Haemolytic activity of sheep complement for two assay systems

Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with rabbit antibody (sheep E-rabbit A) provided serum is used as soon as possible after collection. If left at 4 °C to separate from the clot, serum C activity for sheep E-rabbit A is markedly reduced. Heparinized plasma retains its haemolytic titre for at least 24 h at 4 °C. Plasma from Mg²⁺-ethyleneglycoltetraacetic acid (EGTA) blood is non-haemolytic, but addition of Ca²⁺ partially restores the titre. A high concentration of rabbit A is necessary to sensitize sheep E.Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg²⁺-EGTA. This C activity is stable at 4 °C for 24 h in serum, Mg²⁺-EGTA plasma and heparinized plasma. Haemolytic activity of serum heated at 50 °C for 30 min was restored by a factor B containing CM-cellulose fraction of foetal lamb serum in the presence of Mg²⁺-EGTA for human E-sheep A but not sheep E-rabbit A.These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca²⁺-Mg²⁺-dependent pathway that is labile in vitro for 24 h at 4 °C.     

Variations in sheep serum conglutinating and haemolytic activity for sheep erythrocytes sensitized by rabbit antibody

Based on conglutinating and haemolytic reactions with sheep erythrocytes (E) sensitized by rabbit antibody (A), three types of sheep sera were encountered. Type 1 sera do not conglutinate or haemolyse sheep E-rabbit A. Type 2 sera failed to conglutinate, but are haemolytically active. Type 3 sera have both activities. Serum from one type 1 sheep still failed to conglutinate 5 days after venepuncture but was now haemolytically active (i.e., type 2). Some sheep that initially had type 2 sera had, five days after an intraperitoneal injection of yeast cells, sera with conglutinating activity (type 3 sera). Type 1, 2 and 3 sera all had haemolytic activity with human E-sheep A indicator cells.Pooled type 3 sera have the highest conglutinating titres with sheep E-rabbit A after 10 min incubation at 39 °C. At this stage, the haemolytic titres were very low. From 10 min, the conglutinating titres decreased whereas the haemolytic titres gradually increased until 80 min. Optimal conglutinating activity required less rabbit A to sensitize sheep E than did haemolytic activity.     

Inhibitory activity of heated ewe serum for ewe and ram serum complement

In the presence of an indicator antigen-antibody complex, the complement (C) activity in ewe and ram serum was reduced or abolished by addition of ewe serum that had been heated at 56 degrees C for 10 minutes. Pre-incubation of heated ewe serum at 39 degrees C for 30 minutes with the C source prior to addition of the indicator system or addition at the same time caused similar reductions in C activity. Results from the haemolytic, conglutinating and haemagglutinating activities studied indicate that the ewe serum inhibitor(s) reacts with activated classical or alternative activating pathway components and/or the third component of C (C3). The presence of a potent C inhibitor(s) in ewe serum probably accounts for the low sheep C titres with some assay systems. As the heated ewe serum did not appear to activate ewe or ram C per se, it is more appropriate to regard it as 'inhibitory' rather than 'anticomplementary'.     

The relation between the rabbit potency test and the response of sheep to sheep clostridial vaccines.

Six commercially available clostridial vaccines comprising one oil-emulsion, two alum-precipitated and three aluminum hydroxide adjuvanted preparations, each containing between two and seven antigenic components, were administered to groups of 10 rabbits and eight sheep in accordance with manufacturers' recommendations. Serum antitoxic values to Cl welchii beta, Cl welchii epsilon, Cl septicum, Cl oedematins and Cl tetani toxins were determined 14 days after completion of each vaccination course. The overall pattern of mean antitoxic values was found to be similar in sheep and rabbits, a vaccine eliciting a comparatively high antibody titre to any given antigen component in sheep also inducing a comparatively high titre in the corresponding group of rabbits. Similarly, comparatively poor responses in sheep were associated with poor responses in rabbits. The degree of variation in response within groups of animals was greater in sheep than in rabbits for all five antigenic components assayed. Sheep consistently developed higher titres than rabbits to Cl oedematins component but consistently lower titres to both Cl welchii beta and epsilon components irrespective of the type of vaccine used. The response of both species to Cl tetani antigen was similar in terms of serum antitoxic values. It was concluded that rabbits provide a suitable model for the assessment of potency of sheep clostridial vaccines.     

