Title of Invention

"A PROCESS FOR PREPARATION OF ANTI- THEILERIOSIS VACCINE"

Abstract

A process for the preparation of anti-theileriosis vaccine comprising the steps of isolating lymphoid ceils infected with schizonts of Theilena annulata present In an infected animal, culturing the infected ceils in a growth medium, subjecting said cultured ceils to the step of in-vitro propagation by-serial passaging, developing cloned cultures from a single cell by limiting dHution technique and further passaging these, karyotyping the ceHs in the medium to ascertain the dipoid character, and obtaining viable cells with maximum infectivity rates, determining macroschizont nuclear number, subjecting a number of viable ceHs with diploid character to the step of cryopreservation in liquid nitrogen in the presence of a cryoprotectant in the said growth medium to obtain a suspension of viabie celts, testing these cultures at dirrerent passages and in varying dose levels in groups of calves for ascertaining their pathogenicity / antigenicity, determining passage level and dose for the safety and efficacy of candidate vaccine, its safe use in new born calves and pregnant cows, time taken to induce immunity, serological responses and effect on haematological parameters, and testing its efficacy under wide field conditions in the susceptible stock.

Full Text

FIELD OF INVENTION This invention relates to an anti-thelleriosis vaccine and the process of preparing the same. This vaccine is safe for aH exotic breeds, their cross-breds at al age groups of cattle even including neo born calves and pregnant cows as welt as indigenous calves and is able to protect vaccinated animals from bovine tropical thelleriosis. . PRIOR ART Exotic breeds of cattle, their cross-breeds and young indigenous calves are highly susceptible to bovine tropical theilerlosis. which is a dreadful tick-transmitted disease in cattle. The disease adversely affects the working capacity of bulls, breeding ability of buiis and milk production in cows, causing enormous economic losses. Apart from direct losses, heavy costs are also Incurred on treatment of sick animals. The occurrence of the disease is seasonal during the summer and rainy months i.e. from Aprill to October. The pertinent singns are lymphadenopathy, fever, anaemia, weakness, recumbency and the intriguing feature of continuous feeding and rumination by exotic and cross-bred cattle until the advanced stages of the disease. Unusual signs observed are Haemoglobinuria and cutaneous eruptions mostly in adult cattle as well as protrusive eye balls and cerebral involvement In cross-bred calves. Diagnosis is confirmed by demonitration of macroschizonts in lymph node biopsy smears and piropiasms in erythrocytes. The morbridity rate of the disease approaches nearly 100 per cent and mortality rate up to 60-80 per cent. Young animals mostly below 2 months of age are the main sufferers of this disease. Thereafter, animals keep on getting natural repeated infective inocula through repeated tick Infestations and remain premune to this disease and withstand even virulent chaiierges. However, residual infection in their body may flare up into clinical disease as a result of any stress such as pregnancy, parturition lactation, Intercurrent disease etc. during later part of their lives. The disease threatens an estimated 250 million cattle in the Mediterranean littoral. North Africa, the Middle East, the Indian subcontinent and Central Asia. Various chemotherapeutic agents like berenil, imidocarb, halofuginorne lactate, buparvaquone alongwith supportive therapy have been tried with varying degrees of success. Out of these, buparvaquone has been found to be most efficacious in treating clinical cases of theiieriosis. However, this drug needs to be imported and is very costly. Keeping in view the prevalence of the disease and the impracticable control of vector ticks under fiekl condlttons, the need for the devek)pment of suitable immunoprophytactic measures has been felt in India and In variours other countries where intensive cross-breeding programmes are being carried out to enhance millk production by controing this dreadful disease. Various methods of immunizafion have been fried from time to time with varynig degree of success. These immunization methods include irradiation, non-specific immunization, cell culture vaccine etc. cell culture vaccine is currently being tried in some of the countries, whereas other methods have been used In the past and have their own merits and demerits. Existing product in the market by the name of Rakshavac-T is neither recommended in calves less than 2 months of age nor in pregnant cows. Whereas, epidemlological studies have confirmed that the disease occurs primarily in this stock. Patent no. DE2914813 discloses a live vaccine against theileriosis