Purification and properties of porcine allantoic fluid retinol-binding protein

Retinol-binding protein (RBP) was purified from Day 60 porcine allantoic fluid by a combination of diethylaminoethyl cellulose, G-100 Sephadex, G-50 Sephadex, Phenyl-Sepharose, and Reactive Green 19-dye-agarose chromatography. The yield was 1 to 2 mg of RBP, which generated a single M_r 20,000 band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and up to four isoelectric variants (isoelectric variants 1 to 4) after two-dimensional PAGE (2D-PAGE). The protein cross-reacted with antiserum raised against human RBP. When incubated with [³H]retinol and subjected to G-100 Sephadex chromatography, [³H]retinol coeluted with the protein. These results indicate that the purified protein is an RBP. When purified RBP was subjected to native 2D-PAGE, six forms of RBP were observed. Three native forms were fluorescent, and three were not fluorescent, suggesting that these forms were RBP with and without retinol, respectively. Denaturing 2D-PAGE analysis of each native form of RBP suggested that two of the nonfluorescent and two of the fluorescent native forms of RBP corresponded to isoelectric variant 1 on denaturing 2D-PAGE, whereas the other fluorescent and nonfluorescent forms corresponded to isoelectric variant 2. The incubation of RBP with 50 μM retinol enhanced the amount of both isoelectric variants present as fluorescent RBP, but uptake by isoelectric variant 1 was greater than that by isoelectric variant 2. These data indicate that RBP can be purified from porcine allantoic fluid and suggest that the isoelectric variants may differ in their affinity for retinol.     

Bovine interleukin 2: production and characterization

The production of bovine IL 2 was studied and IL 2 was partially characterized. PMA at 5 ng/ml + Concanavalin A at 5 micrograms/ml treatment of peripheral blood mononuclear cells gave a greater yield of IL 2 activity in the supernatants than Con A, PMA or sodium periodate treatments alone. Macrophage depletion increased yields as did the addition of indomethacin, a prostaglandin E2 inhibitor. Bovine IL 2 was sensitive to trypsin, relatively stable at pH 2-9, 2-ME resistant and sensitive to increasing molar concentrations of urea. The activity of bovine IL 2 was reduced by over 45% at 70 degrees C for 30 min and 95% at 90 degrees C for 30 min. Bovine IL 2 was more stable at 4 degrees C than at room temperature and the stability at room temperature could be improved by inclusion of 1% BSA. Bovine IL 2 eluted from DEAE-Sephadex as a broad peak with 0.1-0.2 M NaCl. Peak activity corresponded to a molecular weight of approximately 16,000 daltons on Sephadex G-100.     

Production of monoclonal antibodies and enzyme immunoassay to bovine retinol-binding protein and determination of retinol-binding protein serum levels and retinol concentrations in serum and liver in dairy cows before and after parturition

To study vitamin A transport in dairy cows and heifers around parturition, an enzyme immunoassay for bovine retinol binding protein (RBP) was developed and serum levels determined. Serum and liver concentrations of retinol were assayed by HPLC. Four weeks before expected calving the cows and heifers were divided into two groups each, and half of the animals received a protein supplementation during the dry period.The mean serum RBP concentration 4 weeks before calving was 42 mg l-1 for the cows and 44 mg l-1 for the heifers. The serum retinol concentrations were 0.53 mg l-1 for the cows and 0.42 mg l-1 for the heifers, and the liver retinol concentrations 0.30 mg l-1 and 0.13 mg g-1, respectively. In the groups without protein supplementation there was a significant decrease in serum RBP at sampling 1 week before parturition compared to initial values. The measurement of serum RBP may prove useful in assessment of amino acid availability in dairy cows.     

禽源多杀性巴氏杆菌(A:1)荚膜保护性抗原提纯及单克隆抗体细胞系建立

用Sep(?)adex G-200分之筛层析、DEAE-cellulose离子交换层析相结合,将禽源多杀性巴氏杆菌(A:1)荚膜抗原粗提物(CE)分为P1.P2两种成分,并经免疫保护试验证明,荚膜中含有保护性蛋白成份P1。P2分子量为129 100,且至少含有分子量分别为46700、42600和39800的3种蛋白亚基。用CE免疫BALB/c小鼠,建立了抗P1的单克隆抗体细胞系1B1和2B7,它们只与多杀性巴氏杆菌反应,而不与其他细菌交叉。     

Isolation of bovine complement factor H

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.     

