Salivary gland transcriptome of Rhipicephalus (Boophilus) microplus

The cattle tick, Rhipicephalus (Boophilus) microplus is a tick of veterinary and health importance globally, transmitting Babesia bovis and B. bigemina. Tick control is important and needed to prevent livestock diseases caused by tick-transmitted pathogens. Traditionally, tick control methods have resulted in development of acaricide-resistant ticks, environmental pollution and meat and milk contamination. Therefore, there is a need for alternative method and vaccines directed against tick feeding. The aim of this study was to identify proteins involved in tick feeding, tickhost-pathogen interactions and tick reproduction. Consequently, these will help in identification of antigens with the ultimate goal of developing anti-tick vaccines. R. (B.) microplus female ticks were collected at five different feeding stages. RNA was isolated from the salivary gland extracts (SGEs). The cDNA libraries were synthesized and sequenced with the Illumina MiSeq technology. Transcriptome data was analyzed with CLC Genomics Workbench, Trinity and Minia. The SGEs were also used to isolate the fractions: membrane, soluble and pellet protein for proteomic analysis. The proteomics data was analysed with Mascot, X!Tandem and Scaffold. Both the transcriptome and proteome analysis revealed the presence of major secretory protein families such as Kunitz, lipocalins, serpins, cement proteins and metalloproteases, while the majority of transcripts coded for housekeeping genes.National Research Foundation (South Africa)Life and Consumer ScienceM. Sc. (Life Science

Bioinformatic and Biochemical Characterization of Amblyomma americanum (Acari: Ixodidae) Serine Protease Inhibitors (Serpins)

Ticks bites are a source of morbidity and mortality for humans and animals. Chemicals have been insufficient for tick control whereas an anti-tick vaccine may be an effective alternative. Hosts use serpins to control their immune pathways therefore ticks may inject serpins into the host for the same purpose. Amblyomma americanum transcriptome data were analyzed to determine extent and diversity of serpins. Candidate serpins were assayed for function against 17 proteses and for ability to affect blood coagulation and platelet aggregation. Spliced serpin constructs consisting of putatively immunogenic regions of selected serpins were designed for expression and vaccine trials. A. americanum expresses 122 unique serpins. The greatest diversity of serpins were in males and in feeding ticks. Forty percent were conserved in other ticks. A. americanum serpins 4 and 8 were selected for characterization as representatives of similar serpin clusters. These serpins bound host antibodies to tick saliva indicating they are immunogenic and used in feeding. Serpin 8 inhibited inflammatory proteases cathepsin G and proteinase 3. Serpin 4 inhibited inflammatory proteases cathepsin G and chymase, and the cysteine protease papain. Neither serpin affected blood coagulation however, serpin 4 delayed platelet aggregation. A spliced serpin construct was cloned in bacteria and yeast cells, however recombinant proteins failed to express. Results indicate serpins 4 and 8 function in the vertebrate host as counter-defense serpins and thus may be useful as anti-tick vaccine candidates, however their relative importance in tick feeding physiology will need to be addressed using an alternative protocol

Purification and Characterization of Pomphorhynchus kashmirensis Somatic Antigens