in cattle consisting of cattle lymphoid cells infected wNh the schizonts of the attenuated strain of theileria annulata. The Vaccine is admfintotered as a dose containing 0.1-3 miltion living infected ceRs. The vaccine is prepared by growing T. anna Ma isolated from the tick hyalomma detritum in In cattle lymphoM cells using a nutrient medium for 20-100 days, preferably over 6-12 passages at 37.5-38.6*C. The schizonts are then collected and frozen in the presence of a suitable cryoprotectant preferably glycerine at temperature less than -60'C. The drawback of the above vaccine is that it contains only 0.1 to 3 million cells as compared to present invention which contains S millon cells. Another difference from the above vaccine Is that cryoprotectant used is glycerine at -60'C, whereas cryoprotectant used in the present invention is dimethyl sulphoxide (DMSO) and the material Is cryopreserved In liquid nitrogen at -1790 C. Moreover, the passage at which present vaccine has been prepared is 200 and it Is a cloned culture developed from a single cell In comparison to the culure used at 6-12 passages in the said vaccine of patent no, DE2014813. Patent no. EP2228 A discloses a therapeutic preparation for the treatment of clinical cases of the ilerlosis in cattle and sheep, wherein the preparation comprises of 2-cycloalcyl-3-hydroxy-1, 4 naphthoquinone. The drawback is that, this is a drug for curative purposes and not a vaccine for prevention and control of the disease. The limitation of the above preparation therefore, is that it is only suitble for treating clinical cases of the lieriosis in cattle and sheep and has no preventive application. OBJECTS OF THE PRESENT INVENTiON: An object of the present invention is to propose an antl-theileriosis vaccine and a process of preparing the same for immunization of exotic and cross-bred cattle as well as indigenous calves against bovine tropical theieriosis caused by Theleria annulata . Another object of the present indention is to propose an anti-thelleriosis vaccine and a process of preparing the same for immunization of cattle against bovine tropical theieriosis. which is safe for al breeds and for al ages and stages of cattle particularly exotic and cross-bred cattle and can protect them from severe tick challenge Still another object of the present invention is to propose an anti-theiieriosis vaccine and a process of preparbig the same for immunization of cattle against bovine tropical theileriosis, which has no side reactions and can be safely used even In a few days old calves as well as in pregnant and high yielding cattle wNhout any adverse effects. Further object of the present invention Is to propose an antitheiieriosis vaccine and a process of preparing the same for immunization of cattle against bovine tropical theHeriosis, which is cost effective as it is based on indigenous ingredients. STATEMENT OF INVENTION A process for the preparation of anti-theileriosis vaccine comprising the steps of isolating lymphoid celts infected with schizonts of theileria annualate present in en infected animal, culturing the infected cells in a growth medium, subjecting said cultured cells to the step of in -vitro propagation by-serial passaging, deveioping cloned cultures from a single cell by limiting dilution technique and further passaging these. karyotyping the cells in the medium to ascertain the diploid character, and obtaining viable cells with maximum infectivity rates, determining macroschizont nuclear number, subjecting a number of viable cells with diploid character to the step of cryopreservation in liquid nitrogen in the presence of a cryoprotectant in the said growth medium to obtain a suspension of viable cells, testing these cultures at different passages and in varying dose levels in groups of calves for ascertaining their pathogenicity / antigenicity, determining passage level and dose for the safety and efficacy of candidate vaccine, its safe use in new born calves and pregnant cows, time taken to induce immunity, seroiogicai responses and effect on haematologicai parameters, and tatting its efficacy under wide field condilions in the susceptible stock . Further according to this invention there is also provided an anti-thelleriosis vaccina comprising schizonts of T. annulata in a cryoprotactant and a growth medium. A process for preparation of anti-thaKeriosis vaccine comprising isolation of lymphoid eels infected with schizonts of Thelleria anbutata from an Infected animal, in-vitro propagation In a growth medium, cryopreservation and preparation of anti-thaileriosis vaccine wherein said in-vitro propagation is carried out by serial passaging twice / thrice weekly till the culture loses its pathogenicity but retains its antlganicily, the said growth medium comprises RPMI-1640 supplemented with 10-20% by