Absence of host proteins from the surface of Trypanosoma vivax of cattle

Trypanosoma vivax (EATRO 1721) organisms were isolated by DEAE-cellulose chromatography from blood of an experimentally infected calf. Attempts to agglutinate the purified trypanosomes with a rabbit antiserum against whole bovine serum or antisera monospecific for bovine IgG1, IgG2, IgM, complement component C3 or albumin were unsuccessful. The trypanosomes, however, were agglutinated by immune sera of four different calves chronically infected with T. vivax (EATRO 1721). It was concluded that T. vivax organisms purified by DEAE-cellulose chromatography from blood of cattle do not have bovine serum proteins on their surface.     

Isolation and purification of a low molecular weight protein from bovine urine

A low molecular weight protein was separated from urine samples obtained from a heifer with spontaneous renal disease and from cows with CaNa2EDTA-induced renal dysfunction. The molecular weight and electrophoretic mobility of the separated protein were examined. The low molecular weight protein collected by gel filtration chromatography was further separated into two fractions by ion exchange chromatography using DEAE-cellulose. One of the two fractions, the lowest molecular weight protein showed a single band in SDS-PAGE, and its molecular weight was approximately 12,000. An antiserum against this protein formed a single precipitin line with the urine from cows with experimentally induced renal dysfunction and a heifer with spontaneous renal disease by the double immunodiffusion technique. However, the antiserum did not form any precipitin line with the concentrated urine of healthy cow and human beta 2-microglobulin. In cellulose acetate membrane electrophoresis, this protein migrated in the same position as that of serum gamma-globulin from healthy cow.     

Milk production of crossbred daughters of high- and low-milk EPD Angus and Hereford bulls.

Milk yield from 273 Angus- and Hereford-sired cows and preweaning performance of their calves were used to determine how accurately milk EPD of Angus and Hereford sires predicted milk production of crossbred daughters and subsequent calf performance. Mean milk EPD (kg) for high Angus (HA), low Angus (LA), high Hereford (HH), and low Hereford (LH) bulls (n = 41) selected as sires were +8.7, -6.2, +7.6, and -4.8, respectively. Cows calved in spring or fall from 1992 to 1997 and yielded a total of 660 records. Twenty-four-hour milk production of the cows was estimated by two weigh-suckle-weigh measurements at monthly intervals. The statistical model included breed, milk EPD level, sire of cow within breed and milk EPD level, year, season, cow age, calf sire, sex, and all two- and three-way interactions. Means were obtained for monthly milk production, total milk production, time and yield of peak production, monthly calf weights, monthly cow weights and body condition scores (1 through 9), and calf birth and weaning data. The least squares means for 24-h milk production (kg) of HA, LA, HH, and LH with P-values for high vs low, across breeds, were, respectively, as follows: mo 1: 6.9, 5.9, 7.1, and 5.7 (P < 0.01); mo 2: 7.2, 6.1, 6.9, and 5.7 (P < 0.01); mo 3: 6.1, 5.1, 5.1, and 4.3 (P = 0.01); mo 4: 6.1, 4.9, 4.9, and 4.8 (P = 0.01); mo 5: 4.8, 4.0, 4.2, and 3.8 (P = 0.01); mo 6: 4.7, 3.4, 3.2, and 3.0 (P < 0.01); and mo 7: 3.7, 2.5, 3.0, and 3.0 (P = 0.05). Least squares means for total milk (kg) were 911.4, 729.6, 758.0, and 664.2 (P < 0.01); for yield at peak (kg/d) were 7.0, 5.7, 6.1, and 5.2 (P < 0.01); for birth weight (kg) were 37.1, 37.9, 38.3, and 38.8 (P = 0.31); for 205-d weight (kg) were 237.3, 218.2, 222.2, and 214.1 (P < 0.01); for final cow weight (kg) were 482.4, 505.4, 509.5, and 511.7 (P = 0.11); and for final cow BCS were 4.9, 5.3, 5.1, and 5.2 (P < 0.01). The correlations of total production with the monthly productions were 0.52, 0.56, 0.52, 0.54, 0.35, 0.37, and 0.31 (P < 0.01) and were 0.12 with birth weight, 0.45 with 205-d weight, -0.12 with final cow weight, and -0.26 with final cow body condition score (all P < 0.01). Daughters of high-milk EPD sires produced more milk and weaned heavier calves than those of low-milk EPD sires at the expense of body condition. These results suggest that sire milk EPD are sufficiently associated with milk yield and calf performance to be useful tools in genetic improvement of preweaning performance.     

