Fractionation on Cross-Linked Dextran, Sephadex G-25, of Sera from Sheep Infected with Johne's Bacilli. Preliminary Report

Ten sera collected during the winter months from six sheep infected with Johne's bacilli were fractionated on Sephadex G-25 columns, and all fractions tested for complement-fixing antibody, anticomplementary properties and for supplementing and inhibitory activities when added to complement-fixation tests of a heterologous antigen-antibody system: bovine or ovine antiserum with Brucella abortus antigen. Serum from a normal sheep was similarly fractionated and examined.

The complement-fixing activity with a carbohydrate fraction of Johne's bacilli was confined principally to supernatant fractions of the earlier eluates containing the greater part of the serum proteins. Some of the unheated earlier and later fractions displayed a limited degree of supplementing effect. Inhibitory activity was demonstrated by certain of the earlier and later eluates after they had been heated, particularly those of antisera with initially low complement-fixing antibody titer.

     

Methods of Preparing Supplementing Fractions from Fresh Unheated Bovine Sera for Use In Modified Direct Complement-Fixation Tests

Two methods of preparing partially purified, relatively stable supplementing factor from fresh bovine serum are described: gel filtration through Sephadex G-25, and anion exchange chromatography on a diethylaminoethyl (DEAE) cellulose column. In both procedures, an active, reconstituted precipitate prepared by dialysis of fresh unheated normal serum in the cold for 18 hours against phosphate buffer pH 6.2, 0.02 M, serves as the starting material.

The Sephadex G-25 column is equilibrated with acetate buffer pH 5.4, 0.2 M. The most actively-supplementing material appears in the eluates in which the pH has risen to 7.5 or higher. For the DEAE cellulose chromatography a gradient system is used: initial phosphate buffer 0.03 M, pH 8.0, limiting buffer Na H₂PO₄, 0.3 M. The greater part of the supplementing activity is eluated between pH 5.6 and 6.0, although some of the earlier fractions are also reactive. Pooled active eluates stored in the frozen state for nine months or longer maintained their supplementing titre in modified complement-fixation tests of two bacterial antigen-bovine antibody systems.

     

The Effect of Unheated Normal Bovine Serum on The Complement Fixing Activity of Heat Inactivated Bovine Antiserum With Homologous Antigen: II. Aggregative Properties

To determine whether the greater fixation of complement observed in “modified” complement-fixation tests is related to an increased aggregation of the antigen-antibody complexes, parallel tests by the two methods have been made with two different particulate bacterial antigens and corresponding bovine antisera. At the end of the fixation period the mixtures were centrifuged, the supernatant fluids removed carefully, the sediments washed twice and re-suspended in a small volume of buffer. Smears of each sediment were examined by immunofluorescence microscopy using a fluorescein-labelled rabbit antibody for a globulin fraction of fresh guinea-pig serum containing first component of complement.

A greater degree of aggregation of the antigen-antibody complexes was observed in the sediments from tests with modified complement, that is complement supplemented with a diluted bovine serum globulin fraction prepared by dialysis. Aggregates from mixtures showing increased fixation of complement, as determined by titration of the residual hemolytic activity of the supernatant, appeared somewhat more brightly fluorescent.

Very faint or no fluorescence was evident in the stained washed sediments from mixtures of antigen and antibody without complement or from mixtures of antigen, heat-inactivated normal bovine serum and complement.

     

The Effect of Unheated Normal Bovine Serum on the Complement-Fixing Activity of Heat Inactivated Bovine Antiserum with Homologous Antigen: III. Immunodiffusion Studies

Examination by immuno-diffusion methods in agar gel plates demonstrated that the supplementing fraction precipitated from normal bovine serum by dialysis against dilute buffer, pH 5.0 to 6.6, contains at least three main antigens, and a weaker fourth. Two of these antigens or antigen determinants appeared to be present in globulin fractions prepared by dilution with distilled water or differential precipitation with ammonium or sodium sulphate.     