volume of bovine serum, selected from foetal bovine serum; neonatal bovine serum and normal bovine serum; an antibiotic solution containing benzyl penicillin sodium @ 100 l.u. per ml, streptomycin sulphate @ 100ug per ml, mycostatin @ 10 I. u. per ml, and 200 mM / ml of L-Giulamina, and said cryopreservation is by using cryoprotactant selected from Dimethyl sulphoxide (DMSO) or glycerol, and said anti-theileriosis vaccine is prepared by adjusting tha concentration of calls at SX 108 viable eels per mi in said growth medium containing 10 % DMSO, aliquoting tha ceH suspension in one- mi pre-labelled sterile cryo-tubes and transferring these to the gas phase of liquid nitrogen. DESCRIPTION OF INVENTION: in the present Invention, the antMhaileriosis vaccine for immunization of cattle was developed by the process comprising of the following steps: (a) Praoaration of Ground-up Tick Supamate (GUTS) from Thelleria annualate The infected adult Hyalomma ticks are pre-fed on a calf for 72 hours and collected In a starllized glass tube. The ticlcs are thoroughly washed wlh water and 1% Cetavton solulton followed by normal saline and RPMI-1640 and the ticks are frozen for 5-10 minutes . A known number of ticks are ground into a Fine paste in a sterilized mortar and pestle by adding sterized sea sand or glass wool. Sufficiant amount of medium RPMI-1640 supplemented with 3.5% bovine abum in powder and antibiotics are added to It. The pasta it collected in a ttarNe culture tube. It It ttlrred wen and centrifuged at 4'C at 2S0g. Supernatant fluid, henceforth referred to at Ground-up Tick Supernate (GUTS) containing sporozoles of T. annulata is alquoted depending upon the number of infected ticks to be taken as infective dose. The OUTS is used for initiating infection or for chalenge of vaccinated animals either fresh or stored In liquied itrogen by adding giycerol/dimethylsulphoxkte (DMSO) as cryoprotectant until used as a StabNata. (b) Establishment of Theilleria annulate infected cullures Susceptible animals were Infected with bovine tropical thaHertosIs by in|actlng GUTS/ StabNata. Blood and lymph node biopsy material were collected when these calves showed cinical l diseasa. White blood ceNs were separated from blood under aseptic : conditons s and cuttured In the growth medium as described bek)w. CeNs coNected from lymph node were washed twice with medium RPMI-1640 and culured in the growth medium Once T. anno fata Infected ceNs started to grow In the tissue culture flasks, their infectivity rates were assessed as descrbed below. Once the growing T. annulata infected ceNs became confluent, they were passaged into a new tissue culture flask as described bek>w. (c) in vitro propagation Prolonged in vitro cultivation 7. annulata is dona by serial passaging at dirferent intervals to achieve the stage wherein the culture loses Ns pathogeniclity and retains Ns immunoganic llty to the desired level. Cultures are passaged either twice- or thrice-weekly and as static cultures wherein medium is changed in the same flask twice weekly. In twice -weekly passaged or static cultures, 9 ml of fresh growth medium is added to 1 mi of old cuture, thus keeping the seeding rate at 1 x 105 ccell per ml. In thrice -weekly passaged culures, 8 ml of fresh growth medium is added to 2 ml of okl culure wNh a seeding rate of 2x105 ccell per ml. The twice and thrice weekly passaged cultures are propagated upto 100 passages and cryopreserved at different passages so as to cryopreserve the stock at all levels. Thereafter, the cloned cultures were devetoped and passaged up to 200 passages and cryopreserved at different passages. The static cultures are processed up to one year and culures are cryopreserved al different time intervals of 3 months, 6 months, 9 months and one year. The cryoprotectant used for these culures is dimethylsulphoxkle (DM80). (d) Cloning of the cell Plating effeciency of the culures was tested and cloned popuiatwns of 7. annutata sehizont infected cell as suspension culures were devetoped fromsingle cell by using Imling diulton technk^ue, whereki, culure at a particular passage is so diuled as to obtain a eel populatton of 1x10^ cell per ml. Culures already passaged up to 90-100 passages elher twice-weekly or thrice-weekly were used for this purpose. Such a diuled culure is then poured into cupark plates and then observed under microscope. Those wells which contained singe cell were marked and others were left as such. Wels contaking single cell were monlored daly and alowed to grow as cloned cell population . These single cell were thus alowed to mulipiy and grow further. When I formed a mass of cell in a few days, the cell along wlh medkim were transferred kito a bigger well of costar plate, medium added and cell alowed to grow further. When their number increased, these were then transferred