The Failure of Purified T-2 Mycotoxin to Produce Hemorrhaging in Dairy Cattle

A Holstein cow was intubated with 182 mg of 97% pure T-2 toxin (0.44 mg/kg of body weight) for 15 days. A dairy ration containing 50 mg/kg (50 ppm) of T-2 toxin was refused. A calf, born four days after onset of maternal treatment, was intubated with 26.2 mg of purified T-2 toxin (0.6 mg/kg of body weight) for seven consecutive days and then on alternate days for a total of 16 days. The calf was severely affected clinically by the T-2 toxin. The T-2 toxin failed to cause bovine hemorrhagic syndrome in either animal. Unspecific gastrointestinal lesions were noted in the cow but none were detected in the calf. In the calf, severe depression, hindquarter ataxia, knuckling of the rear feet, listlessness and anorexia were caused by the T-2 toxin.     

Insulin-like growth factor binding proteins and mitogenic activity of partially fractionated sheep amniotic fluid

Amniotic fluid collected from ewes on various days of gestation was examined for the presence of insulin-like growth factor (IGF) binding proteins. IGF-binding proteins with a molecular mass of 40-45 kDa appeared at day 41 of gestation. The level of these major IGF-binding proteins increased during pregnancy and reached a maximum at day 106. Smaller IGF-binding molecules with an approximate molecular mass of 35 kDa and 25 kDa appeared at day 90, also reaching a concentration peak at day 106. The mitogenic activity of sheep amniotic fluid after chromatography on Sephadex G-50 was separated into two peaks. The peak having lower molecular mass corresponded to an elution profile of 125I-IGF-I. The first peak, having higher molecular mass, was eluted immediately after the void volume of column. Electrophoresis and ligand blotting showed that proteins in the first peak had similar properties as IGF-binding proteins.     

A case of an embryo transfer calf infected with bovine leukemia virus from the recipient cow

A case was discovered where the embryo transfer (ET) calf had been infected with bovine leukemia virus (BLV) from the recipient cow. The embryo was transferred from the BLV-uninfected donor cow to the recipient cow. However, the BLV test had not been performed to the recipient cow before ET was performed. The ET calf was raised in a calf hatch from birth to 1-month old and was given the recipient cow's colostrum and milk artificially. The ET calf was raised with the two other calves from a 1-month old to a 6-month old. The BLV test was performed to the ET calf by agar gel precipitation (AGP) and passive haemagglutination (PHA) assay when the ET calf was 6 months old. Because the ET calf was positive, the BLV test was performed to the recipient cow, the two other calves raised with the ET calf and the two dams of the two other calves. Because the recipient cow only was positive at the time of the first test, we judged that the ET calf had been infected with BLV from the recipient cow. The importance of the BLV test being carried out on the recipient cow for the prevention of enzootic bovine leukemia in a case of ET was recognised.     