A modified indirect immunofluorescent assay for the detection of antibody to Eperythrozoon ovis in sheep

Experimental ovine eperythrozoonosis was studied using Giemsa staining of blood films and a modified indirect immunofluorescent antibody assay (IFAA). The serums of 21 Border Leicester Merino cross lambs between 12 weeks and 7 months-of-age were analysed before and after infection with Eperythrozoon ovis (E. ovis) using the IFAA test. No rise in the IFAA titre was seen until day 7 and this coincided with the first detection of E. ovis organisms in blood smears stained with Giemsa. The percentage of E. ovis infected red blood cells peaked on day 14, but the IFAA titre did not peak until day 35. Titres to E. ovis, on average, had begun to drop by day 63. There was considerable individual variation in response to E. ovis infection as measured by the IFAA. Titres as high as 6,400 were observed in individual sheep at the peak of E. ovis parasitaemia of red cells. One sheep had a titre of 51,200 nineteen days after infection, and titres of 3,000 were maintained for several months in a few sheep. The assay proved reliable, and up to 100 samples per day could be tested. The antigenicity of the slide preparations was found to be satisfactory after storage for 6 months at -20 degrees C and 4 degrees C and for 28 months at -70 degrees C. Temperature fluctuations during storage rendered slides unsuitable for the IFAA after these times. A method of storing E. ovis infected blood in liquid nitrogen is described.     

III. Comparison of Two Complement-Fixation Methods

The complement-fixation test used at Onderstepoort was compared with the method used at A.D.R.I. on infected calf and sheep sera. In the first method, the tests are incubated at 37°C for 90 minutes and the test sera are inactivated at 53°C; whereas in the A.D.R.I. method, the test sera are inactivated at 60°C for 30 minutes, incubation is at 9°C for 18 hours, and guinea-pig complement is supplemented with 5 per cent fresh, non-inactivated, normal calf serum. Serial serum samples from one of six experimentally infected calves were negative in the Onderstepoort test, three calves gave only trace reactions and two showed maximum titres of 1:10 whereas all six had maximum serum titres of 1:10 to 1:80 in the A.D.R.I. test. A good correlation was obtained, however, between the results of the two methods with the sera of experimentally inoculated sheep although titres 3 to 8 times higher were obtained with the A.D.R.I.'s test. Post inoculation bleedings from each sheep reacted in both tests.     

Comparison of insulin-like growth factor I serum binding proteins in sheep and cattle: species differences in size and endogenous binding capacities

Binding proteins (BP) for insulin-like growth factor I (IGF-I) were characterized in sheep and beef cattle serum for molecular weight (Mr) and binding characteristics. Serum was incubated with [125I] IGF-I at 37 degrees C before chromatography over a 1.6-cm X 94.0-cm column of Sephacryl S-300 (pH 7.4, 4 degrees C). Beef serum exhibited a 145 k Mr (mol. wt X 1,000) and a 35 to 39 k Mr BP. Sheep serum possessed a 170 to 190 k and a 35 to 38 k Mr protein. Binding of [125I] IGF-I was inhibited in the presence of excess unlabeled ovine somatomedin, demonstrating specific binding for each BP of both species. The high Mr component was pituitary-dependent in sheep, as evidenced by binding patterns from serum of hypophysectomized sheep. Direct binding studies of the Sephacryl-separated BP demonstrated that the native BP of high molecular weight of both species bound only minor amounts of [125I] IGF-I in a manner unrelated to BP concentration. The BP of low molecular weight of beef cows displayed a bell-shaped dose-response binding curve with maximum binding at 250 micrograms/ml BP, whereas binding to sheep BP of low molecular weight was independent of BP concentration. After chromatography on Sephadex G50 at pH 2.8, both BP from both species exhibited concentration-dependent binding that plateaued at 250 to 500 micrograms/ml of BP of low molecular weight but was curvilinear for the BP of high molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

A myopathy of sheep associated with sarcocystis infection and monensin administration

An outbreak of muscle disease affected approximately 20 of 600 ewes in spring 1987 in south-east Scotland. The clinical signs were a flaccid paralysis of the hind limbs and in severe cases collapse. Serum creatine kinase and aspartate aminotransferase activities were increased. Clinically affected sheep had a mean reciprocal serum antibody titre in a sarcocystis immunofluorescence antibody test of 557 whereas 22 sheep from the same flock, sampled one year earlier, showed a mean reciprocal titre of only 51. Histologically a heavy infestation of sarcocysts, myodegeneration and a non-suppurative myositis centred on degenerating sarcocysts were observed in a wide range of skeletal muscles and myocardium from four affected sheep. Monensin sodium had been inadvertently included in the protein pellet used in the feed for one week before the onset of the disease.     

