Evaluation of Toxocara canis Glycosylated TES Produced in Pichia pastoris for Immunodiagnosis of Human Toxocariasis

Abstract Recombinant proteins are a suggested alternative for the diagnosis of toxocariasis. The current Escherichia coli recombinant protein overexpression system usually produces insoluble products. As an alternative, yeast such as Pichia pastoris have secretory mechanisms, which could diminish the cost and time for production. This study aimed to produce recombinant proteins in Pichia pastoris and verify their sensibility and specificity in an indirect ELISA assay. Two sequences (rTES-30 and rTES-120) of Toxocara canis excretory-secretory antigens were cloned in a pPICZαB vector and expressed in P. pastoris KM71H. Sera samples collected from human adults infected by Toxocara spp. were tested by indirect ELISA using rTES-30 and rTES-120 as antigens. Recombinant proteins were detected at 72 hours after induction, in the supernatant, as pure bands between 60~70 kDa with hyperglycosylation. Regarding diagnosis potential, recombinant antigens had high specificity (95.6%); however, sensitivity was 55.6% for rTES-30 and 68.9% for rTES-120. Further deglycosylation of the P. pastoris antigens did not seem to affect ELISA performance (p>0.05). The low sensitivity in the serodiagnosis diminished any advantage that P. pastoris expression could have. Therefore, we do not recommend P. pastoris recombinant TES production as an alternative for the diagnosis of toxocariasis.

Influence of Pichia pastoris X-33 produced in industrial residues on productive performance, egg quality, immunity, and intestinal morphometry in quails.

This study was conducted in quails to evaluate the probiotic potential of Pichia pastoris X-33, cultivated in parboiled rice effluent supplemented with biodiesel glycerol or in standard medium Yeast Extract-Peptone-Dextrose (YPD). Forty-days-old female quails were divided into three treatments: T1 (Control) received a basal diet without P. pastoris; T2 (Pichia Effluent) received a basal diet supplemented with P. pastoris grown in parboiled rice effluent and biodiesel glycerol, and T3 (Pichia YPD) received a basal diet supplemented with P. pastoris produced in YPD. The birds were vaccinated against Newcastle Disease (NDV), Avian Infectious Bronchitis (IBV), and Gumboro Disease on days 1 and 28. The following parameters were analyzed: performance, egg quality, humoral immune response to the vaccines, organ weight, and intestinal morphometry. P. pastoris grown in YPD increased egg weight (p < 0.05). The lowest liver weight on day 14 was obtained in Pichia Effluent, whereas both P. pastoris supplemented groups had the lowest duodenum weights on day 14. Besides that, livers and duodenums presented no morphological changes in any of the three treatments. Supplementation of P. pastoris modulated the immune system of the birds, increasing anti-IBV, anti-NDV, and anti-Gumboro antibodies levels compared to the Control (p < 0.05). In conclusion, quail's immune response was improved by Pichia pastoris X-33, either it was grown in YPD or industrial residues, and the egg weight increased with Pichia pastoris X-33 grown in YPD, thereby demonstrating to be a promising probiotic for poultry.