Anti-Tick Vaccine Development in the Domestic Cows of District Quetta Balochistan and to Detect the Efficacy of Vaccine Through Immunological Parameters

Abstract

The present study was carried out in different local cattle farms of Quetta (Balochistan province Pakistan). The first experiment was focused on the collection and identification of various species of ticks. During this study ticks were isolated from cows and buffaloes of various farm houses of the Distract Quetta and from the vicinity during the time period of April 2012 to June 2014. It was concluded that very large number of cows and buffaloes were infested and affected by three species of ticks including Hyalomma aegyptium (Linnaeus, 1758), Hyalomma anatolicum anatolicum (Koch, 1844) and Dermacentor andersoni (Stiles, 1908). It was revealed that Local Sahiwal and Friesian Sahiwal cows and buffaloes were less susceptible to the infestation of these tick species as compared to Holstein Friesian which were more infested and affected by these ticks. Milk productivity of Holstein Friesian and Sahiwal cows and buffaloes were also found to be reduced and this might be due to the infestation of cows and buffaloes by these ticks. In the second experiment of the present study was emphasized on the effect of Rhipicephalus (Boophilus) microplus (Canestrini, 1988) was also given in term of the yield of milk production in cattle studied in various farm houses of Quetta. The obtained results revealed that milk production appeared to decline in different species of infested cows due to R. microplus. Breeds of Red Sindhi, Sahiwal and Dahni cows when infested with R. microplus showed 0.5 liter decrease in milk production, while infested Walaeeti cow (foreign cow) breed revealed a significant decrease, at 2 to 3 liters decline in milk production. Thus, our present study proved that heavy load of tick R. microplus could be responsible for decline in milk production and the study will be useful to estimate the economic loss caused as result of the effect of ticks on dairy cattle. In the third experiment of this study, cows were found to be more susceptible to severe babesiosis because out of a total 82 samples 54.8% were infected. Furthermore, among these infested cows 31.1% suffered from severe babesiosis while 42.2% were suffered from mild babesiosis. Whereas in contrast, out of 28 observed bull samples, 42.8% were found to be infected. Out of these infected bulls 33.3% suffered from severe babesiosis while the remaining 33.3% were suffering from mild babesiosis. 3 A forth experiment conducted immunological studies against ticks (H. a. anatolicum (Koch) and H. aegyptium) in domestic breeds of cow Bovis permiginus (Linnaeus, 1758) randomly selected from different farms of the Quetta district. Cows were allocated five groups of twenty animals each for immunization and control experiments. Each group was used to assess the effect of immunization against ixodid ticks H. a. anatolicum and H. aegyptium using extracts from attachment cones made up of cement proteins taken from ticks as the source of antigen to detect immunogenicity, cross reactivity, stage reactivity, consistent intensity and antiserum reactivity against unfed, partially fed and fully fed ticks and also to perform immunological tests (ELISA) for the detection of immunity against ticks in domestic cow breeds in these study areas. The process of extracting cement cones was performed, using mouthparts of tick species H. a. anatolicum and H. aegyptium. In this process common (23 KDa) cement cone proteins were recognized on western blots. Putative (23 KDa) tick cement cone proteins (Anatolin and Aegyptin) were extracted using SDS (PAGE) from larval cement cones of H. a. anatolicum and H. aegyptium respectively which showed immunogenic, stage reactive, cross reactive immune response during larval and post larval stages of tick feeding and its intensity remained constant throughout parasitic stages (p <0.01). Common immunological cross reactivity was established among cement cone proteins of H. a. anatolicum and H. aegyptium ticks against anti-Hyalomma anatolicum anatolicum cow serum using SDS (PAGE) and immunoblotting. During feeding of larvae on tick hosts, the first (23 KDa) protein in the cement cone was detected with anti H. a. anatolicum and anti H. aegyptium cow antisera after SDS-PAGE using western blots. Western blotted cement proteins of both tick species revealed common bands which proved to be an immunogen of importance while immunizing the crossbred domestic cow breed Bovis permiginus of the Quetta district (Balochistan).The level of significance was p < 0.01. Based on immunoblot assays, cross reactivity among the tick hosts H. a. anatolicum (Koch) and H. aegyptium established that (23 KDa) cement cone extract antigen molecule was immunogenically identical for both ticks. Species studies during the present investigation; intensity of this immunogen through larval, nymphal and adult stages of ticks as well as its antiserum reactivity with unfed, partially and fully fed stages was also studied to detect consistent 4 intensity. There was consistent stage reactive immunogenic response throughout developmental stages of ticks under study (p<0.05). Immune response was evaluated through different immunological parameters. Both cellular and humoral immune responses were demonstrated through using humoral (ELISA), cellular, cutaneous and hematological responses as well as changes in tick behaviour due to immunization. Antibody titres in ELISA were positively related to the number of adult ticks feeding