Real-time PCR detection of Toxoplasma gondii in tissue samples of wild boars (Sus scrofa) from southern Italy reveals high prevalence and parasite load

Toxoplasmosis is a zoonotic disease caused by the protozoan Toxoplasma gondii. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on T. gondii tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable PCR positivity. This protocol was then used to analyze milk samples form sheep from three different farms in southern Italy, including Real Time PCR for DNA quantification and PCR-RFLP for genotyping. The pre-treatment protocol using EDTA and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, Real Time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of T. gondii transmission through consumption of raw milk and its unpasteurized derivatives.

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Ability of Real-time PCR in diagnose Differentiation Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

10.21203/rs.2.12885/v1 ◽

2019 ◽

Author(s):

Maryam Fekri Soofi Abadi ◽

Meisam Fekri ◽

alireza moradabadi ◽

Reza Vahidi ◽

Simin Shamsi Meymandi ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

Relative Number ◽

Parasite Load ◽

Detection Methods ◽

Tissue Samples ◽

Microscopic Evaluation ◽

Histopathological Evaluation ◽

Histopathological Studies

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

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Frequency and parasite load of Toxoplasma gondii in hemodialysis patients based on RE gene by real-time PCR

Gene Reports ◽

10.1016/j.genrep.2021.101145 ◽

2021 ◽

Vol 23 ◽

pp. 101145

Author(s):

Hadi Mirahmadi ◽

Batol Nozari ◽

Vahid Raissi ◽

Ebrahim Alijani ◽

Soudabeh Etemadi

Keyword(s):

Toxoplasma Gondii ◽

Real Time ◽

Real Time Pcr ◽

Parasite Load ◽

Hemodialysis Patients

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Real-time PCR-based quantification of Toxoplasma gondii in tissue samples of serologically positive outdoor chickens

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02762010000700018 ◽

2010 ◽

Vol 105 (7) ◽

pp. 935-937 ◽

Cited By ~ 13

Author(s):

Cleiton Paulo Aigner ◽

Aristeu Vieira da Silva ◽

Fabiano Sandrini ◽

Paulo de Sá Osório ◽

Lilian Poiares ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Real Time ◽

Real Time Pcr ◽

Tissue Samples

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Ability of Real-time PCR for Differential Diagnosis of Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

10.21203/rs.2.12885/v4 ◽

2019 ◽

Author(s):

Maryam Fekri Soofi Abadi ◽

Meisam Fekri ◽

alireza moradabadi ◽

Reza Vahidi ◽

Simin Shamsi Meymandi ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

Relative Number ◽

Parasite Load ◽

Detection Methods ◽

Tissue Samples ◽

Microscopic Evaluation ◽

Histopathological Evaluation ◽

Histopathological Studies

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

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Ability of Real-time PCR for Differential Diagnosis of Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

10.21203/rs.2.12885/v2 ◽

2019 ◽

Author(s):

Maryam Fekri Soofi Abadi ◽

Meisam Fekri ◽

alireza moradabadi ◽

Reza Vahidi ◽

Simin Shamsi Meymandi ◽

...

Keyword(s):

Cutaneous Leishmaniasis ◽

Real Time ◽

Real Time Pcr ◽

Relative Number ◽

Parasite Load ◽

Detection Methods ◽

Tissue Samples ◽

Microscopic Evaluation ◽

Histopathological Evaluation ◽

Histopathological Studies

Abstract objective: Histopathological studies suggest that parasite load is different between acute and chronic forms of cutaneous leishmaniasis (CL). However, highly sensitive detection methods are still needed to distinguish different forms of leishmaniasis. In the present study, we developed a quantitative real-time polymerase chain reaction (PCR) to detect and quantify leishmania tropica parasites in paraffin-embedded tissue samples. Results: The ability of real-time PCR for leishmania detection was higher than histopathological evaluation. The parasite loads were quantified by qPCR assay and microscopic evaluation were highly correlated ( r =0.598; P <0.001). Among patients, the parasite load was inversely correlated with disease duration (acute CL lesions had very higher parasite loads than chronic CL lesions), but there was no difference in parasite load according to the patients’ age and sex as well as location of the lesions. In contrast to Ridley scoring system (P<0.001), there were no statistically significant differences in the relative number of parasites among the lupoid and non-lupoid forms of chronic lesions in real-time PCR (P=0.549), which indicates the superiority of histopathological evaluation in CL forms differentiation.

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Leptospira fainei Detected in Testicles and Epididymis of Wild Boar (Sus scrofa)

Biology ◽

10.3390/biology10030193 ◽

2021 ◽

Vol 10 (3) ◽

pp. 193

Author(s):

Giovanni Cilia ◽

Fabrizio Bertelloni ◽

Domenico Cerri ◽

Filippo Fratini

Keyword(s):

Wild Boar ◽

Real Time ◽

Reproductive System ◽

Real Time Pcr ◽

Sus Scrofa ◽

Rrna Gene ◽

Pathogenic Leptospira ◽

Wild Boars ◽

First Time ◽

Opening Up

Leptospirosis is a re-emerging and worldwide diffused zoonosis. Recently, the high importance of their epidemiology was explained by the intermediate Leptospira strains. Among these strains, Leptospira fainei was the first intermediate strain detected in domestic and wild swine. Wild boars (Sus scrofa) are well known as a reservoir, as well as all swine, for pathogenic Leptospira, but very little information is available concerning intermediate Leptospira infection. The investigation aim was to evaluate if intermediate Leptospira can infect the reproductive systems of wild boars hunted in the Tuscany region (Italy), as previously demonstrated for pathogenic ones. The reproductive system tissue (testicles, epididymides, uteri), and placentas and fetuses, were collected from 200 regularly hunted animals. Bacteriological examination and real-time PCR were performed to detect intermediate Leptospira DNA. Unfortunately, no isolates were obtained. Using real-time PCR, in six (3%) male organs (both testicles and epididymis), intermediate Leptospira DNA was found. The amplification of the 16S rRNA gene identified that all DNA obtained belong to Leptospira fainei. The results of this investigation highlighted for the first time the localization of Leptospira fainei in the male wild boar reproductive system, opening up a new avenue to further investigate.

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