The inactivation of mycoplasmas and bacteria in calf serum by 60Co gamma rays

Reported in this paper is the use of 60Co gamma radiation to inactivate mycoplasmas in calf serum, newborn calf serum, and fetal calf serum. A dose of 3 kGy, independent of dose rate, was found to be sufficient for inactivation in the above sera of several mycoplasmas, including Acholeplasma laidlawii, Mycoplasma (M.) orale, M. arginini, M. hyorhinis, and M. bovis. The critical dose proved to be at 2 kGy. No difference was found to exist between the above species in susceptibility to irradiation in diluted sera (50%) and 10% in Eagle MEM). Sensibility of wild mycoplasma strains was found to be identical with that of laboratory strains. Hence, 60Co gamma irradiation of sera appears to be a safe method by which to make sera mycoplasma-free. Bacillus subtilis in calf serum was inactivated by doses above 18 kGy, with the critical dose being 15 kGy.     

Cell lines from sheep and cattle blood

A simple and reproducible method of establishing cell lines from the blood of sheep and cattle is described. Buffy coat cells were allowed to adhere to plastic culture flasks in media containing 20 per cent autologous plasma overnight. The fluids were then replaced with growth medium supplemented with non-inactivated foetal calf serum, lamb serum or autologous serum. Ovine cell lines were established with any of the serum supplements but bovine cell lines were established more readily if unheated autologous serum was used.     

Detection and molecular characterisation of bovine polyomavirus in bovine sera in New Zealand

AIM: To investigate the prevalence of bovine polyomavirus (BPyV) DNA in commercial batches of bovine serum products, cell lines and cattle in New Zealand and to characterise the viral DNA detected. METHODS: Two nested polymerase chain reaction (PCR) assays were applied to detect BPyV in bovine sera. One was used to screen for the VP1 gene of BPyV DNA in: 140 batches of commercial bovine serum products, including 66 batches of fetal bovine serum (FBS), 34 batches of calf serum, and 40 batches of adult bovine serum (ABS)/plasma; 112 individual adult bovine sera; and 16 cell lines of various species origin. Fifty batches of serum samples were also tested, using the second nested PCR assay that screened for the Large T gene. Restriction fragment length polymorphism (RFLP) was conducted with 36 PCR products amplified from the VP1 gene of BPyV using EcoRI. Five selected VP1 PCR products were subjected to DNA sequencing and phylogenetic analysis. RESULTS: BPyV DNA was detected in 46 (70%) batches of FBS, 11 (32%) batches of calf sera and two (5%) batches of ABS/plasma, an overall prevalence of 42%. None of 112 adult bovine sera was BPyV-positive. RFLP analysis demonstrated a uniform digestion pattern in the majority (31/36) of amplicons tested, while the remaining PCR amplicons did not show enzyme cleavage. Sequence analysis of the PCR products (a 263 base pair (bp) fragment of the VP1 gene) obtained from five batches of FBS showed 96.2-98.9% homology to that of published sequences of BPyV. CONCLUSION: BPyV is a frequent contaminant of commercial bovine serum in New Zealand. The incidence of BPyV in adult bovine serum products is much lower than in FBS and calf serum. Genomic variations exist among different viruses. The clinical significance of the high prevalence of BPyV DNA in bovine serum products is yet to be determined.     

A paravaccinia virus isolated from cattle.

Isolation of viruses from calves with acute respiratory tract disease were attempted on bovine embryonic lung cell cultures. An isolate obtained from one calf with oral lesions and respiratory disease, designated 44-M-E482, was characterized as a paravaccinia virus on the basis of biological and physical properties. The calf from which the paravaccinia virus 44-M-E482 was isolated did not possess serum neutralizing antibody in its convalescent sera; neither did experimentally inoculated calves possess serum neutralizing antibody to the isolate. However, a low titer of serum neutralizing antibody was produced in one calf after several intravenous injections of the virus. Inoculation of calves with 44-M-E482 into the oral mucosa, skin, nasal cavity and pharynx did not cause any noticeable illness or lesions. The relation of 44-M-E482 to the viruses which cause bovine papular stomatitis and pseudocowpox is discussed.     

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.     

