Naturally occurring Anaplasma marginale infection in cattle: Molecular prevalence and associated risk factors, haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels.

The present investigation was undertaken to map the distribution of Anapalsma species infection in cattle from the Aizawl region of Mizoram, India, in relation to various risk factors, and to study the haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels in cattle with naturally occurring Anapalsma marginale infection. The study was carried out over 31 months from June 2019 to December 2021. A total of 401 cattle blood samples were collected and screened for the presence of Anaplasma spp. by microscopic examination and polymerase chain reaction (PCR). Non-infected clinically healthy cattle (n = 21) served as control. Blood samples were collected to study the haemogram and serum samples were used for the evaluation of biochemical parameters, oxidative stress indices and trace minerals. During the study period, an overall prevalence of 15.71% was recorded for A. marginale infection in cattle. The prevalence of A. marginale infection was highly associated with age, sex, breed and tick infestation status of animals, floor system and management of farms, and season. The mean values of total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), total platelet count, total protein, albumin, superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (TAC), copper (Cu) and zinc (Zn) were significantly (P < 0.05) lower, whereas the mean values of mean corpuscular volume (MCV), aspartate aminotransferase (AST), total bilirubin, indirect bilirubin, blood urea nitrogen (BUN), creatinine and lipid hydroperoxide (LPO) were significantly (P < 0.05) higher in cattle infected with A. marginale. A negative correlation of TEC with LPO, and a positive correlation with SOD, GSH, TAC, Cu and Zn suggest a possible link between oxidative stress and the haemolytic crisis noticed in bovine anaplasmosis. Incorporation of antioxidants and organ protective drugs as an adjunct therapy may result in better prognosis.

RNA interference in Fasciola gigantica: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke.

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170-223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / µl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Quantification and expression studies of IL-13 gene in response to experimental infection with Haemonchus contortus in Garole and Sahabadi sheep.

To evaluate the role of IL-13 gene in Garole/Sahabadi sheep against resistant/susceptibility to Haemonchus contortus five Garole sheep (Group I) with consistently showing low egg per gram of faeces (EPG) (≤150) and another five Garole sheep (Group II) with high EPG (≥500) were selected and fed orally with active H. contortus third stage (L3) larvae at the dose rate of 500 larvae/kg body weight. Five Sahabadi sheep (Gr-III) known susceptible to H. contortus were also challenged with the same dose as the previous groups. Blood was collected from each group on day 0, 7 and 14days post infection (dpi). The serum level of IL-13 cytokine was evaluated by ELISA at different days of post infection. The ovine IL-13 was partially isolated and the RNAm level of IL-13 expression was studied by real-time PCR at different days post infection. In the resistant Garole sheep (Gr-I) the concentration of IL-13 cytokine increased significantly (P≤0.05) 14days PI compared to susceptible Garole (Gr-II)/Sahabadi (Group-III). In similar way the relative expression of IL-13 in group I (Gr-I) was found more than seven fold higher than Gr-II and Gr-III on 14 dpi. The Th2 type immune response to H. contortus characterized by higher serum level of IL-13 cytokine and IL-13 gene expression indicated that IL-13 gene plays a vital role for the reduced establishment and/or survival of established H. contortus in resistant Garole sheep.