The effect of selenium supplementation on immunity, and the establishment of an experimental Haemonchus contortus infection, in weaner Merino sheep fed a low selenium diet

SUMMARY: Immunity in 12 weaner Merino sheep fed a low selenium (Se) diet (low Se sheep) was compared with that in 10 matching sheep fed the same diet but each given an intraruminal Se pellet (high Se sheep), while the sheep were housed in individual, sheltered pens. All sheep were challenged with killed Brucella abortus cells (days 0 and 28), rabbit red blood cells (days 0, 7 and 28) and corynebaclerium pseudotuberculosis toxoid (days 0 and 28), and serum antibody titres were measured weekly for 8 weeks from day 0. The sheep were then experimentally infected with Haemonchus conforfus, and slaughtered 8 weeks later. The mean antibody titre to B. abortus, measured by 4 different tests, was significantly higher in the high Se sheep on occasions during the primary immune response phase (Rose Bengal test - day 21 (p < 0.05), day 28 (p < 0.025); complement fixation - day 7 (p < 0.05); enzyme-llnked immunosorbent assay - day 14 (p < 0.01); serum agglutination - no differences), but not during the secondary phase. The mean antibody titre to rabbit red blood cells, measured by haemagglutination test, was marginally higher in the high Se sheep on day 49 (p = 0.049). The mean antibody titre to C. pseudotuberculois, measured by enzyme-linked immunosorbent assay, was not significantly different between the groups at any time during the trial. In addition, the mean invitro responsiveness of peripheral blood lymphocytes to stimulation with phytohaemagglutinin in the high Se sheep was significantly greater than that in 10 sheep from the low Se group on day 22 (p < 0.01), but not day 50. However, there were no significant differences in the mean number of sheep in which the infection with H. contortus established, time to first shedding of eggs in faeces, abomasal worm burdens at necropsy, or inflammatory response in the abomasal mucosa in the sheep in each group. The results showed that the low Se sheep produced strong overall immune responses that were largely comparable to those in the high Se sheep.     

Measurement of vitamin B12 in the livers and sera of sheep and cattle and an investigation of factors influencing serum vitamin B12 levels in sheep

Modifications of a radioassay method for the analysis of vitamin B12 using chicken serum as the binder are described. This obviates the need to use individual serum blanks to correct for non-specific binding in vitamin B12 assays of the sera and livers of sheep and cattle. Samples with high vitamin B12 levels can be diluted prior to assay without loss of linearity. Recoveries of added cyanocobalamin or hydroxocobalamin were better than 95% and results correlated significantly with those obtained using a microbiological assay (Poteriochromonas malhamensis). Sera and liver samples stored for four weeks at temperatures ranging from -20 degrees to 22 degrees showed no change in vitamin B12 levels. Withholding food from sheep for 44 hours led to a marked increase in serum vitamin B12. This effect was also evident in sheep eating a limited amount of cut grass. In sheep at pasture there was no evidence of a diurnal variation in serum vitamin B12 levels. Serum vitamin B12 levels in sheep at pasture were shown to be an unreliable indicator of liver vitamin B12.     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

Freezing mouse blastocysts. The influence of the preparations prior to freezing on the survival rate of the blastocysts

A good survival rate in culturing mouse blastocysts can be obtained in Ovum Culture Medium, enriched with 20 per cent inactivated Foetal Bovine Serum or Sheep Serum under air. The transfer of fresh blastocysts gives the best results if the recipients are on day 3 of the pseudo-pregnancy, but with 20 hours' cultured blastocysts it is better to use recipients on day 4. Exposure to 1.5 M DMSO has no harmful effect, provided that the DMSO is added at 5 degrees C in 6 steps and is removed, again in 6 steps, at 35 degrees C. The crystallization of the medium containing the embryos at -5 degrees C to -6 degrees C doet not appear te have a harmful influence on culture results of the blastocysts.     

Thermostability of the vaccination strain of infectious bursal disease virus

The vaccination strain of infectious bursal disease virus, multiplied in cultures of chick embryo cells, was very resistant to heat. At a temperature of 56 degrees C the infection titre of the virus (TCID50) decreased by 0.9 log10 within two hours and by 1.2 log10 within five hours, but the virus remained infective still after 24 hours. At a temperature of 37 degrees C, a slight decrease in infection titre was recorded only after two days and a decrease by 1.2 log10 was recorded within ten days. After the 21st day the virus was almost inactivated. At a temperature of about 20 degrees C the infection titre of the virus decreased linearly from the third to the twelfth weeks. The control samples kept at +4 degrees C retained their infectivity for three months and at -20 degrees C even for six months. The discussion deals with the effect of the concentration of protein and magnesium chloride in the medium on the thermostability of infectious bursal disease virus.     

The effects of Leptospira serotype pomona in sheep of different haemoglobin types

A warm complement fixation test that will detect antibody in sheepserum in the virtual absence of anti-complementary activity is described. Sheep antibody-antigen complexes were detected by fixation of sheep complement. Sheep serum, heparinized or Mg⁺⁺—EGTA plasma was used as the source of sheep complement. Sheep-antibody-sensitized human erythrocytes were used as the haemolytic indicator cells for sheep complement. As the modified complement-fixation test was performed in the presence of Mg⁺⁺—EGTA, sheep C probably reacts with sheep Ab-Ag complexes by a different mechanism than does guinea-pig complement.     