Neonatal calf serum immunoregulation of lymphocyte blastogenic responses

The ability of lymphocytes from newborn calves to undergo blastogenic responses to the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA), or Pokeweed Mitogen (PWM), and immunomodulation of these responses by neonatal calf serum was assessed as a function of age. Lymphocytes were obtained from thymus, spleen, and mesenteric lymph nodes of 1-, 2- to 3-, 5- to 7-, and 9- to 10-d-old calves, aliquoted and incubated (+/- mitogens) in sera from 1-, 2-, 3-, or 7- to 10-d-old calves. Lymph-node lymphocytes responded least when cultured in sera from 1-d-old calves, regardless of mitogen or age of cell donor; the response increased as age of serum donor increased (P less than .05). Splenic lymphocytes responded similarly (P less than .005). However, when cultured in sera from older calves, splenic lymphocytes from older calves responded greater to PWM than did those from younger calves. Thymic lymphocytes responded minimally to PWM and PHA. Their response to Con A increased (P less than .005) with age of serum donor calf, but the effect was greatest on lymphocytes from 5- to 7-d-old calves. Mixing experiments with varying ratios of 1-d-old calf serum: 10-d-old calf serum suggested that serum from 1-d-old calves contained suppressive activity. Serum cortisol level (measured by radioimmunoassay) was 30 +/- 4.6 ng/ml in calves at 6 h of age and declined to 5.5 +/- 1.1 ng/ml by 10 d. Charcoal treatment to remove steroids did not enhance blastogenesis. Addition of cortisol (50 ng/ml) to charcoal-treated sera resulted in inhibition of response to PHA, but no change in response to Con A or PWM. Further investigation is indicated to characterize this immunosuppressive activity and to establish its relationship to disease susceptibility.     

Classical and alternate complement pathway activities in paired dairy cow-newborn calf sera

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.     

Human serum for in vitro cultivation of Babesia bovis.

Various combinations of human serum (from blood of groups A and Rhesus positive) with bovine serum, i.e. 20% + 20% (Medium I), 30% + 10% (II), 40% + 0% (III) and 0% + 40% (IV) and Medium-199 (60%) were used in the propagation of Babesia bovis. Babesia bovis stabilate revived by inoculation in a bovine calf was used at a level of 6% parasitized erythrocytes (PPE). The medium was replenished every 24 h. The medium changed from bright red to dark-coffee color every 24 h. The increase in PPE was maximal between 24 and 48 h. It was also observed that the increase in PPE was significantly higher in a 1:2 dilution compared with a 1:1 dilution. The increase in PPE was highest in Medium I with a 50% replacement of bovine with human serum. However, the parasites could not be subcultured and maintained continuously. Fifty percent replacement was thus optimal for the human-bovine serum combination in a microaerophilous stationary phase (MASP) system. The results indicate that B. bovis can also multiply in a human/bovine serum MASP culture system at least for a period of 48 h, and this is consistent with the zoonotic nature of Babesia species.     

Isolation and culture of parenchymal and nonparenchymal cells from neonatal swine liver

Procedures for the isolation and monolayer culture of hepatocytes and nonparenchymal (Kupffer and endothelial) cells from livers of neonatal pigs (1 to 15 d of age) are described. Cell suspensions were obtained by a modification of the two-step collagenase perfusion technique. Hepatocytes were collected by low-speed centrifugation and nonparenchymal cell populations were purified by centrifugal elutriation. Hepatocytes were readily maintained in arginine-free medium fortified with either fetal calf serum or bovine serum albumin and oleate for periods as long as 6 d. The ability of cultured hepatocytes to incorporate 3H-leucine and 3H-thymidine into protein and DNA, respectively, demonstrated that cells were metabolically active for at least 3 d in culture. The 3H-leucine incorporation into total cell protein was constant regardless of animal age at the time of cell isolation, while incorporation of 3H-thymidine was influenced by animal age. Incorporation of both precursors was dependent upon duration of culture period in vitro and the type of medium (serum-free vs serum-containing) in which the cells were maintained. Morphological observation and analysis of the DNA and protein levels of hepatocyte monolayers suggest that cells did not replicate during the 3-d incubation period. The ability to isolate and culture metabolically active, nonreplicating hepatocytes from neonatal pigs in a serum-free medium affords opportunities for investigation of the influence of specific hormones and specific growth factors on the uptake and metabolism of nutrients by the liver. Similarly, the neonatal pig will serve as a useful model for the characterization of hepatic nonparenchymal cell metabolism during the neonatal period.     