Infection of the Bovine Udder with Bovine Herpesvirus

Infectious bovine rhinotracheitis — infectious pustular vulvovaginitis (bovine herpesvirus) grown in tissue culture was used as inoculum in trials to infect the lactating bovine udder. Six experiments were undertaken in which one or more quarters were infused with 1 ml. of tissue culture fluids containing 10⁶ to 10⁷ tissue culture infectious doses (TCID) of virus. In four of the experiments the inoculated quarters showed marked evidence of infection in the form of acute inflammation, swelling, reduced milk secretion and profound changes in the physical appearance of the milk. In each case virus was recovered in high titres in the milk from about the second until the tenth to fifteenth days following exposure. Uninfected quarters remained normal in appearance and virus could not be recovered from the milk.

In three of the experiments it was shown that serum and milk antibodies appeared shortly after the disappearance of virus from the milk. One experiment involving two animals showed that about 1000 TCID of virus were required to produce infection. In one experiment a cow having a pre-inoculation serum titre for bovine herpesvirus proved resistant to infection.

The experiments indicate that the bovine udder is readily susceptible to bovine herpesvirus in non-immune animals, and that the virus produces an acute, limited infection leading to a temporary disfunction of the gland. It appears that natural invasion of the udder through the teat canal is not readily accomplished by the virus.

     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera.

Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.

     

Study of serum drug levels in calves following intramuscular administration of three tetracycline drug preparations

Three antibiotic formulations, oxytetracycline (A) in propylene glycol and oxytetracycline (B) in polyvinyl pyrrolidine and pyrrolidino-methyltetracycline in an oil suspension were given to calves by the intramuscular route. Only oxytetracycline (A) appeared to cause much pain after injection.

The half-time elimination (t_½cl) for oxytetracycline (A) was 14.000 ± 3.580 h for oxytetracycline (B) 10.290 ±5.159 h and for pyrrolidinomethyltetracycline 8.160 ± 0.920 h. The rate of elimination `beta slope” for oxytetracycline (A) was 0.052 ± 0.012 h⁻¹ for oxytetracycline (B) 0.077 ± 0.261 h⁻¹ and for pyrrolidinomethyltetracycline 0.086 ± 0.010 h⁻¹. The Y intercept of the “beta” elimination slope C_os (μg/mL) for oxytetracycline (A) was 2.490 ± 1.040, for oxytetracycline (B) 3.463 ± 1.874 and for pyrrolidinomethyltetracycline 2.852 ± 1.360.

Pyrrolidinomethyltetracycline appeared to have a two component elimination curve, however, only the “beta slope” was used for the calculations.

     

Immunofluorescent Studies of Bovine Hypersensitivity Pneumonitis

Antigens of Micropolyspora faeni were located by immunofluorescence in apparent association with alveolar macrophages within sections of lung from cases of bovine interstitial pneumonia. Pretreatment of frozen sections with pH 2.8 glycine-HC1 buffer was a necessary prerequisite for specific staining. Bovine immunoglobulin and bovine complement were identified in similar locations.     

Chromatographic Studies of Sera from Calves Vaccinated with Brucella Abortus, Strain 19

The presence of antibody was detected by agglutination tests in the serum of calves four days after vaccination with Brucella abortus strain 19. Titres had reached a maximum by seven to ten days post-vaccination. Sucrose density-gradient ultracentrifugation demonstrated that the earliest antibodies were macroglobulins, IgM (19Sγ; γM)-globulins. Lighter antibodies, IgG (7Sγ₂; γG)-globulins, appeared a few days later. With time, antibody titres fell, IgM declining somewhat more quickly than IgG. After revaccination some seven months later, there was a rapid rise in both IgM and IgG.

Anion-exchange column chromatography (DEAE-cellulose) and gel filtration (Sephadex G-200) were applied in separating the two forms of antibody. The former method, in which a gradient buffer system was used, proved to be the more efficient; the IgG antibodies apeared in early eluates at pH 7.8 to 8.0 and low ionic strength, 0.03M, whereas IgM was eluted late when the pH had fallen below 6.0 and the molarity had increased to beyond 0.2. DEAE cellulose chromatography detected IgG as well as IgM sera collected as early as five days after vaccination.

