In vitro generation of cloned populations of Toxoplasma gondii.

Cloned populations were generated from Indian isolates of Toxoplasma gondii by transferring single tissue cysts from the brains of chronically infected mice to confluent murine macrophage (J774A.1) monolayers. The clones were then maintained continuously as tachyzoites in culture. Physical rupture of the tissue cysts and release of bradyzoites prior to seeding was found to be necessary for establishment of the parasite in culture. Although intact tissue cysts seeded over monolayers released bradyzoites spontaneously, they did not succeed in setting up an infection in the monolayers. Random amplified polymorphic DNA (RAPD)-PCR, which revealed distinct patterns for a clone and its progenitor, further confirmed the efficiency of the technique. The cloning technique was found to be simple and rapid compared to those involving limiting dilutions.

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata.

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.

Detection of Theileria annulata carrier cattle by PCR.

A simple method for treating bovine blood samples for direct detection of T. annulata in carriers, after polymerase chain reaction (PCR) amplification of small subunit ribosomal RNA (SSU rRNA) gene is described. The threshold of detection of the PCR-assay was an erythrocytic parasitaemia of 0.00008% corresponding to 16 infected bovine erythrocytes. In 50 known carriers, 42 were positive in PCR, in which 8 cattle revealed presence of T. annulata in stained blood smear under microscope.

Prophylactic efficacy of buparvaquone in experimentally induced Theileria annulata infection in calves.

The antitheilerial activity of buparvaquone (BW 720C) was evaluated in experimentally induced Theileria annulata infections in cross-bred male calves. T. annulata infections were induced by injecting a suspension of infected ground tick tissue suspension (GUTTS) equivalent to two ticks subcutaneously into each calf. Buparvaquone at a dose of 2.5 mg kg-1 body weight was given as a single injection (intramuscularly) on Day 0 (Group 1), Day 8 (Group 2) and Day 12 (Group 3) post-infection. The animals in Groups 4 and 5 were untreated and challenged controls, respectively. All of the recovered animals from Groups 1-4 were challenged with a lethal dose of T. annulata at 6 weeks post-infection. The immunized animals were resistant to the homologous challenge, which killed three of four control animals (Group 5); the controls showed typical antemortem and post-mortem lesions of theileriosis.

Comparative pathogenicity of Theileria annulata strains.

Twenty three susceptible crossbred calves were inoculated with five Indian strains of Theileria annulata, collected from natural cases of tropical theileriosis at Ludhiana, Hissar, Jaipur, Uruli-Kanchan and Bangalore. All the strains produced clinical disease with typical symptoms and lesions of acute theileriosis. The strains were considered highly virulent and equally pathogenic.

Efficacy of parvaquone and long-acting oxytetracycline in Theileria annulata infection.

Therapeutic and prophylactic efficacies of parvaquone and long-acting oxytetracycline were tested against Theileria annulata infection, induced by injecting a suspension of infected ground tick tissues (GUTS) into groups of 4 or 5 calves. This infection killed two of four control calves, while all the animals given a single intramuscular dose of 20 mg kg-1 parvaquone or long-acting oxytetracycline on the day of infection underwent mild reactions and recovered. Two separate doses of parvaquone of 10 mg kg-1 administered on the first and second days of fever protected four out of five calves. All the recovered animals from both treated and control groups resisted a homologous challenge with GUTS on Day 45 post-infection which killed three out of four susceptible unimmunized control calves.

Observations on histamine, serotonin, cortisol and cortisol binding globulins in bovine anaplasmosis.

Changes in histamine, serotonin, cortisol and cortisol binding globulin (CBG) levels were observed in Anaplasma marginale-infected bovine calves. In addition, packed cell volume (PCV), platelet number and percentage parasitaemia were also recorded. The whole blood histamine and serotonin values rose significantly (P less than 0.01 and P less than 0.025, respectively) during the acute stage of Anaplasma infection. Higher serum cortisol and CBG levels (P less than 0.05) were observed in acute and carrier infections, respectively. A sharp drop in thrombocyte count (59%) and PCV (33%) was also noticed in clinical anaplasmosis. The results suggest that the higher levels of biogenic amines which are known to produce increased vasodilation, capillary permeability and tissue anoxia and hypercortisolaemia to protect animals from stress and cell damage may play a similar role in the pathogenesis of acute anaplasmosis.