Treponema hyodysenteriae growth under various culture conditions

The influence of various culture conditions on the growth of Treponema hyodysenteriae was determined. Six different anaerobically prepared culture broths were tested for the ability to support growth of strains B78, B204 and B169. Each medium contained glucose (0.2%) and 10% (v/v, final concn.) heat-inactivated fetal calf serum. Brain-heart infusion (BHIS), heart infusion (HS) and veal infusion (VS) broths gave the highest cell yields of the spirochete with the shortest incubation times. Vigorous mixing of the cultures and the introduction of O2 (1%, final concn.) into the culture atmosphere were necessary for optimum growth. Although BHIS broth was found to be the best for routine cultivation of the 3 strains, HS broth was more suitable for investigating the physiology of growing cells, inasmuch as cell growth in this medium was limited unless a growth substrate was added. Glucose, fructose, sucrose, galactose, trehalose, N-acetyl-glucosamine, glucosamine, mannose, maltose and pyruvate were growth substrates for all 3 strains. During the growth of B204 cells in HS broth under N2:O2 (99:1), glucose and O2 were consumed and CO2, H2, acetate and butyrate were produced. In HS agar-containing medium, cells of strains B78 and B204 formed spreading colonies typical in appearance to those of other spirochetes.     

In vitro culture of bovine embryos in murine ES cell conditioned media negatively affects expression of pluripotency-related markers OCT4, SOX2 and SSEA1

Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels.     

In vitro comparison of bovine mastitis and fecal Escherichia coli isolates

In vitro methods were used to test the hypothesis that Escherichia coli from bovine mastitis are essentially no different from isolates from bovine feces. Fifty E. coli isolates from bovine mastitic milk, 50 from feces of mastitic cows and 50 from feces of healthy cows were compared with respect to biochemical properties and certain potential virulence factors. There were no significant differences among the groups in tests for biotype; production of colicins, colicin V, or Vero cell cytotocity; and growth in 90% gnotobiotic calf serum or 90% normal milk whey. Resistance to killing in 90% gnotobiotic calf serum varied from 66 to 84%. Most isolates grew in normal whey: the percentage in a group varied from 86 to 96. Mastitic milk isolates were significantly different from the fecal isolates in adonitol fermentation (P0.006), production of aerobactin (P0.026), and ability to grow in 90% mastitic whey (P0.00004). However, only 40% of mastitis E. coli fermented adonitol and only 20% produced aerobactin. Ninety-six percent of mastitic milk E. coli grew in mastitic whey, whereas 64% and 60%, respectively, of mastitic fecal and normal fecal isolates grew in this medium. It is concluded that none of the properties that were investigated constitute potential virulence factors or markers for ability to induce mastitis; the data are consistent with the hypothesis that mastitic E. coli are simply opportunistic pathogens.     

Heterogeneity of Hanganutziu-Deicher antigen glycoproteins in different species animal sera.

A heterophilic Hanganutziu-Deicher (HD) antigen is present in many animal sera except human and chicken sera. To visualize the antigenic molecules, nine species animal and human sera were analyzed by SDS-PAGE, followed by Western blotting and immunostaining with avain anti-N-glycolylneuraminyl-lactosyl ceramide antibody which recognizes the terminal N-glycolylneuraminic acid moiety of glycoconjugates as an epitope of the HD antigen. Several HD antigen-active glycoprotein bands were detected in the sera of fetal calf, calf, horse, goat, monkey, rabbit, guinea pig, rat and mouse, except for human serum. The HD antigenic proteins showed heterogeneities in their molecular weights and were not identical with any major band visualized with silver-staining, indicating that they are minor components of serum proteins in each animal. Neuraminidase treatment destroyed the antigenicity of all proteins, confirming that N-glycolylneuraminic acid (NeuGc) at the non-reducing terminal of carbohydrate chains is the antigenic epitope in serum glycoprotein molecules as already confirmed in glycosphingolipid (GSL) antigens. The finding of HD-antigenic glycoproteins in animal sera suggests that they also stimulate HD antibody production in patients who received animal antiserum for therapeutic aim.     

Prevalence of neutralizing antibody to the calf rotavirus in New York cattle.

