Viability of DNA detection and genetic sequencing of Toxoplasma gondii in beef milk and fresh cheese samples

• Grant number: 25/13487-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2025
• End date: September 30, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Gustavo Felippelli
• Grantee: Gabrielle Jacobs de Lima
• Host Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

The diagnosis of Toxoplasma gondii through raw cow's milk is controversial and has been little studied, as it is a zoonotic disease of great relevance in the context of public health, the objective of this study is to investigate the seroprevalence and molecular detection of Toxoplasma gondii in lactating cows on farms with low sanitary control in the north-western region of São Paulo, which produce fresh (raw) cheese as their main dairy product. Small to medium-sized farms whose production activity is dairy farming will be selected, totalling an average range of 10 to 200 animals per farm. A questionnaire will be conducted covering the health profile of the farms. Immunoparasitological tests (indirect immunofluorescence) will be performed to detect the presence of animals with IgM and IgG titres. Milk aliquots will be collected from each mammary quarter (50 to 200 samples) per animal and stored in DNAse-free Falcon tubes. Fresh cheeses (100 samples) made artisanally with milk from these animals from these properties will be purchased. DNA extraction will follow the methodology performed by (Pokorska et al., 2016): For the detection of Toxoplasma gondii, PCR is performed, targeting the 529 bp non-coding fragment (Primers: TOX4 and TOX5). The amplicons from the positive samples will be extracted and purified from the agarose gel using the GenElute¿ Gel Extraction Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's protocol. Afterwards, the amplicons will be sent for bidirectional Sanger sequencing, using the secondary reaction primers as initiators. After sequencing, consensus sequences will be determined using Codoncode Aligner version 4.0.1 (CodonCode Corporation Dedham®, MA, USA) software and aligned with homologous sequences available in GenBank using Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree will be inferred by the maximum likelihood method and based on the Tamura 3-parameter genetic distance model. Branching will be performed with bootstrap support, based on 1000 replications, and the phylograms will be drawn using Mega sequence align version 11. The sequences obtained in this study will be deposited in Genbank. The data resulting from the PCR protocol will be analysed using the chi-square test with the aid of Jamovi 2.3.28 and Epi Info 7.2.6.0 software to determine the prevalence of the variables.