     

Occurrence of Blood Cells and Serum Proteins in Bovine Fetuses and Calves

Hematological values of peripheral blood were determined for bovine fetuses and calves of various ages. Erythrocyte values increased through gestation. Fetuses 100 days or older had total values within the ranges of those reported for normal adult cattle. Mature erythrocytes were not observed in embryos and only a few were observed in fetuses 40 days of age. Fetuses 250 days or older had only a few rubricytes (<10/100 WBC). Leukocytes were first identified in the peripheral blood of a 45-day old fetus. Absolute leukocyte values increased through gestation and reached maximum values shortly before parturition. Granulocytes were first observed at 130 days of gestation and reached maximum values near parturition.

Total serum protein and gamma-globulin concentrations of colostrum-deprived calves were similar to serum protein and gammaglobulin concentrations of fetuses older than 265 days and were lower than values for the colostrum-fed calves. The immunoelectrophoretic pattern of 59-day old fetuses, the earliest age at which serum samples were obtained, demonstrated albumin, an α1 globulin and a β globulin, possibly transferrin. Additional α and β globulins appeared in the older fetuses and by 175 days of gestation serum electophoretic patterns of the fetuses were similar to patterns normally found with adult bovine serum except for the absence of the gammaglobulins in fetal serum. Immunoglobulin M was detected in 39 of 95 fetal serum samples by radial diffusion and in 13 of 95 samples by immunoelectrophoresis. Immunoglobulin G was detected in ten of 95 fetal serum samples by radial diffusion and in six of 95 samples by immunoelectrophoresis.

     

Epizootiologic Studies of Shipping Fever of Cattle: I. The Microbial Agents Isolated

Nineteen strains of Pasteurella spp., but no viruses cytopathogenic for bovine embryonic kidney cells were isolated from pneumonic lesions present in “normal” veal calves at slaughter.

In studies on two herds of native cattle and six lots of western feeder calves, Pasteurella spp. were isolated from nasal swabs from healthy cattle and those with shipping fever. Viruses of the psittacosis-lymphogranuloma group were isolated from nasal swabs from animals in five groups. Viruses provisionally identified as bovine enteroviruses were isolated from nasal swabs of calves in two lots.

There was serologic evidence of a temporal association of myxovirus para-influenza 3 (PI3) with shipping fever in three lots of calves. From two of these three lots, strains of PI3 were isolated from ten animals, four of which had clinical shipping fever at the time of virus isolation.

     

The Properties and Classification of Bovine Viral Diarrhea Virus

Examination of the C24V (Oregon) and MAC A (Ontario) strains of bovine viral diarrhea viruses have shown them to be ribonucleic acid containing viruses, with essential lipid and having compound helical symmetry with the diameter of the helix being in the neighbourhood of 180 A. Because of these properties it is suggested that the virus should be considered a member of the Myxovirus group. Hog cholera virus is related to bovine viral diarrhea virus by means of a “soluble” antigen, and also possesses essential lipid. It is therefore suggested that hog cholera virus represents still another veterinary myxovirus.     

Bovine Fetal Inoculations with Calf Rotavirus

The serological and histopathological responses of bovine fetuses to in utero inoculation with virulent and attenuated strains of the calf rotavirus (reovirus-like agent of neonatal calf diarrhea) are described. Thirteen bovine fetuses, 63 to 190 days of gestation, were inoculated in utero with attenuated (three fetuses) or field strain virus (nine fetuses) or both (one fetus).

Serum-neutralizing antibody titers ranging from 1:16 to > 1:256 were detected in six of eight fetuses tested, demonstrating the ability of the bovine fetus to respond immunologically to this agent. The youngest fetus in the series was inoculated at 63 days of gestation and developed a titer of 128 in 64 days. This represents the earliest stage of gestation at which a bovine fetus has been inoculated with a bovine virus and found to produce antibody to it. Serum neutralizing titers in six of the eight dams tested increased significantly following the inoculations of their fetuses in utero.