In March 1973, a modified live virus vaccine was released for sale in the United States for the protection of neonatal calves from infection with calf rotavirus. At that time no published evidence existed that this agent was present in New York or the New England states. Serum neutralizing antibodies for the calf rotavirus (reovirus-like agent of neonatal calf diarrhea) were detected in serum from 108 of 110 dairy cattle in New York State representing 78 different herds. To exclude the possibility that the demonstrated serologic response may have been stimulated by vaccine virus, 36 samples were included that had been collected prior to the date of vaccine release. Neutralizing antibodies were demonstrated in 108 of the 110 sera ranging from titers of 4 to greater than 1024, thereby offering indirect evidence of the ubiquitous nature of calf rotavirus in New York.     

Rhodococcus equi: equine neutrophil chemiluminescent and bactericidal responses to opsonizing antibody

The opsonic capacity of serum containing R. equi-specific antibody was compared with antibody-deficient sera using luminol-dependent chemilumenscence (LDCL) and bactericidal assays. These assays incorporated peripheral blood polymorphonuclear neutrophilic leukocytes (PMNL) exposed to R. equi opsonized with neonatal equine pre-colostral serum (control) or serum from foals with R. equi infections (principal). All sera were complement inactivated at 56 degrees C for 30 min. Bacteria were obtained from the lung of a foal with R. equi pneumonia. Neutrophils were obtained from one adult horse for LDCL and another for bactericidal assays. Chemiluminescence of PMNL exposed to R. equi opsonized with control or principal sera was measured in a liquid scintillation counter. Mean peak LDCL within 1 h was significantly (P less than 0.01) higher with principal sera (2.4 X 10(5) cpm) than with control sera (0.018 X 10(5) cpm). A radioisotope bactericidal assay was used to determine the effect of control or principal sera on PMNL capacity to kill R. equi. Mean peak percent kill of R. equi by PMNL within 2 h was significantly (P less than 0.01) higher with principal sera (95.2%) than with control sera (54.6%). Enzyme-linked immunosorbent assay (ELISA) values for R. equi-specific antibody were determined on all sera. Mean ELISA values were significantly (P less than 0.01) higher for principal sera (71.8) than for controls (0.0). This investigation documents the presence and biological effectiveness of opsonic activity in complement-inactivated sera from foals with R. equi infections and R. equi-specific antibody.     

Immunization and culture of rainbow trout organ sections in vitro

Splenic and anterior kidney sections or whole organs were excised from large (1 kg) or small (200 g) rainbow trout (Salmo gairdneri) and placed in sterile 60 mm plastic plates containing 10 ml of Eagle's minimal essential medium (EMEM) supplemented with normal or fetal calf serum for in vitro culture. The organ samples were immunized in vitro by direct injection or by mixing in the medium Yersinia ruckeri O-antigen or dinitrophenyl-Ficoll. The medium was changed once during the 10-day incubation at 15 C. The passive hemolytic plaque assay demonstrated antibody production from the plaque-forming cells (PFC); passive hemagglutination was used to measure antibody titers in the media. High numbers of PFC occurred in cultures of either kidney or spleen, demonstrating that these organs can function independently for antibody production. Splenic sections from large fish produced more PFC than comparable whole organs from small fish. EMEM supplemented with 2% normal calf serum was a satisfactory culture medium. 2-hydroxyethyl-mercaptan an ingredient used in mammalian cell culture, inhibited antibody production in trout cells. These techniques are being used in the culture of organs and cells to elucidate pathways and sequences of antigen uptake and delivery of the immunopoietic tissues in trout.     

Age-related variations in serum concentrations of the third component of complement in swine

Single radial immunodiffusion was used to determine the concentration of the third component of complement (C3) in serum from swine in the following age groups; 36 to 60 hours (neonates), 6 to 7 weeks (weanlings), 4 to 5 months (adolescents) and greater than 1 year (adults). Mean serum C3 concentrations, expressed as the percentage of pooled reference sera from 15 adult swine for the 4 groups were 23, 123, 119, and 98%. With the exception of mean values for weanlings and adolescents, all comparisons of group means were significantly different. Regression models were developed to estimate serum C3 concentration in neonates as a function of litter size, birth weight, and total serum IgG concentration. Increases in birth weight and litter size were accompanied by increased serum C3 concentration, possibly reflecting ontogenic variation of the complement system at the end of gestation. Passive transfer of colostral immunoglobulins, as measured by total serum IgG concentration, was inversely related to serum C3 concentration in neonatal swine, suggesting that colostral absorption of C3 has minimal impact on complement activity in neonatal swine.     

