Title : Phylogenetic relationship of toxoplasma gondii in dolphins stranded in sicily (South Italy)
Abstract:
Toxoplasma gondii, a protozoan pathogen causing zoonosis, represents a serious threat for aquatic mammals including dolphins, as it causes severe brain lesions in dolphins leading them to stranding and death. This study aimed to investigate T. gondii DNA presence in organs collected from dolphins stranded along Sicilian coasts and to carry out sequence analysis based on gra6 gene.
From 2021 to 2023, n.16 dolphins were analysed, found stranded along the coast of Sicily. The animals belonged to different species, including Stenenella coeruleoalba, Delphinus delphis and Tursiops truncatus. Different organs were collected, including brain, spleen, liver, lung, lymph node, muscle and heart, for a total of 87 examined organs. One gram of tissue sample, diluted in 9 mL of saline solution, was homogenized in the Stomacher. Genomic DNA was extracted from the homogenate using a commercial kit. T. gondii DNA was amplified by both a nested PCR targeting the B1 gene and a TaqMan Real Time PCR targeting the 529 bp repeat element.
A fragment of 773 bp of the gra6 gene was amplified in positive samples by nested PCRs and sequenced. Obtained sequences were analysed using BioEdit and MEGA version 7.0. Phylogenetic analyses were performed by neighbour-joining using the Maximum Composite Likelihood method. Phylogenetic tree was constructed with several valid type I (RH), II (Beverley and ME49) and III (NED, TONT and C56) T. gondii strains.
Two dolphins resulted positive for T. gondii DNA. The nucleotide sequences of gra6 fragment was obtained from all the different organs of one of them, a S. coeruleoalba found in the Western coast of Sicily and all the examined organs of this animal (brain, lymph nodes, spleen, hearth, liver and muscle) were positive, including those of choice for this pathogen. Gra6 sequence revealed high percentage of sequence similarity with the published sequences of T. gondii gra6, thereby establishing its specificity.
In silico analysis of the consensus obtained sequence of gra6, its subsequent phylogenetic analysis and pairwise distance calculations revealed the closest genetic relationship of the T. gondii strain from Sicilian stranded dolphin with that of type III strains (NED, TONT and C56).
T. gondii Type II lineage as well as a Type II-atypical isolate have been previously characterized from dolphins stranded along the northern coasts of Italy. Further studies will be addressed to the phylogenetic characterization of T.gondii detected in the second dolphin.
The positive animal belonged to cetacean species living in the open sea, making interesting better understanding the transmission routes of T. gondii for such animals in this area. Further studies are necessary to better characterize the genetic configuration of the strain and to clarify its possible origin and transmission routes in aquatic mammals.
Audience Take Away:
- The study provides new insights into the spread of T. gondii infection in dolphins.
- Phylogenetic analysis revealed the closest genetic relationship of the obtained T. gondii strain with that of type III strains
- The study provide information to better understand the transmission routes of T. gondii in aquatic mammals in Sicilian sea
