A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Determination of extracellular traps structures from sheep polymorphonuclear leukocytes to Echinococcus granulosus protoscoleces.

It was aimed to detect extracellular traps structures from sheep polymorphonuclear leukocytes (PMN) after being confronted with Echinococcus granulosus protoscoleces in vitro. Also, the effect of cyst fluid was examined on the development of extracellular traps. At the end of the incubation for 1 h, the extracellular traps augmented with neutrophil elastase, histone (H3) and myeloperoxidase were visualized in the protoscoleces-PMN co-culture microscopically. Some protoscoleces lysed and the chitinous hooks released were surrounded by the extracellular traps. The other protoscoleces were still intact and the extracellular trap structures were observed around them. The relationship between the extracellular DNA contents and the protoscoleces concentration was not found statistically significant (P > 0.05). The extracellular DNA amount in the co-cultures diluted in RPMI-1640 increased with the incubation time (P < 0.05). However, the time-dependent relationship was not found in the co-cultures diluted in the cyst fluid (P > 0.05). The difference in the extracellular DNA amount was detected as statistically significant (P < 0.05) between the two co-culture groups (diluted in RPMI-1640 or the cyst fluid), except for 30 min incubation. To the Author's knowledge, NETosis reaction was firstly observed in sheep PMN after being confronted with protoscoleces in vitro. The cyst fluid had some negative effects on the development of extracellular traps from sheep PMNs at the 1-h incubation time. It should be investigated which molecules are responsible for NETosis inhibition in hydatid cyst fluid. Future studies may clarify whether neutrophils fight with protoscoleces by using their different mechanisms.