Chemoprophylaxis of Anaplasma ovis infection in sheep with a long-acting oxytetracycline.

The chemoprophylactic efficacy of long-acting oxytetracycline was determined in 13 susceptible ewes infected with Anaplasma ovis. The drug was administered intramuscularly at a dose rate of 20 mg kg-1 body weight during the prepatent period. When the ewes were exposed to an equivalent homologous challenge on Day 45 post-infection, each showed a mild or inapparent reaction. The host reactions, i.e., body temperature, parasitaemia, packed-cell volume (PCV), haemoglobin, humoral and cell mediated immunity (CMI) were also studied. The results indicate that the use of oxytetracycline during the incubation period would minimise clinical signs of an Anaplasma infection; this may be due to an increasing CMI response.

Chemoprophylaxis with diminazene aceturate in experimental Babesia bigemina infection in cattle.

Four-month-old fully susceptible cross bred calves were immunised against Babesia bigemina infection by treating them, during the prepatent period, with one or two doses of diminazene aceturate at 3 mg (kg bodyweight)-1. After 45 days the recovered calves were challenged with the homologous strain of B bigemina infected blood. The ensuing host responses, body temperature, parasitaemia, haemoglobin, packed cell volume, haemoglobinuria, capillary agglutinating antibodies and mortality, were studied. The four untreated control calves developed a severe form of the disease with classical symptoms and two died. Calves of both groups which recovered resisted the challenge infection with a mild relapse in the immunised group, thus indicating a state of premunition. The effect of one dose of diminazene was sufficient to suppress all the clinical effects of the disease.

Gastro-intestinal nematodes in sheep in Kashmir.

Examination at necropsy of the abomasum and intestinal tract of 62 sheep (including both sexes) of Karnah breed, aged one to two years over a period of one year was conducted at Handwara, situated in the north-west of Kashmir Valley. The following species of nematodes were found: Haemonchus contortus, Trichostrongylus axei, Trichostrongylus colubriformis, Bunostomum trigonocephalum, Chabertia ovina, Nematodirus spathiger, Skrjabinema ovis and Trichuris ovis. Infections with Trichostrongylus axei and T. colubriformis were, respectively, 57.7 and 40.2% of the total of all nematodes recovered. Faecal examination of adult sheep of Karnah breed, aged two to four years, comprising mostly pregnant and lactating ewes, showed a rise in strongyle egg counts during the months of March and April, coinciding with the lambing season in the area. The lambs showed a rise in faecal strongyle egg output from May onwards. The counts reached their peak during the month of September. It is presumed that pastures contaminated by pregnant and lactating ewes during the spring season were responsible for the acquisition of heavy nematode infections by lambs in the area.

Immunological relationship between strains of Theileria annulata Dschunkowsky and Luhs 1904.

The immunological relationship between the Ludhiana and Hissar strains, and between the Uruli-Kanchan, Bangalore and Jaipur strains of Theileria annulata was studied. Calves were immunised against the Ludhiana strain by the infection-treatment method, against the Uruli-Kanchan strain by the infection-treatment method or by untreated low-grade infection and against the Jaipur strain by untreated low-grade infection. After 45 days, groups of immune calves and susceptible controls were challenged with 10-tick equivalent stabilates. The ensuing host responses, body temperature, swelling of the regional lymph node, appearance of schizonts in the regional lymph node and piroplasms in blood and mortality, were studied. All five strains proved very virulent and produced severe disease in susceptible calves. Immunisation with the Ludhiana, Uruli-Kanchan and Jaipur strains conferred absolute protection against severe homologous challenges. Immunisation with the Ludhiana and Uruli-Kanchan strains conferred substantial protection against heterologous challenges with the Hissar strain, and the Bangalore and Jaipur strains, respectively. Immunisation with the Uruli-Kanchan strain gave less protection against heterologous challenge with the Jaipur strain and vice versa as some of the challenged calves showed slight swelling of the regional lymph node, two had fever, all exhibited a low-grade parasitaemia and four developed anaemia.