per domestic cow breed at the time samples collected (Correlation coefficient r = 0.99).The relative concentration of eosinophils in “skin eosinophil index” and mast cells in the skin of immunized cows was detected, showing significantly higher concentrations in immunized as compared to control cows (p< 0.05). There was significant (p<0.05) rise in basophils against H.a. anatolicum antigen, observed in the dermis of immunized cows as compared to control animals. Packed cell volume (PCV) in immunized cows was high which reflected decreased blood loss. Post immunization cutaneous responses were detected showing significant increase (p<0.01) in mean skin lesion area at the site of inoculation. Cutaneous hypersensitivity was induced by infestation by larvae and nymphs on the ears of immunized domestic cow breeds. There was more skin thickness in immunized as compared to control. Post immunization haematological studies showed a gradual decrease in haemoglobin (Hb) levels in control cows as compared to vaccinated and there were significantly more mean weekly WBC (white blood corpuscles) and RBC (red blood corpuscles) counts within vaccinated groups compared to controls (p< 0.05). Similarly, tick challenge responses were evaluated through changes in tick reproductive and feeding behaviors and there was a significant (p<0.01) decrease in tick development, feeding performance, counts, engorgement, fertility, egg counts, egg weight in immunized as compared to control cows. A considerable (p<0.01) decrease in tick mean weight in immunized domestic cow breeds was observed as compared to controls. There was a considerable (p<0.05) difference in non viable eggs, oviposition period and egg incubation period among immunized and controls. The moulting of larvae on vaccinated domestic cow breeds was reduced by 81.8 % (ANOVA, F1, 2 = 18.51, p< 0.05). There was progressive mortality of ticks (46.6 % increases) throughout the feeding stages of ticks parasitized on immunized cows. In contrast, the ticks on control showed negative responses. Many of the 5 ticks failed to engorge, or if they did, died before egg lying. There was a significant fall in egg mass (p<0.01), feeding and engorgement performance (p<0.01) in immunized cows. There was great reduction in the weight of unfed, partially and fully fed ticks parasitized on immunized cows compared to controls. The mean duration of feeding time of ticks on immunized and control cows was not significant (p< 0.05). There was 34.9 % reduction in failure of the ticks parasitized on immunized cows to lay eggs. In the fifth experiment, a putative tick cement protein (D. andersoni (Stiles) 30 KDa) was extracted from the cement cone of D. andersoni ticks which elicited a strong stage reactive immune response during larval and post larval stages of tick feeding parasitized on the domestic cow breed Bovis permiginus. During feeding of D. andersoni tick larvae the first (30 KDa) protein in the cement cone was detected when probed with anti- D. andersoni cow sera after SDS-PAGE. Western blotted cement protein of D. andersoni larvae, nymphs and adults revealed common bands of (30 KDa) which proved to be an immunogen of importance while immunizing domestic cow breed Bovis permiginus. In a sixth experiment effects of vaccine doses emulsified with different adjuvants were analyzed using five groups, each cohort comprising 20 domestic cow breeds. The first group was immunized with alum, 2nd with Freund’s complete adjuvant (FCA), 3rd with Incomplete Freund’s adjuvant (IFA) and 4th with Montanide ISA-50 (Incomplete Seppic adjuvant) adjuvants. Cow breeds administered with antigen + Montanide adjuvant, proved to be the most potent adjuvant in this case. In seventh experiment examined the seasonal effect on immune responses and the correlation of seasonal changes and nutritional stress with host immune response. Numbers of tick bites on immunized cows were significantly (p<0.01) lower due to seasonal changes and nutritional stress as compared to controls. Antibody titers were higher during the spring compared to the autumn periods. In addition for conducting an immunization programme in Balochistan, potential constraints were also analyzed. Shelf life and safety profile for vaccine production were also studied in the final experiment and recommendations to streamline industrial constraints were examined. 6 This research concludes that (23KDa) cement cone extract from larval ticks Hyalomma anatolicum anatolicum as well as (30 KDa) cement cone extract from larval ticks Dermacentor andersoni should be recommended for commercial development as an anti tick vaccine against domestic cow breeds of Balochistan (Pakistan). Hyalomma anti-tick vaccine has a significance impact on livestock of Balochistan. Proper commercial launch of the product by using the sufficient marketing channels, will serve the agrarian societies and dairy farmers. The development of Hyalomma vaccine is significant contribution for the province and it has the potential to export to neighboring countries, such as; Afghanistan and Iran. Hyalomma vaccine (Hyalomma anti-tick antigen) developed in the present study will be sent to veterinary Pharmaceutical Industry for patent right and application also will be submitted to Pakistan Intellectual Property Organization (PIPO) [application No. PO.2016/1244 app.] to patent the vaccine. This vaccine then might be made accessible for cattle owners, farmers and marketing in and outside the country. It is suggested to further purify the antigens or recombinant antigens may also be used in future