牛白细胞髓过氧化物酶的分离纯化

应用分子筛层析(Sephadex G-200凝胶)和亲和层析(ConA-Sephrose 4B)从奶牛全血中分离纯化髓过氧化物酶(myeloperoxidase,MPO)。分离出的MPO活性为0.068 U/mL,分子质量为64.7、47.9、13.4 ku,纯度为94.2%,蛋白质浓度为147.3 μg/mL。本研究建立了MPO分离纯化方法,对于深入研究MPO具有重要意义。     

A protective antigen for Turkeys purified from a type 1 strain of Pasteurella multocida

A protective antigen was purified from a saline extract of a Type 1 strain of Pasteurella multocida by chromatographic methods, and its chemical and immunological ccharacteristics were studied. Three protein peaks were obtained from crude extract by gel filtration with Sephadex G-200. A bacteria-specific antigen was detected only in the first peak fraction, which, after passing through an immunoadsorbent column to remove any components originating from the growth medium, was adsorbed onto DEAE-cellulose followed by elution with a gradient of NaCl. From the first peak fraction of the gel filtration, 4 protein peaks were obtained, the second and third peaks being the major ones. Carbohydrate/protein ratios of the peak fractions varied from 0.06 to 1.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that 2 proteins of molecular weights 44 000 and 25 000 were present in all the fractions. The 4 DEAE-cellulose fractions (DP-1 to DP-4) contained a single antigenically identical material, and induced protective immunity in turkeys against challenge exposure. The second peak fraction from DEAE-cellulose (DP-2) protected turkeys when subcutaneously injected as 2 doses of 10 μg protein with a 14-day interval between doses. The DP-2 fraction induced antibodies in rabbits which formed a single precipitin line against the crude extract. The purified antigen (DP-2) from a Type 1 strain was antigenically distinct from a similar antigen purified from a Type 3 strain; there was no significant cross protection in turkeys between the 2 antigens. These results indicate that protective antigens purified from soluble extracts of a Type 1 or Type 3 strain possess similar physicochemical properties, but that they are immunologically distinct from each other.     

Retinol and estradiol regulation of retinol binding protein and prostaglandin production by porcine uterine epithelial cells in vitro

Secretion into the uterine lumen follows a precise pattern during early pregnancy. Near the end of the second week of pregnancy and coincident with elongation of conceptuses, retinol, retinol binding protein (RBP), estradiol (E2), and prostaglandins E (PGE) and F (PGF) increase in the uterine lumen, and RBP mRNA increases in the endometrium. In the present studies the potential for E2 (0.1 microM) and retinol (10 microM) to regulate RBP and PG production by cultured luminal (LEC) and glandular (GEC) epithelial cells collected from postpubertal females and LEC from prepubertal gilts was examined. Endometrial tissue was collected surgically from cyclic and pregnant females (n = 8) on d 10 and 13 postestrus (first day of estrus = d 0) and from 120- and 150-d-old prepubertal gilts that were treated with progesterone (P4) (2.2 mg x kg(-1) x d(-1), n = 6) or corn oil (n = 6) for 14 d prior to tissue collection. The LEC from postpubertal females responded to retinol with increased (P < 0.05) RBP, PGE, and PGF in culture medium and increased (P < 0.07) RBP mRNA but E2 decreased (P < 0.05) RBP and RBP mRNA and had no effect on prostaglandins. No E2 or retinol effects on secretions of GEC occurred in vitro, but a day x pregnancy status interaction (P < 0.06) affected PGE output by the GEC. Secretion of PGE was greater when GEC were collected on d 10 of pregnancy than from d-10 cyclic or d-13 pregnant or cyclic females. Both E2 and retinol stimulated (P < 0.05) secretion of RBP by LEC isolated from prepubertal gilts, but their effects were not additive. In vivo treatment of prepubertal gilts with P4 increased (P < 0.05) RBP and decreased (P < 0.05) PG production by LEC in vitro. Therefore responses to E2 and retinol differ between pre- and post-pubertal females, and retinol may function in the regulation of endometrial RBP and PG secretion.     

犊牛维生素A缺乏造成骨骼发育异常的探讨研究

一牛场出现犊牛腿部骨骼发育异常、多数母牛胎衣不下的情况，开展现场临床检查，实验室检测犊牛和母牛的血清Ca、P、碱性磷酸酶，犊牛骨碱性磷酸酶，母牛和犊牛血清维生素A含量。结果表明，部分犊牛Ca、P、碱性磷酸酶偏低，其他犊牛以上指标正常，所有检测的犊牛、母牛血清维生素A含量均远低于参考值，处于严重缺乏状态。通过临床检查、实验室检验及药物试验，证明该牛场此次犊牛腿部骨骼发育异常及母牛胎衣不下是以维生素A缺乏为主因造成的。     

