Preliminary verification of molecular techniques to more accurately assess the risk from Toxoplasma gondii in pork

Toxoplasma gondii is a two-host meat borne protozoan parasite, with felines being the primary host and all other warm-blooded animals (including humans) as secondary hosts, in which it causes lifelong infection (Tenter et al. 2000). Secondary hosts can become infected from either faeces of an infected cat or from consumption of undercooked infected meat (including pork). The ability of T. gondii to cross the placental barrier and infect the foetus, as well as its ability to emerge from hibernation within muscle cysts during periods of immune suppression and its association with schizophrenia, has led to increasing public health concern (Flegr 2013). Traditional serological diagnosis of infection has proved problematic when attempting to assess the risk of human exposure through the consumption of undercooked meat, with different tests giving widely varying results (Dubey 2009). Molecular methods such as polymerase chain reaction (PCR) have low sensitivity, particularly in pigs/pork, as there is a low density of cysts in muscle tissue. This preliminary study investigated a method to (1) concentrate the diffuse Toxoplasma bradyzoites in meat for identification by both nested and qPCR, followed by (2) a bioassay to determine both the accuracy of quantification, and (3) the continued infectivity of the concentrated bradyzoites. The aim of this project was to trial and verify methods developed overseas for use in Australia on meat, to support assessment of the risk of consumer exposure.

Infected brain material was obtained from Swiss-Webster mice injected subcutaneously 12 weeks earlier with an inoculum of tissue culture Toxoplasma tachyzoites. The PCR and qPCR estimated a concentration of 2.7 × 10⁵ T. gondii/20 mg of brain. A 50 g sample of previously frozen pork mince was spiked with 300 mg of mouse brain, then digested with pepsin, filtered, centrifuged and re-suspended in 5 mL of 0.9% saline as described by Dubey (1998). After quantification by qPCR, three pairs of fresh mice were then subcutaneously injected with an estimated 10⁵, 10³ or 10¹ T. gondii. Clinical signs and cysts were observed in both the 10⁵ and 10³ tachyzoite-infected mice, and one of the 10⁵ infected pair died. The five surviving mice were euthanised after 12 weeks. Infection was confirmed in the 10⁵ and 10³ infected mice by qPCR with estimated levels of 2.3 × 10³ and 4 × 10³ T. gondii/20 mg of brain, respectively, but not in the 10¹ mouse, suggesting the infective mouse dose for this post-digestion Toxoplasma pig strain lay between 10¹ and 10³ organisms.

An opportunistic preliminary estimate of the recovery rate of T. gondii from spiked pork mince following acid/pepsin digestion was conducted by preparing two 50 g pork mince samples: one as a control, and one spiked with an estimated 4.6 × 10⁴ T. gondii organisms. Both mince samples were processed by pepsin digestion/centrifugation and the re-suspension examined by qPCR. The re-suspension from the control mince contained no detectable Toxoplasma DNA, while that from the spiked mince contained an estimated 1.1 × 10⁵ T. gondii. Despite the acknowledged lack of replicates, this single result suggests recovery of the majority of the spiking organisms is possible.

In conclusion, a refined molecular test was established to concentrate and detect T. gondii in meat samples. The Dubey (1998) digestion/centrifugation technique was verified to improve sensitivity and the T. gondii recovery rate investigated. An effective mouse bioassay was developed to enable verification of the continued infectivity of T. gondii detected in meat samples following the digestion-PCR method, and multiplication of detected strains to allow future genotyping if required. Standard test methods and laboratory procedures that cover extraction and detection of T. gondii in meat samples are now available to allow the pork industry to determine risks to human health associated with the consumption of undercooked pork products that has not undergone a kill step for T. gondii.