Novel molecular tools for the diagnosis of avian coccidiosis

TOMAZIC, ML; ARIAS, LUCIANA; BARBANO, PABLO; PALACIOS, LUCIANO; GONZALEZ, MATIAS; SCHAPIRO, JAVIER; RODRIGUEZ, ANABEL E

CABA

Jornada; I JORNADAS INCLIVET; 2021

Avian coccidiosis is caused by protozoan parasites of the genus Eimeria. It is a highly widespread disease that negatively affects productivity, causing economic losses to the poultry industry worldwide. It is a threatening disease when the control measurement fails such as management of chickens and farms, chemoprophylaxis (anticoccidial drugs), and/or live coccidia vaccines. The production of meat and eggs faces new challenges, such as the projected increases in demand, food security, and quality, environmental impact, which in post-pandemic this scenario has become even more relevant. Thereby, increasing productivity under better control strategies and diagnosis is necessary. Seven species infect poultry, having different pathogenicity and, as a consequence, their productivity impact, mortality, vaccination, and drug susceptibility vary depending on the involved species. In Argentina molecular epidemiological data farms are scarce, and knowing the species commonly infecting poultry, where mixed species infections are common, is the first step to improve diagnosis and, thus, the control measurements. The goal of the present work was the implementation of multiplex polymerase chain reaction (PCR-multiplex) for the simultaneous differentiation of Eimeria spp. from different types of samples.Sampling was carried out in intensive and extensive farming systems from Buenos Aires province, and fomites, fecal or intestinal samples were obtained. From fomites and fecal samples, oocysts were floated in a hypertonic sodium chloride solution while oocysts from gut tissue samples were scrapped from intestinal mucosa, homogenized in PBS buffer pH 8 and incubated in 1.5 % w/v trypsin at 41°C for 60 min. The eimerian oocysts from the 3 types of samples were then allowed to sporulate in 2% w/v potassium dichromate solution at 28°C for three days and, after washed and pelleted, they were broken with glass beads. Total genomic DNA was isolated from purified and sporulated oocysts using a standard phenol/chloroform extraction. Two PCR-multiplex (M1 y M2) were performed to simultaneously differentiate 3 or 4 species. PCR M1 contains specific primers to E. acervulina, E. brunetti, E. tenella and E. maxima, and PCR M2 to E. mitis, E. praecox and E. necatrix. For the setup of PCR M1 and M2 DNA from commercial vaccines were used. Amplicons were solved in 2% agarose gels stained with bromide ethidium and visualized under UV light. Results obtained so far for the first time in Argentina shown that 6 of the 7 species were detected in chickens, having most of them at least three mixed species. The most frequent species found were E. acervulina and E. tenella, the latter being one of the most pathogenic.In conclusion, the current work demonstrates that molecular tools are feasible to apply in different types of samples, allowing the specific and simultaneous detection of Eimeria spp. These are powerful tools that complements traditional diagnosis, contributing to molecular epidemiology and allowing to improve current control measurements.