Effects of chronic renal disease on the transport of vitamin A in plasma and urine of dogs

OBJECTIVE: To determine plasma and urine concentrations of retinol, retinyl esters, retinol-binding protein (RBP), and Tamm-Horsfall protein (THP) in dogs with chronic renal disease (CRD). ANIMALS: 17 dogs with naturally developing CRD and 21 healthy control dogs. PROCEDURE: A diagnosis of CRD was established on the basis of clinical signs, plasma concentrations of creatinine and urea, and results of urinalysis. Concentrations of retinol and retinyl esters were measured by use of reverse-phase high-performance liquid chromatography. Concentrations of RBP and THP were measured by use of sensitive ELISA systems. RESULTS: Dogs with CRD had higher plasma concentrations of retinol, which were not paralleled by differences in plasma concentrations of RBP. Calculated ratio of urinary total vitamin A (sum of concentrations of retinol and retinyl esters to creatinine concentration) and ratio of the concentration of urinary retinyl esters to creatinine concentration did not differ between groups. However, we detected a significantly higher retinol-to-creatinine ratio in the urine of dogs with CRD, which was paralleled by a higher urinary RBP-to-creatinine ratio. Thus, in dogs with CRD, the estimated fractional clearance of total vitamin A, retinol, and RBP was increased. Furthermore, dogs with CRD had a reduced urinary THP-to-creatinine ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study documented that CRD affects the concentrations of retinol in plasma and urine of dogs. Analysis of the data indicates that measurement of urinary RBP and urinary THP concentrations provides valuable information that can be helpful in follow-up monitoring of dogs with CRD.     

Effect of increasing proportion of supplemental N from urea in prepartum supplements on range beef cow performance and on forage intake and digestibility by steers fed low-quality forage

Four experiments were conducted to evaluate the influence of changing the proportion of supplemental degradable intake protein (DIP) from urea on forage intake, digestion, and performance by beef cattle consuming either low-quality, tallgrass prairie forage (Exp. 1, 2, and 4) or forage sorghum hay (Exp. 3). Experiments 1, 2, and 3 were intended to have four levels of supplemental DIP from urea: 0, 20, 40, and 60%. However, refusal to consume the 60% supplement by cows grazing tallgrass prairie resulted in elimination of this treatment from Exp. 1 and 2. Levels of supplemental DIP from urea in Exp. 4 were 0, 15, 30, and 45%. Supplements contained approximately 30% CP, provided sufficient DIP to maximize digestible OM intake (DOMI) of low-quality forage diets, and were fed to cows during the prepartum period. In Exp. 1, 12 Angus x Hereford steers (average initial BW = 379) were assigned to the 0, 20, and 40% treatments. Forage OM intake, DOMI, OM, and NDF digestion were not affected by urea level. In Exp. 2, 90 pregnant, Angus x Hereford cows (average initial BW = 504 kg and body condition [BC] = 5.0) were assigned to the 0, 20, and 40% treatments. Treatment had little effect on cow BW and BC changes and calf birth weight, ADG, or weaning weight. However, pregnancy rate tended to be lowest (P = 0.13) for the greatest level of urea. In Exp. 3, 120 pregnant, crossbred beef cows (average initial BW = 498 kg and BC = 4.6) were assigned to the 0, 20, 40, and 60% treatments. Prepartum BC change tended (P = 0.08) to be quadratic (least increase for 60% treatment), although BW change was not statistically significant. Treatment effect on calf birth weight was inconsistent (cubic; P = 0.03), but calf ADG and weaning weight were not affected by treatment. Pregnancy rate was not affected by prepartum treatment. In Exp. 4, 132 pregnant, Angus x Hereford cows (average initial BW = 533 and BC = 5.3) were assigned to the 0, 15, 30, and 45% treatments. Prepartum BC loss was greatest (quadratic; P = 0.04) for the high-urea (45%) treatment, although BW loss during this period declined linearly (P < 0.01). Prepartum treatment did not affect pregnancy rate, calf birth weight, or ADG. In conclusion, when sufficient DIP was offered to prepartum cows to maximize low-quality forage DOMI, urea could replace between 20 and 40% of the DIP in a high-protein (30%) supplement without significantly altering supplement palatability or cow and calf performance.     