Micro-epidemiology and micro-plaque titration of infectious bovine rhinotracheitis virus on primary calf kidney cells by means of immunofluorescence]

Immunofluorescence was used to study the virus of infectious bovine rhinotracheitis and the course of infection in cell cultures of calf kidney. An indirect relationship was found to exist between the magnitude of inoculation and the onset of specific fluorescence. Fluorescent plaques are formed as a result of inoculate dilution. The plaques will grow along with incubation time. Release of virus into the culturing medium will first lead to the formation of secondary plaques, then followed by generalised infection of the cell culture. The time at which the infection will begin to be disseminated was found to depend on both multiplicity of the infection and quality of the cell culture. Therefore, no limitation is possible of the time during which only primary infectious foci are recordable. Antibody present in the culturing medium prevent propagation of the infection, but this does not inhibit the course of primary infection nor intercellular virus transmission. The conditions are defined for the microplaque fluorescence method and its use on quantitative virus assay. While reproducible results are offered by that method, its sensitivity is below that of the tubule method, when it comes to identifying the infection due to the cytopathic effect. The microplaque fluorescence method was used to study the conditions for absorption of virus of infectious bovine rhinotracheitis by calf-kidney cell cultures. Absorption was tested under temperatures of 20 degrees C, 37 degrees C, and 40 degrees C and found to be accelerated by higher temperatures. Yet, the total quantity of virus absorbed in 120 minutes was found to be almost the same in all three temperatures. The degree of virus absorption was found to depend on the kind of medium, with the rate of absorption having been strongly increased by adding to the medium serum of different animal species. About 70 per cent of the virus present in the inoculate were absorbed by the calf-kidney cell cultures under defined experimental conditions.     

低血清培养PK-15细胞及其增殖猪圆环病毒2型的研究

依次采用含60、30、20、10mL/L血清浓度的低血清培养基驯化PK-15细胞,并给驯化好的细胞上接种猪圆环病毒2型(PCV-2),以确定PCV-2在该培养体系下的生长情况。结果用含60、30、20mL/L血清浓度的低血清培养基各进行3代次驯化,PK-15细胞能完全适应且生长状态良好;当血清浓度降至10mL/L时,传代1次细胞无法保持良好状态,细胞出现贴壁较差、生长停滞等现象。各血清浓度培养体系中细胞生长曲线测定结果显示,在细胞培养的0、24、48、72、96h各组细胞密度与常规培养的对照组差别不明显。因此用该低血清培养体系培养PK-15细胞时,血清最低添加量为20mL/L。用该体系培养的PK-15细胞接种PCV-2后,通过荧光抗体染色测定病毒滴度。结果显示,低血清培养的PCV-2病毒滴度为10~(6.5)TCID_(50)/mL,与常规条件培养的PCV-2对照(病毒滴度为10~(6.375 )TCID_(50)/mL)差别不明显,表明该体系可用于PCV-2的增殖。表明研究建立了PK-15细胞低血清培养PCV-2体系,为PCV-2的相关研究奠定了基础。     

Kinetics of neutralization of two strains of Myxovirus Parainfluenza 3 in the presence of complement and anti-gammaglobulin-serum.

Using the kinetics of neutralization, it was established that two strains of Myxovirus Parainfluenza 3 of cattle and sheep origin which are immunologicaly identical have a different sensitivity to early neutralizing antibodies of reconvalescent calf sera. Complement-dependent neutralizing antibodies, supplied by adding 5% guinea pig serum to the calf serum, appear earlier than the usual neutralizing antibodies. Goat anti-cattle gammaglobulin serum decreases the neutralizing activity of early reconvalescent serum and release some of the neutralizing virus from the virus-antibody complex. It is believed that the lability of the virus-antibody complex depends not only on the activity of the antibody, but also on some properties of the strains. The addition of complement stabilizes this complex. The previous addition of adenovirus antigen to bivalent cattle serum against Myxovirus Parainfluenza 3 and cattle adenovirus, decreases the complement-dependent neutralizing antibody of the serum against Myxovirus Parainfluenza 3. It is suggested that this is the possible mechanism for the mutual activation of mixed virus infections.