Fish fauna is ecologically very important and plays a vital role in food chain. There is a great scope for the development of fishery resources in the valley of Kashmir so as to overcome the prevailing animal protein defiency in the diet of the local people. In country like India, intake of meat & milk is low, so fish has special importance as a supplement to ill- balanced cereal diet. Fish flesh is also a highly perishable commodity constituted by 60-80 %water & 13-20 % protein & being low in cholesterol & free from fats. Fish protein hydrosylates also have antioxidant properties. The flesh also contains phosphorus & vitamins. Freshwater fishes form one the important food sources in both the developed as well as underdeveloped countries. A lot of research work has been done on various aspects of these vertebrates but a meagre work is available on the molecular aspects particularly immunogenicity of parasites of the fish host. Therefore, study on the characteristics of the protein profile of the fish parasite Pomphorhynchus kashmirensis was undertaken. The fishes were collected from Dal Lake and River Jhelum and it forms the first study of its type in this part of the country. In order to have a glimpse of the related work done in the past, an attempt was made to review the available literature on the subject. Parkhouse, et al., (1987) characterized and studied the protective effect of nematode antigens. Bunyatova and Elchiev (1989) constructed electrophoretic spectrum of proteins of an acanthocephalan Leptorhynchoides plagicephalus. Coscia and Oreste (2000) investigated the presence of antibodies against protein antigens of the nematode parasite Pseudoterranova decipiens in the plasma and bile of the Antartic Teleost, Trematomus bernachii. Knopf, et al., (2000) studied the humoral response of the European eel Anguilla Anguilla elicited by an experimental infection with a swim bladder nematode Anguillicola crassus. Noga, et al., (2000) purified antimicrobial proteins from rainbow trouts and sunshine bass by RP-HPLC and SDS-PAGE. Saifullah, et al., (2000) worked on the excretory /secretory (ES) metabolic products released by Gastrothylax crumonifer (trematode:Digenea) during in vitro incubations and the somatic extract of the adult parasites were analyzed using PAGE. As obvious, it was not possible to review all the available literature on the said topic, the important ones were penned down in the dissertation. Studies on the fishes has showed that Pomphorhynchus kashmirensis is usually found in the intestine by boring its proboscis into it and thereby produces lesions and makes it prone to further secondary infections. Besides, it is also present in the body cavity, liver and spleen of its fish host. It causes tremendous damage to the intestinal walls at the site of its attachment. The lamina propia gets thickened and goblet cells in this region become more prominent and their number also gets increased (Yildiz, et al, 2004; Ahanger, et al., 2008).This parasite is considered to be one of the most dangerous parasite responsible for fish mortality and morbidity (Ahanger, et al., 2008). Hence the need was felt to study this parasite at molecular level so that our fish fauna are spared from them. A total of 363 fish specimens of Schizothorax species were collected and out of which, 203 fishes were collected and examined from Dal Lake and 160 fishes were collected and examined from River Jhelum during the present study. The host was collected with the help of local fishermen in live condition. Fishes were dissected & body cavity was thoroughly examined for any parasite. Intestines were placed in Petri dish containing normal saline (0.75%Nacl, Cable 1958) to allow adhering parasites to be released from the lumen. Pomphorhynchus was carefully removed from the intestines with the help of brush and needle. A regular record of this parasites was recorded and then subjected to various immunological and biochemical techniques in order to understand the nature of somatic antigens. Out of 203 specimens examined from the Dal Lake only 42 specimens were found infected with the Pomphorhynchus kashmirensis which constitutes the prevalence of 20.68%. Similarly out of 160 specimens examined from the River Jhelum only 52 specimens were infected with the Pomphorhynchus kashmirensis which constitutes 32.5% prevalence. Also, Pomphorhynchus kashmirensis showed a wide host range and was successfully establishing in various species of Schizothorax. The highest prevalence were found in S. niger (30 %) (26.19% in Dal and 34.85% in Jhelum) followed by S. curvifrons (27.11 %) (19.11% in Dal and 38% in Jhelum) and least prevalence were found in S. esocinus (17.89%) (13.72% in Dal and 22.73% in Jhelum). Pomphorhynchus infection also revealed definite seasonal prevalence of infection in all the three species of Schizothorax, with highest infection in summer and lowest in winter. There was a gradual increase in the prevalence rate from spring to summer and falls down with onset of autumn and least observed prevalence during winter season. In summer the prevalence was 34.54% (S. niger 46.34%, S. curvifrons 31.11% and S. esocinus 30%) and the least prevalence was found during the winter season 8.16% (S. niger 14.28 %, S. curvifrons 6.89 % and S. esocinus 9.52%). Gender wise observations were also made which revealed that the sex wise differences were not much prominent but in most cases males 30.30% (S. niger 32.50%, S. curvifrons 27.65% and S. esocinus 28.94%) were found to be more infected than females 27.27% (S. niger 27.14%, S. curvifrons 36.61% and S. esocinus 15.78%). To characterize the somatic antigens from Pomphorhynchus kashmirensis, the sera of the fish was used as a source of antibodies. For this purpose affinity chromatography is the most appropriate technique to be utilized. From this technique the eluted bound protein was dialyzed extensively against 20 mM Tris-saline buffer, pH 7.4, concentrated with PEG 20,000 and was designated as affinity purified P. kashmirensis somatic antigen (Aff-PSAg). The protein content of the antigen was determined spectrophotometrically. SDS-PAGE confirmation of purified antigen Aff-PSAg was done by SDS-PAGE followed by staining with Coomassie Brilliant blue. Electrophoretic separation of Aff-PSAg resolved into 5 prominent polypeptides of molecular weight ranging from 29 to 66 kDa which is inferred to the presence of 5 or more number of active somatic antigens of P. kashmirensis. To measure the antigenicity of the purified antigens the commonly used serotest viz., Ouchterlony double diffusion (ODD) was used. Crude somatic and partially purified pooled fractions of Pomphorhynchus kashmirensis were subjected to double immunodiffusion against rabbit hyper immune sera. Ouchterlony gel diffusion test of somatic antigens showed one precipitation arch against heterogeneous hyper immune sera and many precipitation arches against homogenous hyper immune sera. In nutshell, it is observed that the somatic antigens derived from the Pomphorhynchus kashmirensis can be used as good immunogens and hence can be exploited for mounting the protective immune response in fish. The results of the present study suggest that low molecular weight antigens of Pomphorhynchus kashmirensis deserve further investigation