Histological changes associated with viral replication and antigenic stimulation of the lymphoreticular system were observed. Pneumonic lesions consisting of both local and diffuse lymphoreticular proliferation were present in five of the nine fetuses that were alive at slaughter. Gliosis and perivascular cuffing were noted in the brains of two of these fetuses and meningitis was seen in one. No evidence of teratogenic change was found.

     

Nitrofuran Production Efficiency In Chickens

Two nitrofuran feed additives, 0.011% nihydrazone and a combination of 0.0055% nitrofurazone and 0.0008% furazolidone, improved weight gains and feed conversions in chickens with “air sac infection.” Both nitrofurans caused a significant reduction in the total chickens condemned at the dressing plant from this disease, but nihydrazone gave the best results.

Nihydrazone^*, a new nitrofuran feed additive for chickens, was found by Wolfgang et al. (1) to be effective against coccidiosis due to Eimeria tenella and E. necatrix. In chickens nihydrazone was shown by Edgar et al. (2) to result in fewer chickens condemned from “air sac infection” than with any other drug used. Rosenberg et al. (3) found nihydrazone caused significant reduction in condemnations due to this disease. Cosgrove (4) showed that nihydrazone prevented an outbreak of cecal coccidiosis, reduced the incidence of “air sac infection,” improved weight gains, feed conversions and livability. Bierer (5) found nihydrazone active against fowl typhoid.

Harwood et al. (6) reported bifuran^** effective against E. tenella and E. necatrix coccidiosis and it has been used commercially for this purpose. Bierer (5) (7) found Bifuran^*** active in prevention of pullorum disease and fowl typhoid in chicks.

The mode of action of nihydrazone and nitrofurazone against E. tenella coccidiosis was shown by Johnson and Van Ryzin (8).

This production efficiency study was undertaken to evaluate nihydrazone and bifuran in the presence of “air sac infection” and concomitant diseases under field conditions. Camden (9) states that the only satisfactory test of a drug is the performance it gives under field conditions.

Nihydrazone (1) (5) and Bifuran (5) (6) have both antibacterial and antiprotozoal activity and under field conditions, normal densities of bacterial and other parasitic organisms are encountered (9). Thus a coccidiostat having antibacterial and antiprotozoal properties is desirable (7).

     

Studies on Bovine Leukosis

Summary

An enzyme linked immunosorbent assay (ELISA) and the agar gel immunodiffusion test with bovine leukosis virus glycoprotein as antigen (AGIDT‐BLV gp) were further used to test 633 bovine sera for antibodies to BL V. Both tests detected the same number of sera positive (149) or negative (464) for antibodies. Nine sera were negative in the ELISA but found to be weakly positive (2 sera) or bending the control line (7) in the AGIDT‐BLV gp. On the other hand 11 sera were scored negative in the AGIDT‐BLV gp but were weakly positive (9 sera), positive (1), and strongly positive (I) in the ELISA. Both tests are used routinely in this Institute as they complement each other, specially if sera with low antibody titers are under investigation. It is concluded that ELISA can fully replace radioimmunoassays in the serodiagnosis of enzootic bovine leukosis.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC₅₀ broth value of 0.025 µg ml⁻¹ and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC₅₀ of 0.025 µg ml⁻¹ and 0.1 µg per disc, and for M. dispar with 0.05 µg ml⁻¹ and 0.03 µg per disc.Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml⁻¹. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

