Application of Real-time PCR to Recognize Atypical Mycobacteria in Archival 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 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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Validation of swab sampling and SYBR Green-based real-time PCR for the diagnosis of Cutaneous Leishmaniasis in French Guiana

Journal of Clinical Microbiology ◽

10.1128/jcm.02218-20 ◽

2020 ◽

Author(s):

Romain Blaizot ◽

Stéphane Simon ◽

Marine Ginouves ◽

Ghislaine Prévot ◽

Denis Blanchet ◽

...

Keyword(s):

South America ◽

Cutaneous Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

French Guiana ◽

Laboratory Diagnosis ◽

Sybr Green ◽

Non Invasive ◽

Skin Biopsies ◽

Sampling Procedures

Recent studies have highlighted the interest of non-invasive sampling procedures coupled to real-time PCR methods for detection of Leishmania species in South America. In French Guiana, sampling method still relied on skin biopsies. Non-invasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of Cutaneous Leishmaniasis. Therefore, we evaluated the performances of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR Green real-time PCR of Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with Cutaneous Leishmaniasis were included in the Cayenne Hospital and its remote health centres. Each patient underwent scrapings for smear, skin biopsies for parasite culture and PCR-RFLP (RNA pol II) and cotton swabs for SYBR Green PCR. The most accurate diagnostic test was the SYBR Green PCR on swab sampling, showing a 98% sensitivity. Mean PCR cycle threshold (Ct) was of 24.4 Ct (min=17 Ct, max=36 Ct) and was inferior to 35 Ct in 97.6% of samples. All samples positive with real-time PCR SYBR Green were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of Cutaneous Leishmaniasis in South America and especially for remote areas, as non-invasive collection tools are easier to use and require less precautions for transportation.

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