Serological survey for Toxoplasma infections in sheep

SUMMARY: A factorial-design survey was performed to determine the prevalence of specific antibody against Toxoplasma in young and adult sheep from 6 areas in 3 different geoclimatic zones in South Australia. Serum samples obtained from 1,159 sheep belonging to 59 flocks were tested by a conventional indirect haemagglutination test (IHAT) as well as by an enzyme-linked immunosorbent assay (ELISA) which used class-specific conjugates to detect both IgG and IgM against Toxoplasma. Titres > 64 were detected In 7.4%, 9.2% and 25.2% of the sheep by the IHAT, IgG-ELISA and IgM-ELISA respectively. A significant positive correlation was found between the results of the IgG-ELISA and the IHAT with identical results obtained for 1,050 samples. Antibody detected by all 3 tests was more prevalent and higher in titre in adult sheep than in lambs as well as in sheep from Kangaroo Island than in those from mainland South Australia. Although the regional differences in prevalence suggested a relationship with climate, no significant correlations were detected between the prevalence results and any single climatic factor; namely, average annual rainfall, average annual evaporation or mean temperature range.     

Cytotoxin from an ovine strain of Pasteurella haemolytica: characterisation studies and partial purification

A cell-free, water-soluble cytotoxin from an ovine strain of Pasteurella haemolytica biotype A serotype 1 killed sheep bronchoalveolar macrophages at 37 degrees C, but not at 4 degrees C or 22 degrees C. The cytotoxin was stable over the pH range 2-12, resistant to heat at 60 degrees C but inactivated at 100 degrees C or by autoclaving. Trypsin also destroyed the cytotoxin, which is therefore thought to contain a protein component essential for biological activity. A preliminary purification of the crude cytotoxin using gel-filtration column chromatography resulted in the isolation of a biologically active fraction which resolved as a single protein band and one carbohydrate band on non-dissociating polyacrylamide gels. However, this fraction resolved into approximately 16 component bands on a sodium dodecyl sulphate polyacrylamide gel.     

Enhancement of Leptospira hardjo agglutination titers in sheep and goat serum by heat inactivation.

Heat inactivation of sheep serum samples resulted in the detection of an additional 9% reactors to Leptospira hardjo that were negative on the initial test of fresh samples. Treatment with EDTA gave results generally similar to heat inactivation suggesting that complement was responsible for the inhibition of agglutination. Tests on heat inactivated serum from experimentally infected sheep and goats revealed enhanced titers or reactions which were not detected in fresh serum.     

Rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep.

The mechanism of onion-induced hemolytic anemia in ruminants was investigated. The ether-extract obtained from the mixture of rumen fluid and onion juice incubated at 38.5 degrees C for 9 hr induced oxidative damage in sheep erythrocytes in vitro, indicating the production of certain oxidants in the mixture. The increase of the oxidative effect in the mixture was inhibited completely by the removal of rumen microorganisms and partly by treatment with antibiotics and by oxygen gas. The sheep fed onions (50 g/kg body weight/day) for 15 days developed more severe Heinz body hemolytic anemia than did the sheep fed the equivalent amount of onions with 5 g/day ampicillin sodium salt. The results indicated that certain rumen bacteria appear to be involved in the onset of onion-induced hemolytic anemia in sheep.     

An apparatus for the restraint of sheep during infestation with schistosomes.

With this adjustable apparatus sheep aged from five months to five years and varying in mass from 16-95 kg could be restrained effectively. Cercarial suspension was split by only 2/44 sheep during infestation with Schistosoma mattheei. Using a battery of seven cages, it was possible to infest 23 sheep, each for 30 minutes, in one day. No cercariae were more than 4-1/2 hours old at the end of exposure.     

Effect on systemic antibody concentrations of topical application of choleratoxin to skin of sheep

OBJECTIVE: To examine the ability of a vaccine formulation combining choleratoxin with an experimental antigen to induce a systemic antibody response when applied topically on unbroken skin of sheep. DESIGN: Seven treatment groups of five adult sheep received systemic or topical priming followed 4 weeks later by systemic or topical boosting with choleratoxin and/or bovine serum albumin. Topical vaccines were administered to clipped skin on the ventral abdomen for 2 h. Booster immunisations were repeated 8 weeks after initial boosting. Serum antibody titres to choleratoxin and bovine serum albumin were determined by ELISA. RESULTS: An antibody response to choleratoxin was observed in serum, but no antibody response to bovine serum albumin was detected. CONCLUSION: Transdermal delivery may be feasible for livestock vaccines, however, further work is necessary to develop formulations that induce protective immunity by this route.