to a 25 cm2 flask and monlored further with the addition of fresh medium daly. When such culures grew to ful proportion, these were then passaged to a new flask. When transformed, these were passaged to bigger flasks and bulked up as herein described and cryopreservedin liquied nitrogen at different passage levels. (e) Growth medium The growth medium used is RPMM-1640 supplemented initialy with foetal bovine serum and later on substituted by neonatal bovine serum or normal bovine serum taken in quantity of 10-20% by volume. To this medium, antibiotics in the form of benzyl penicIlin sodium @100 l.u. per ml, streptomycin sulphate @ 100 µg per ml and mycosfatin @ 10 l.u. per ml were added. Bessies, 200mM of L-glutamine is also added. All the contents are pre-sterillsed by filtration and made free from any micro-organism contamination by standard procedure. (f) Bulking UP of cultures The cryopreserved seed culture Is thawed immediately to 7 0C from liquied nitrogen, washed by resuspending in 10 ml of growth medium and centrifuged at 400g for 10 minutes. The supernatant is discarded. The sediment containing cells is resuspended in 10 ml growth medium seeded in a 25 cm2 tissue culture flask and Incubated In an incubator at 37°C. The culture is in exponential growth phase after 24 hours. Following cell count, the culture Is seeded in 80 or 250 cm2 tissue culture flask at 2x105 cells per ml in 25 or 100 ml growth medkan respectively and incubated at 370C. After 48 hours, equal amount of growth medkim is added in each flask. On the following day, flasks are examined for the growth of schizonts of 7. annulate: Two alk|uots of 100 µl from each flask are taken out for counting of viable and non-viable cells and Infectivity rates, infectivity rates in cultures were more than 99 percent. Mitotic indices were recorded and macroschizont nuclear numbers were counted. Karyotyping revealed that cells in cultures maintained their dipioid character. Assesment of infectivity rate infectivity rates of monouck cells with T. anulata schiaonts in a flask is assessed by preparing a cytospin smear from 100 µl culure. The smear Is immediately air dried, fixed wNh methanol, stained wUh Giemsa and examined under the oil immersion lens of the microscope. More than 99% cells have T. annulata schizonts in their cytoplasm. Vialbility counts The number of viable cells In each flask are assessed by standard 0.1 per cent trypan blue dyeexclusion method and counting in a haemocytometer. Other modern automatic or semi-automatic methods like Coulter Counter etc. can also be used if available. Viability counts showed higher viability counts by day 2 to 3. (g) Preparation vaccine doses cell from aH flasks are pooled and their cell concentration adjusted to 10x108 viable cell per ml. The total volume is measured and mixed with equal quantity of growth medium containing 20% dimethylsulphoxide (DM80) so as to make the final concentration of cell at 5 x 106viable cell per mi in growth medium containing 10% DM80. The cell suspension is alkfuoted in one-ml pre-tabelled sterle cryo-tubes and immediately transferred to gas phase of liquid nitrogen container. The vials can be transferred to Iqukt phase of Iquid nitrogen container next morning for indefinite storage ti use. EVALUATION OF CULTURES FOR DEVELOPMENT OF VACCINE (a)Testing of cultures for Dathogenictiy/ anttentecity Various cell lines infected with the schizonts of T. annulata devetoped by different ways of propagation were tested at variours passages i.e 10, 20, 50, 100, 150 and 200 with varying dose levels Inoculated subculaneously as a single injection for ascertaining their pathogenicity and/or protective efficacy in groups of susceptible calves kept under controled tick-free conditions in the experimental animal sheds. The calves were inoculated with cell cultures after Immediate thawing at 37°C from liquid nitrogen and the control calves were injected with the placebo. A it calves were later chaHenged wlh infective Ground -Up Tick Supernates (GUTS)/ Stabttates so as to obtain a candklate cell culture line at appropriate passage and dose level, where It to unable tp produce disease but generates immunity in the susceptible stock. Using several animal trials involving groups of calves, proper passage level of cultures,titirastion of dose of cell for immunization, route of injection and time taken for the development of immunity etc. were establshed where the vaccine gave desired level of profection against lethal chaltenge wlh tick material. Thus, a candklate cell culture vaccine from a cloned culture at passage 200 was developed which did not produce any disease in vaccine-irijeeted animals but protected them from tropical thelleriousis when ehalenged with GUTS /Stablate. This group was compared to placebo-irijected animals which showed severe clinical