Prevalence of avian coccidiosis in Peshawar Khyber Pakhtunkhwa, Pakistan: its control using medicinal herbs and iron nanoparticles of Verbena officinalis including in-silico molecular docking

Abstract

Coccidiosis is an economically important disease that leads to $ 3 billion in annual loss globally, caused by several Eimeria species. It is prevalent throughout Pakistan, according to the poultry research institute’s annual reports, but only a small number of cases were published online. The current thesis can be summarized into four study schemes. In the first scheme of the current research, the prevalence of coccidiosis was reported for the first time in Peshawar, Pakistan. The postmortem data of coccidiosis effected farms and coccidiosis-associated risk factor data were statistically analyzed using the chi square test, binary logistic regression, and graphically presented in maps using arc-GIS to evaluate the impact of Eimeria spp. on poultry. The village, age, and year-wise distribution of coccidiosis in relation to humidity and temperature revealed 25.99% of the towns with cocci-positive farms, with many cases in between 16 to 30-day-old broiler chicks, i.e., 64.49%. The postmortem reports revealed 23.37% of farms had severe modes of infection. The survey of cocci-associated risk factors in 2020, concludes that vaccination (P = 0.008), disinfection (P = 0.04), footwear (P = 0.02), flooring (P = 0.03), and feeder hygiene (P = 0.03) have significantly affected the coccidial emergence. In the second scheme of the current study, five pharmaceutically active herbs were used against avian coccidiosis in-vitro for sporulation inhibition (SPI) assay to calculate the inhibitory concentration (IC50) and revealed V. officinalis & P. glabrum with maximum SPI % of 81.04% and 72.47% and minimum IC50 of 0.14, and 12 mg/ml, respectively. The same plants were selected for in-vivo anticoccidial activity in E. tenella-infected chicks. The mean weight gain, oocyst count, diarrhea, biochemical tests, hematology, and histopathology of all groups were analyzed using the Tukey test. The active herb was characterized by antioxidant assay, FT-IR, UV-Vis spectroscopy, and GC-MS analysis. The GC-MS’ identified phyto-compounds of V. officinalis were docked with S-Adenosyl methionine (SAM) synthetase, revealed the best binding of strychane, 1-acetyl-20a hydroxy-16-methylene with the lowest binding score (-6.4 Kcal/mol), suggested their anticoccidial potential in poultry. As in the previous study scheme, V. officinalis was found effective against E. tenella. For this reason, various fractions of V. officinalis were tested for SPI and anticoccidial XIV assay. In the first step, fractions F1 to F9 were tested in the SPI, with F1, F3, F5, and F8 exhibiting good maximal (> 70%) inhibitory activity at 40 mg/ml concentration and the lowest IC50 of 16.83, 8.59, 10.65, and 10.32 mg/ml, respectively. The active fractions were in-vivo tested for trail of anticoccidial activity with F3 and F8 fraction as highly effective in case of higher concentration with significant mean weight gain at 14th post-treatment, respectively, with FCR < 1. All the biological parameters were analyzed using the Tukey test (P < 0.05). The active functional groups and purity and composition of active fractions was characterized via UV-Vis and FT-IR spectroscopy, and GC-MS. The ligands of active fractions were docked against S-adenosyl methionine synthetase, resulting in the best binding with a-sitosterol, 1,2 benzenedicarboxylic acid, mono(2 -ethylhexyl) ester, and 3,9-epoxypregnane-11,14,18-triol-20-one, 16-cyano-3-methoxy-, 11-acetate with lowest binding energy, which suggests its best compatibility with the target protein and can be used as an inhibitory substance against avian coccidiosis to alter the methylation of DNA, which will interfere with the gene expression of parasite. In the fourth study scheme, the iron oxide nanoparticles of Verbena officinalis were synthesized and characterized to confirm their purity, size, and crystalline structure using XRD, FT-IR, and SEM. The in-vivo antiprotozoal iron nanoparticle of V. officinalis was found to be effective against E. tenella-infected chicks. Which revealed significant weight gain in iron nanoparticle-treated groups with maximum weight gain at 15 mg/ml dosage with the lowest FCR of 1.0 (P < 0.05), least oocyst count (0.53×103/g), and cured from diarrhea. The means of all studied parameters were analyzed statistically using ANOVA (tukey test) to confirm the significant difference (P < 0.05) between the treated and untreated groups. The higher concentration of nanoparticle treated chicks showed normal hematological, biochemical, and histological profile.