Glycogen in Leukocytes from Bovine Blood and Milk

Glycogen content was determined quantitatively by the Anthrone reagent method in leukocytes obtained from blood and milk of five cows. Distribution of glycogen in leukocytes was studied by microscopic examination of slides stained by Periodic acid-Schiff (PAS) reaction. Blood glucose concentrations were investigated in these animals by standard procedures. In two of five cows both blood glucose levels and blood leukocyte glycogen levels on the same day were determined for six consecutive days. One hundred and two blood leukocyte samples from five cows had a mean glycogen content of 1.32 ± 0.04 (S.E.) mg/10⁹ WBC, and 6.11 ± 0.17 (S.E.) mg/10⁹ PMNs. Leukocyte preparations from 80 samples of milk comprising 97 to 98% PMNs contained 3.81 ± 0.18 (S.E.) mg glycogen/10⁹ milk leukocytes. In PAS preparations of blood and milk leukocytes glycogen was found almost exclusively in PMNs. Glycogen granules, present frequently in PMNs and occasionally in monocytes and large lymphocytes from blood, were not observed in those from milk. The glycogen level in milk leukocytes was significantly lower (P = <0.01) than that of the blood PMNs in every cow, and the overall mean difference between levels for milk leukocytes and blood PMNs was highly significant (P = <0.001). Mean blood glucose concentration in the five cows was 44.46 ± 0.66 (S.E.) mg%. There was no significant relationship between blood glucose and blood leukocyte glycogen levels in the five corresponding cows; nor between blood glucose and blood PMN glycogen levels on the same day in either of two cows investigated. Leukocyte preparations from milk samples obtained on the second day following intramammary infusion of endotoxin consistently contained markedly less glycogen than the leukocyte preparations from first day post-infusion samples.

These tended to level off and became intermediate between first and second day levels. It is postulated that the poor phagocytic competence of leukocytes from bovine mammary glands compared to their counterparts in blood observed by various workers may be due partially to low energy reserves in these cells.

     

Bovine costimulator. I. Production kinetics,partial purification,and quantification in serum-free Iscove's medium

Bovine peripheral blood leukocytes were examined for blast transformation in response to T-cell lectins in serum-containing RPMI 1640 medium and serum-free Iscove's medium. Phytohemagglutinin-induced blastogenesis was significantly greater in Iscove's medium than in RPMI containing ten percent fetal calf serum. Concanavalin A-induced blast transformation was equivalent in both media. However, the kinetics of lectin response and the quantity of lectin required for optimum blastogenesis was considerably different in the two culture media. Concanavalin A-induced blast transformation of bovine thymocytes in Iscove's medium revealed that at a concentration of 10⁶ cells/ml, inconsequential blastogenesis ensued; but at 10⁷ cells/ml blast transformation was significant and dose-dependent. Therefore, conditioned media from concanavalin A-stimulated bovine peripheral blood leukocytes, prepared in serum-free Iscove's medium, were assayed for costimulator activity using bovine thymocytes at 10⁶ cells/ml in Iscove's medium as indicator cells. Both optimum lectin requirements and cell concentrations for production of costimulator activity were found. Conditioned medium, generated with the total exclusion of serum and with optimal costimulator activity, was fractionated via gel exclusion chromatography. A quantitative assay was described, and results indicated that bovine costimulator had an approximate molecular weight of 20,000 daltons.     

Complement Titres of Naturally and Artificially Raised Piglets: I. In piglets of different birth weights

Titrations of haemolytic complement, C¹, activity have been carried out on serial weekly bleedings from four litters of piglets, a total of 39 animals. One of these litters was raised naturally on the sow, the other three litters were removed by hysterectomy and fed artificially without colostrum supplement. At one day of age the nursing piglets had considerably higher C¹ titres than the colostrum-deprived animals, owing presumably to their ingestion of maternal complement components from the colostrum. The naturally-raised piglets grew more rapidly and continued to have higher C titres during the first three weeks. After that time, although the artificially-raised piglets were still gaining weight more slowly, their C¹ titres began to compare favourably with those of the naturally-raised animals. Considerable variation was noted in the complement activity of sera of littermates. Deaths occurred among animals with high or with low C¹ titres.     

The ERA Strain of Rabies Vaccine

An antigenic extinction trial in cats showed that the ERA rabies vaccine had superior antigenic properties over Flury H.E.P. C.E.O. and killed tissue culture rabies vaccine.

Dogs and cats on a duration of immunity study of ERA rabies vaccine were challenged with fox salivary gland “street” rabies virus. The results of this challenge show a duration of immunity of five years in dogs and four years in cats.

Vaccination of dams in late pregnancy with ERA rabies vaccine resulted in transference of maternal antibody to the newborn, in both cattle and dogs. This maternally derived antibody interfered with the successful active immunization of the young calf. Calves free of antibodies for rabies could be successfully vaccinated as early as 17 days of age and were able to withstand a challenge with virulent “street” rabies virus two years later.