l disease when chalenged wlh GUTS/ Stablate. It was therefore, ascertained that a cloned cidture at passage 200 containging 5 X 10° cell as a dose given as a single injection by sub-cutaneous route was safe and immunogenk: as a vaccine. (b) Candidate Vaccine A cloned culture at its passage 200 was used @ 1.0 ml containing 5 X 108 cells as candklate vaccine given as a single dose by sub-cutaneous route. (c) Vaccination experiments AH the vaccinated animals remained clinically normal after iniection of the vaccine. There were no clinical symptoms in the animals vaccinated with the schizont cell cultures at the dose rate of 5X 108 cells per mi given subcutaneously at passage 200. Haematological values were within normal range in al the vaccinated animais. These animals showed devetopment of antibodies against the organism in their serum. Even afler a very potent challenge with GUTS/ 6tabilate, that produced clinical disease in placebo-iinjected animals, did not Induce any symptom of the disease or any change hi haematological values in animals iri|ected with schizont ceil cultures at passage 200. Rare schizont and piroplasms may be seen in a smaH percerriage of animals afler vaccation. his parasitaemia being extremely tow, may indicate the establishment of cell mediated immune response in vaccinated animals. Serotogically, antibody titres were detected as early as one week post-vaccinatton and peak antibody titres were observed afler four weeks of vaccinatton. Protective immunity was induced within 15 days time afler vaccinatton. The vaccine was used successfufly even in newborn calves and pregnant cows, which are the main susceptible stock to this disease and was found safe and protective. (d) Testing of the vaccine bv Indian Counci of Agricultural Research The cell culture vaccine thus devetoped was also tested by the Coordinating unN of the Indian CouncH of Agricultural Research (ICAR)'s All India Coordinated Research Project (AICRP) on Btood Protista by blind-fold method. This independent agency also reported that the vaccine was safe for susceptible stock and induced protective immunity against bovine tropical theHertosis. EXAMPLE Twelve young cross-bred male cow calves aged about 2 to 4 months, reared under tick-proof conditions were included in this exercise. By blind-fold method, 6 of them were injected with Theileria schizont cell culture vaccine developed by HAU centre. One ml of the vaccine was injected s/c on the left side of the necic on 30.10.90 and the remaining 6 were kept as controls . AH the 10 suving calves (the remaining two from the control group died of other causes) were challenged on 17.12.90 by inoculating 7. annulata infective Ground-Up Tick Suspenskins, on the right side of the neck. The vaccination and challenge was done by the staff of H.A.U. Centre. The host responses to vaccination and challenge were monitored by Project Coordinator and his staff as per the protocol developed by the project. OBSERVATIONS All 6 vaccinated calves survived. The vaccine has not produced any clinteat and parasitological reaction except fever for a day in 2 calves. Host responses to challenqe infection: a) Vaccinated group: One calf showed fever for a day and died on 12th day with no visible parasitaemia. No typical lesions of theilerioisis were found on post mortem. The death of this calf was, therefore, attributed to any other cause than theiieriosis. The remaining 5 calves survived. b) Control group: For al practical purposes only 2 calves have participated In this exercise. Out of these one calf suffered severe thelleriosis and died of it on 15thday. Several punched necrotk: ulcers typical of thelleriosis were seen on the abomasal mucosa during post-mortem. The second calf suffered miild the ilerioisis but died on 33rd day with anemia. Haemorrhagk: punched necrotic ulcers were not present or. the abormasal mucosa. The remaining 4 control calves died -2 of them before challenge and 2 calves on 2nd and 9th day after challenge. The details are presented in the following table: Table showing host responses to challenge infection: (Table Removed) 'Death not due to theiieriosis. No ParasKaemla. Conclusion: The vaccine can be corrsidered safe for vaccination to 2 to 4 months old crossbred calves since it has not produced ciinical disease. The vaccine has conferred sufficient protection to withstand severe challenge infection with 10 tick-equivalent GUTS. (e) Field vaccination studies The ceH culture vaccine thus developed against bovine tropical theiieriosis was used under field conditions at organised livestock farms and also at farmers' doorsteps in various parts of Haryana, Punjab, Madhya Pradesh and Maharasthra. Cattle of all exotic breeds and their cross-breads of aN age groups were included in these trials. The vacckie was found safe and effective under field