Toxoplasma gondii and pre-treatment protocols for polymerase chain reaction analysis of milk samples: a field trial in sheep from Southern Italy

Abstract

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 polymerase chain reaction (PCR) positivity. This protocol was then used to analyse milk samples from sheep of three different farms in Southern Italy, including real time PCR for DNA quantification and PCR-restriction fragment length polymorphism for genotyping. The pre-treatment protocol using ethylenediaminetetraacetic acid 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 unpasteurised derivatives.

Occurrence of Toxoplasma gondii in raw goat, sheep, and camel milk in Upper Egypt

“…Many different molecular techniques are used for diagnosis of T. gondii in milk samples as conventional PCR [ 18 , 19 ], nested PCR [ 23 ], PCR-RFLP technique [ 24 ], and qPCR [ 25 , 26 ]. qPCR assay is the most rapid, accurate, and effective molecular technique that can be used for Toxoplasma detection [ 27 , 28 ].…”

Public health risks associated with food‐borne parasites

“…The main detection methods used on raw milk samples involve the detection of T. gondii DNA using PCR‐based tests, most commonly targeting the 529 bp repeat element (Bezerra et al., ; de Santana Rocha et al., ; da Silva et al., ; Vismarra et al., ) or the B1 gene (Dehkordi et al., ).…”

“…T. gondii has been detected in raw milk from infected animals using a variety of techniques, mainly using PCR, tissue culture, and in vivo bioassays (Dehkordi et al., ). A recent study sampled 21 milk samples taken from three different sheep farms in southern Italy and found one milk sample to be positive using a PCR assay (Vismarra et al., ). A previous study in Italy looking at goat milk found 13% of 77 samples to be positive using a T. gondii ‐specific PCR test (Mancianti et al., ).…”

Apicomplexan Protozoa Responsible for Reproductive Disorders: Occurrence of DNA in Blood and Milk of Donkeys (Equus asinus) and Minireview of the Related Literature

“…The presence of T. gondii DNA in camel milk has been reported in several studies [ 38 , 39 , 54 ], even though in another study all specimens tested negative [ 53 ]. The literature dealing with potential infection of ovine milk has been recently reviewed [ 57 ], and T. gondii DNA and viable tachyzoites occurrence has also been reported [ 38 , 39 , 48 , 53 , 54 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. Camossi et al [ 59 ] reported the presence of T. gondii DNA in milk from naturally infected ewes and detected T. gondii DNA twice in the milk of two sheep, suggesting a possible resurgence of tachyzoites from cysts, which can circulate again and be excreted through the milk.…”