Studies on some blood parameters of crossbred calves with experimental Theileria annulata infections.

Subcutaneous inoculation of 1 ml of ground Theileria annulata tick tissue stabilate (0.75 tick equivalent) into crossbred calves (n = 6, average age 53 days) resulted in the development of acute theileriosis. The percentage parasitaemia was 71.7% +/- 3.3% on day 20 after inoculation. Macroschizonts were observed in lymphocytes and monocytes. Phagocytosed schizonts were observed in neutrophils, along with cytoplasmic vacuolation in monocytes and neutrophils. There was progressive decrease (p < 0.05) in the haemoglobin and packed cell volume, along with a marked reticulocytosis. Serum analysis revealed a decrease (p <0.05) in the concentrations of calcium, cholesterol and triglycerides, while there was an increase (p < 0.05) in the concentrations of blood urea nitrogen as compared to day 0 values. The total serum proteins, albumin and serum immunoglobulin concentrations and the albumin-to-immunoglobulin ratio showed marked decreases (p<0.05). Coagulopathies included thrombocytopenia and an increased prothrombin time, along with a non-significant increase in the bleeding time and activated partial thromboplastin time during the terminal stages of the disease. There was an increase in the osmotic fragility of erythrocytes during the disease. Morphological alterations in the erythrocytes were observed with the developing parasitaemia.

Conservation skills among preschool children in Umtata, South Africa.

To 41 boys and 29 girls (M age = 5.2 yr.) in a South African preschool a test of conservation of liquid was given. Analysis showed conservation skills had not been acquired as only 5 children showed such skills and only one knew why.

Effectiveness of lipopolysaccharide as an intrauterine immunomodulator in curing bacterial endometritis in repeat breeding cross-bred cows.

Antibiotics are usually used to combat microbial infections of the uterus, responsible for hindering establishment of pregnancy in cross-bred cows. The major disadvantages of antibiotics are: development of bacterial resistance, high costs and diminishing uterine defense mechanisms (UDM). As an alternative therapy, intrauterine application of Escherichia coli Lipopolysaccharide (E. coli LPS) as a uterine defense stimulator was used in this study in confirmed clinical cases of repeat breeding associated with bacterial endometritis. In the treated group (n=12), on the day of estrus, 100 microg of E. coli LPS dissolved in 30-ml sterile phosphate buffer saline (PBS) was infused intrauterine; while in the control group (n=12), only 30 ml of PBS was infused. Six-hour post-treatment, in the treatment group uterine washings showed a 100-fold increase in the total leucocytic count (TLC). Out of the cellular contents, more than 80% of the cells were recognised as neutrophils; above 60% were alive and their phagocytic activity was five bacteria/neutrophil. Such a cellular response was maintained until 72-h post-treatment. At the subsequent estrus period, the cervicovaginal mucus (CVM) became clear in 9 out of 12 cows (75%) and showed no bacterial growth. In the control group, similar micro-organisms were present in CVM of all the 12 cows before and after the PBS infusions. During the subsequent estrus, all nine cows with sterile CVM in the treatment group conceived while only one cow conceived from the control group. It was concluded that, administration of intrauterine E. coli LPS as single infusion in cows with bacterial endometritis stimulated UDM and cleared the infection within one estrous cycle, and thereby restoring fertility.

Haematological and biochemical studies on experimental Theileria annulata infection in crossbred calves.

Administration of ground-up tick tissue stabilate (0.75 tick equivalent) by the subcutaneous route to crossbred calves aged 1 week to 1 month led to the development of acute theileriosis. Haematological studies revealed significant progressive decreases in haemoglobin concentration, packed cell volume and red blood cell count, whereas the total leukocyte count showed an initial non-significant leukocytosis followed by a significant leukopenia. Analysis of serum revealed significant increases in levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatinine kinase and gamma-glutamyltransferase, and in the concentrations of uric acid, blood urea nitrogen and bilirubin. The concentrations of total protein, albumin, glucose, cholesterol and calcium showed non-significant decreases, while phosphorus decreased significantly during the terminal stages of the disease.

Lowered vaccine dose for immunisation of calves against tropical theileriosis using Theileria annulata lymphoblasts.

Bovine tropical theileriosis is of high incidence in calves below 2 months old in India. The Theileria cell culture vaccine evolved by the Punjab Agricultural University (PAU) was evaluated previously for its efficiency and safety in younger calves, both experimentally and in the field. The dose of cells used for vaccination was regulated at 1.75 to 2 x 10(6) cells. This study, which was undertaken to assess the minimum number of cells which would be effective and safer for vaccination, showed that optimal protection was given by a dose of 1 x 10(6) cells.

Enhanced immunity to Hyalomma anatolicum anatolicum ticks in cross-bred (Bos indicus x Bos taurus) calves using ascaris extract immunomodulator with the tick salivary gland extract antigens.

