extracellular Dna ◽

Economic Losses ◽

Besnoitia Besnoiti ◽

Cattle Industry ◽

Histone 3 ◽

Bovine Besnoitiosis ◽

Skin Biopsies ◽

First Time

AbstractBesnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.

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