Antigenic and immunogenic studies on cell culture-derived Babesia canis

Babesia canis antigens derived from cell culture reacted specifically with immune serum from dogs convalescing from babesiosis. The antigens were heterogenous as compared to antigens elaborated in vivo. The major antigenic moiety from cell culture eluted in the first peak of Sephadex G-200 is indicative of a molecular weight around 900 000. In contrast, in vivo-derived antigen coeluted with albumin and hemoglobin suggesting a molecular weight of 67 000. The major antigenic mass is proteinacious and contains disulfide bonds as indicated by thermolability and sensitivity to 2-mercaptoethanol. Both particulate and soluble B. canis antigens were immunogenic, particularly when emulsified in Saponin as an adjuvant. Such antigens conferred a considerable degree of protection in susceptible dogs and it suggested that immunoprophylaxis to B. canis may be feasible.     

The effects of degradable and undegradable intake protein on the performance of lactating first-calf heifers

Two 60-d experiments were conducted to evaluate the effects of supplementing degradable (DIP) and(or) undegradable (UIP) intake protein on the performance of lactating first-calf heifers. Diets were formulated to meet the requirements for either DIP, metabolizable protein (MP), or both when diets contained low-quality grass hay and an efficiency of microbial protein synthesis estimate of 10%. In Exp. 1, 32 individually fed first-calf heifers (avg 395 kg) were allotted to a 2 x 2 factorial arrangement of treatments (main effects of DIP, MP, and DIP x MP interaction) 1 d after calving. Cows consumed a basal diet of chopped crested wheat grass hay (4.3% CP, 67% DIP) ad libitum. Supplemental DIP and UIP were supplied by varying the ratios of soybean meal (75% DIP) and a heat-treated, protected soybean meal (70% UIP). Cow weight gain was better (P < 0.01) when adequate DIP was supplied than when DIP was deficient. However, calf weight gain was not increased by supplementing the cow with DIP. Supplemental UIP did not (P > 0.40) improve cow or calf weight gain. Blood urea N levels were higher (P < 0.01) for cows receiving supplemental DIP and UIP. However, milk production estimates were similar among treatments, as were digestibilities of OM and ADF. Nitrogen digestibility was greater when supplemental DIP was fed, but providing additional UIP did not (P = 0.15) change N digestibilities. Experiment 2 evaluated similar supplements using the same experimental design to determine changes in cow and calf weight gain, body condition score, and pregnancy rate. Seventy-two first-calf heifers (avg 441 kg) were allotted to supplement treatments 1 d after calving and were fed grass hay (5% CP, 53% DIP, 10% microbial efficiency) for ad libitum consumption for 60 d. Supplements were individually fed three times/week. Varying the ratios of soybean meal, heat-treated soybean meal, and corn gluten meal provided additional DIP and UIP. Unlike in Exp. 1, supplemental UIP improved (P < 0.05) cow weight gain. Calves from dams supplemented with DIP gained 5 kg more weight after 60 d than calves from dams deficient in DIP. Pregnancy rates in the fall were similar (P = 0.90) among treatments. These data suggest that DIP was more limiting in Exp. 1 than was UIP. Supplementing UIP in Exp. 2 improved cow weight gains but did not improve calf gains. Data suggest that the efficiency of microbial protein synthesis for this forage-based diet was probably less than 10%.     

Transmission of bovine brucellosis from dam to offspring

Transmission of bovine brucellosis from dam to offspring under natural circumstances was demonstrated in a 2 1/2-year study of an infected cow and her calf. The cow delivered a full-term heifer calf after her first gestation. The calf remained with its dam until 7 months of age, then was placed in isolation until bred. During her first gestation, the second-generation heifer became seropositive for Brucella abortus. She later gave birth to a calf with B abortus infection, as determined by isolation of B abortus biotype 1 from stomach contents, heart blood, lung, and spleen of the calf. The same organism was isolated from the placenta and milk of the dam.