Development of acquired immunity in cross-bred (Bos indicus x Bos taurus) calves against Hyalomma anatolicum anatolicum, a tick vector of bovine tropical theileriosis was studied using ascaris extract (AE), an immunomodulator of IgE responses, along with the tick salivary gland extract (SGE) antigens in Freund's incomplete adjuvant (FIA) emulsion. Calves immunised with SGE + FIA showed significant rejection (47.4 +/- 2.8%) of larvae, whereas only marginal rejection (12.47 +/- 1.5%) of nymphs was observed. In contrast, calves immunised with the immunomodulator AE in addition to SGE + FIA showed significant enhanced rejection of nymphs (50.25 +/- 5.2%), while the rejection of larvae was only slightly higher (55.8 +/- 11.4%), and not statistically different. In addition, incorporation of the immunomodulator AE also resulted in significant enhancement in the percentage recovery of abnormally fed larvae and nymphs. This abnormal feeding was characterised by their white to pale-yellow colour instead of the dark-grey colour of normally fed ticks. It may possibly be attributed to their inability to gain access to the blood vessels owing to the host immunological reaction at the bite sites. Consequently, the ticks fed on extravascular fluid devoid of red blood cells resulting in their white to pale-yellow colour. Difference in the disease transmission potential of normally and abnormally fed ticks is of future interest. Sera from all the immunised calves after 2 weeks of immunisation were positive for anti-tick SGE antibodies in the dot enzyme immunoassay (DOT-EIA). The immunised calves were positive for immediate type hypersensitivity (ITH) skin reaction on intradermal inoculation of the tick SGE antigens. There was no apparent difference in the DOT-EIA antibody titres between the two immunised groups. However, ITH skin swelling was significantly higher in AE + SGE + FIA immunised calves. The study indicates that use of the AE as an immunomodulator along with the tick SGE antigens enhanced anti-tick immunity.

Immunological cross-reactivity between salivary gland proteins of Hyalomma anatolicum anatolicum and Boophilus microplus ticks.

Immunological cross-reactivity was established between salivary gland extract antigens of Hyalomma anatolicum anatolicum (H.a.a) and Boophilus microplus (B.m.), using native-polyacrylamide gel electrophoresis (native-PAGE), immunoblotting, DOT-enzyme immunoassay (DOT-EIA) and in vivo intra-dermal skin test revealing immediate type hypersensitivity swelling reaction. Native-PAGE revealed six proteins in common of molecular weights of 60, 66, 148, 264, 300 and > 300 kDa in the salivary gland extract (SGE) of both the ticks. Four additional bands of molecular weights of 56, 64, 120 and 220 kDa were present in H.a.a. but absent in B.m. Immunoblot assay of native-PAGE transblotted proteins of H.a.a., using mice anti-H.a.a. SGE and anti-B.m. SGE immune serum revealed a common protein of 66 kDa. In DOT-EIA, anti-H.a.a. or anti-B.m. SGE immune serum gave similar antibody titres against the SGE antigens of either tick species. Intradermal inoculation of the SGE antigens of either species on cross-bred calves (Bos indicus x Bos taurus) immunized to H.a.a. or B.m. ticks produced a strong, immediately hypersensitivity skin swelling reaction.

Significance of detection of immune-complexed 8 kDa hydatid-specific antigen for immunodiagnosis of hydatidosis.

Three micro-enzyme-linked immunosorbent assay (micro-ELISA) systems were developed and evaluated for detection of specific free circulating antigen and circulating immune-complexes (CICs) of 8 kDa antigen in the sera of patients with hydatidosis. All (100%) the sera of 30 confirmed positive cases of hydatidosis had detectable levels of antigen in the acid-treated sera. However, 23 (77%) and 26 (87%) sera of 30 confirmed cases had free as well as CICs of 8 kDa antigen in the untreated and in the polyethylene glycol (PEG) precipitated sera, respectively. None of the sera from other patients with parasitic infections or viral hepatitis had any detectable levels of 8 kDa antigen in the untreated, acid-treated or PEG-precipitated serum samples. The investigations, therefore, suggested that the demonstration of circulating antigen employing monospecific antibodies to affinity purified 8 kDa antigen in acid-treated sera is more efficient as compared to detection of free circulating antigen or CICs in the untreated or in the PEG-precipitated sera which could provide a specific immunodiagnostic tool for ongoing hydatid infection.

Caprine retrovirus infection in New South Wales: virus isolations, clinical and histopathological findings and prevalence of antibody.

Caprine arthritis-encephalitis virus (CAEV) was isolated by explant cultures of carpal synovial membranes and lung from 7 goats in New South Wales. These goats were clinically affected with the arthritic, neurologic, and pneumonic forms of CAEV infection either singly or in combination. CAEV antibody was detected by the gel immunodiffusion precipitin (GDP) test in 5 of the 7 goats. Serum samples from 2,708 goats, from 115 herds, were examined for CAEV antibody using the GDP test. Approximately one-third of the animals and 82% of the herds tested had CAEV antibody. The infection was common in all breeds of dairy goats with an indication of a significantly lower prevalence in the Saanen breed (24.4%) compared to Nubians, British Alpines and Toggenbergs (43.8%, 38.7% and 39.1% respectively). CAEV antibody was also demonstrated in 11 of 230 Angora goats. The infection was equally common in all age groups, with slightly higher prevalence in males (83 of 230, 36%) compared to females (648 of 2,232, 29%). Among seropositive animals 85% were clinically normal. Of 280 clinically affected goats tested only 42% had detectable antibody. One of 5 sheep that had been in contact with infected goats in one herd had CAEV serum antibody.