conditions as the vaccinated animals were protected from bovine tropical theileriosis, whereas clinicai cases were observed in the non-vaccinated animals in those areas. It was observed that calves born from vaccinated dams in later stages of pregnancy, were susceptible to the disease. WE CLAIM 1. A process for the preparation of anti-theiieriosis vaccine comprising the steps of isolating lymphoid cells infected with schizonts of theileria annulate present in an infected animal, culturing he Infected cells In a growth medium, subjecting said cuttured cells to the step of in-vitro propagation by-serial passaging, developing cloned cultures from a single cell by limiting dilulion technique and further passaging these, karyotyping the cells in the medium to ascertain the diploid character, and obtalning viable cells with maximum infectivity rates, determining macroschizont nuclear number. subjecting a number of viable cells with diploid character to the step of cryopreservation in liquid nitrogen in the presence of a cryoprotectarnt in the said growth medium to obtain a suspension of viable cells, testing these cultures at different passages and In varying dose levels in groups of calves for ascertaining their pathogenicity / antigenicity, determining passage level and dose for the safety and efficacy of candidate vaccine, Its safe use In new born calves and pregnant cows, time taken to Induce immunity , serological responses and effect on haematoiogical parameters, and testing its efficacy under wide field conditions in the susceptible stock. 2. The process as claimed in claim 1 wherein the said growth medkim used is RPMI 1640: 10 to 20 % neonatal or normal bovine serum; antibtottos solutton containing benzyl penicillin sodium @100 I. u. per ml. streptomycin sulphate 0 100 µg per mi. and mycostatin @10 i.u. per ml; and also 200 mM of L- glutamlne per ml. AH the contorts shoukl be pre-sterilllised by filtratton and shoukt be free from any micro-organism contamlnatton by standard procedures. 3. The process as claimed in claim 2, wherein the said growth medium is pre-sterilized by filtration and made free from any micro-organism contamination by standard procedures. 4. The process as claimed in claim 1, wherein said cryoprotectani is selected from DM SO and glycerol. 5. The process as claimed in claim 4 wherein preferably said cryoprotectani used is DMSO. 6. The process as claimed in claim 1, wherein the step of in vitro propagation of various cell lines of the parasite developed were tested at various passages i.e. 10, 20, 50, 100, 150 & 200 with varying dose levels and K was found that the cloned culture at its passage 200 with 5x10° cells per ml as a single dose was safe and Immunogenic as a vaccine. 7. A process for the preparation of Ground-Up Tick supernatant (GUTS) comprising: collecting infected ticks sterilizing the ticks with cetavlon solution , and normal saline and RPMI- 1640, freezing the sterilized ticks, grinding a known number of frozen ticks to obtain a fine paste , subjecting the paste to the step of sterllization adding antibiotics and albumin powder to the paste. subjecting the paste to the step of cenlrifugalion to separate the supernate fluid. stiring the supernatant containting sporozoltes of T. annuleta in cryoprotectarnt to be used as Stabllte. 8. The process as claimed in claim 1, wherein the ceH concentration in said cell suspension is adjusted to 10x108 viable cells per ml, the total volume is measured and mixed with equal quantity of growth medium containing 20% DMSO so as to make the final concentration of cells at 5x10° viable cells per ml in growth medium containing 10% DMSO. 9. An anti theilertosis vaccine comprising schizonts of T annulate in a cryoprotectants and In growth medium . 10. The antl-theHertosis vaccine as claimed in claim 9, wherein the growth medium contains 20% DMSO so as to make the final concentration of 5x108 viable cells per ml in growth medium containing 10% DMSO. 11. The anti-theileriosis vaccine as claimed in claim 9 is protective and safe for the exotic and cross-bred cattle includtng new born calves and pregnant cows.

Documents:

880-DEL-2004-Abstract-(19-03-2012).pdf

880-del-2004-abstract.pdf

880-del-2004-assignment.pdf

880-DEL-2004-Claims-(19-03-2012).pdf

880-del-2004-claims.pdf

880-DEL-2004-Correspondence Others-(19-03-2012).pdf

880-del-2004-correspondence-others.pdf

880-del-2004-correspondence-po.pdf

880-del-2004-description (complete).pdf

880-del-2004-description (provisional).pdf

880-del-2004-form-1.pdf

880-del-2004-Form-13-(07-03-2006).pdf

880-del-2004-form-13.pdf

880-del-2004-form-18.pdf

880-del-2004-form-2.pdf

880-DEL-2004-Form-3-(19-03-2012).pdf

880-del-2004-form-5.pdf

880-del-2004-form-6.pdf

880-DEL-2004-GPA-(19-03-2012).pdf

880-del-2004-gpa.pdf

880-DEL-2004-Petition-137-(19-03-2012).pdf