Vulvovaginitis of goats due to a herpesvirus.

Two concurrent outbreaks of genital disease in goats were associated with infection by a herpesvirus that was isolated from vulval and vaginal lesions of affected does. Serum neutralising antibody to the virus was present both in goats with the clinical disease and some unaffected goats. Of 19 goat herds examined only 4 had serum neutralising antibody positive goats with low (5%) to high (60%) incidence of infection. The virus isolate was characterised as a herpesvirus on its physico-chemical and morphological features. It contained DNA and was inactivated at low pH and by treatment with lipid solvents and trypsin. The virus particles were icosahedral, consisting of a nucleocapsid surrounded by an envelope membrane and measured approximately 150 nm in diameter. The virus was serologically related to a New Zealand isolate of caprine herpesvirus (NZ-CpHV), associated with similar genital disease, and was distinct from bovine herpes virus-1 (BHV-1) showing a one way neutralisation pattern.

Evaluation of a rosette inhibition test for pregnancy diagnosis in pigs.

A rosette inhibition test was developed using pig lymphocytes and sheep red blood cells. Antilymphocyte serum (ALS) in the presence of complement inhibited rosette formation by greater than 95% at 1/250 declining to no inhibition at 1/8000. Sera obtained from a total of 14 pregnant sows before and 1, 2, 3 and 4 wk after mating were tested for their ability to augment the rosette depression caused by ALS. In one experiment in which the responses of 4 pregnant sows were compared to 4 non-pregnant sows by discriminant analysis, sera were classified correctly in 83% of the samples taken from either pregnant or non-pregnant sows. When the more usual method of calculating the rosette inhibition titre was used, the responses of sera from pregnant pigs were classified with 31% accuracy and those from non-pregnant pigs with 80% accuracy. In a second experiment, sera from 10 pregnant sows were classified with 25% accuracy using the rosette inhibition titre. Thus 4 of these pigs were classified as non-pregnant by this method. Data from the second experiment were not suitable for discriminant analysis. It was concluded that there was some factor present in the sera of pregnant pigs, particularly by 3 or 4 wk post-mating, which could be detected by the rosette inhibition test. However, the test is not sensitive enough to allow specific diagnosis of early pregnancy in pigs.

Complement-dependent, polymorphonuclear neutrophil-mediated cytotoxicity of herpesvirus-infected cells: possible mechanism(s) of cytotoxicity.

Highly enriched bovine polymorphonuclear neutrophils (PMN) were able to destroy herpesvirus-infected cells in the presence of complement. Our results suggest that some viral antigens are capable of activating the alternate complement pathway directly which results in cytotoxicity however, the addition of PMN plus complement resulted in high levels of cytotoxicity. Chelation of Ca2+ or Mg2+ indicated that Ca2+ was involved in PMN--target cell interactions but Mg2+ was involved in activation of the alternate complement pathway. Cytotoxicity in the presence of PMNs was shown to be under bidirectional control of cyclic nucleotides. Thus agents that elevated cAMP reduced cytotoxicity whereas agents that elevated GMP increased it. If the PMN is secreting some product during the cytotoxic reaction it must be performed since chemical shown to inhibit DNA, RNA and protein synthesis had no influence on CDNC. Our results are discussed in terms of the mechanism of CDNC lysis as well as in the vivo significance of such a defence mechanism.

Polymorphonuclear neutrophil-mediated antibody-dependent cell cytotoxicity of herpesvirus-infected cells: ultrastructural studies.

Bovine polymorphonuclear neutrophils (PMN) can mediate antibody-dependent cell cytotoxicity (ADCC) of herpesvirus-infected cells. Since cytotoxicity occurs only in the presence of PMN and specific antiviral antibody, but not until viral membrane antigens are expressed on the target cell, it is concluded that antibody must recognize viral membrane antigens before cytotoxicity can occur. Cytotoxicity also requires very close contact between the target cell and the PMN cell. These interactions occur as early as 1 h after incubating antibody, infected cells, and PMN, but the actual lysis and release of intracellular components occur over an extended period. It was assumed that degranulation was not involved in the initiation of cytotoxicity, but was involved in the final stage of destruction. The mechanism of lysis is proposed to involve the interaction of PMN membranes with target cell membranes with subsequent reorganization and activation of the PMN plasma membrane at points of contact with the target cell. This results in possible production of transmembrane channels which allows for the release of target cell contents.

Destruction of virus infected cells by neutrophils and complement.

The paper describes an antibody independent mechanism of cytotoxicity whereby virus infected but not uninfected cells are destroyed by the combined presence of neutrophils and complement.