Donor Calf Research Articles

Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus. 1. Cell culturing requirements

The production of hybridoma cell lines secreting antibody against foot-and-mouth disease virus (FMDV) was more difficult than the production of similar cell lines secreting antibody against vesicular stomatitis virus or measles virus. A rapid and efficient protocol for the selection and culturing of 'anti-FMDV' hybridoma cultures was therefore developed and is described. This required the determination of the optimal culture medium (commercially available), source of serum supplement, line of myeloma cells, type of culture and routine for the subculturing of the hybridoma cells. The protocol consisted of fusion between immune splenocytes and NS-1 mouse myeloma cells, seeding into the wells of 24-well (24W) plates, culturing in RMPI 1640 medium supplemented with either foetal or donor calf serum, and passaging through 24W plates, 6W plates and 100 ml flasks (20 ml medium), respectively. The time at which aminopterin was added to kill unfused myeloma cells was also critical, with the optimum time being 24 h after fusion. In contrast, the B lymphocyte growth stimulant (2-mercaptoethanol) had no beneficial effects on the growth of the hybridomas.

Serological methods to detect calves infected in utero with bovine leukemia virus.

Calves born to dams infected with bovine leukemia virus (BLV) were investigated serologically and virologically before and/or after ingetion of colostrum to develop effective methods to detect BLV-infected calves in an early postnatal life. Of 50 precolostral sera, 10 (20%) and 13 (26%) were BLV-antibody positive by immunodiffusion (ID) and virusneutralization (VN) tests, respectively, indicating in utero BLV infection of the donor calves. In 18 postcolostral sera, ID antibody titers against BLV correlated well with serum ID antobody titers of the respective dams. The colostral antobodies decayed with a half-life of about 20 days in 76 BLV-uninfected calves. The antibody became undetectable in almost all of the calves at 6 months of age. On the other hand, in 16 calves infected in utero with BLV, ID antibody was often detected even at 6 months of age and later. The pattern of change in individual sequential ID antibody titers of these BLV-infected calves could be divided into three types. In type 1, ID antibody titers were maintained from birth to 7 months of age or older. In Types 2 and 3, the antibody titers decreased at a rate similar to those in uninfected calves. The titers then increased again at 5 to 7 months of age in Type 2 but they did not appear again after their disappearance in Type 3. Calves with antobody changes of Types 1, 2 and 3 were 6 (37.5%), 6 (37.5%) and 4 (25.0%), respectively. By testing sequential antibody titers, it was revealed that 7 (11.7%) of 60 calves which could not be tested for precolostral antibodies showed antibody changes of Types 1 and 2, indicating BLV infection in utero. Methods to detect BLV-infected calves in an early postnatal life are discussed.

Estimation of viability of bovine granulosa cells.

Granulosa cells harvested from non-atretic, antral follicles of cow (and pig and sheep) ovaries were incubated over glass cover slips in medium containing 20% (v/v) donor calf serum. Cell attachment to the cover slips was rapid, being in most cases complete within 3 h at 37 degrees C. There was little further change over the next 20 h. The number of bovine granulosa cells which attached to a cover slip was proportional to the volume of cell suspension added to the medium; and the amount of oestradiol secretion by attached cells in a testosterone-enriched medium increased in parallel with their number. Granulosa cells which did not attach within 3 h produced little oestradiol. There was no clear relationship between the number of nigrosin-impermeable cells in suspensions prepared from different follicles and plating efficiency. It is concluded that the 3-h attachment of granulosa cells in culture is a useful measure of the number of viable cells in a suspension and is to be preferred to less direct techniques based on dye exclusion.

Exposure of cattle immunised against redwater to tick challenge in the field: challenge by a homologous strain of B divergens.

A field trial was conducted in Northern Ireland to determine whether calves could be protected against babesiosis by the prior inoculation of irradiated blood infected with Babesia divergens; The trial involved 30 yearling calves. Ten were inoculated with infected blood from a donor calf after the blood had been irradiated at 25 kilorads, and 10 with blood irradiated at 30 kilorads. Their reactions to the inoculation were observed daily for a month. They were then released, along with a control group of 10 susceptible calves, into an area heavily infested with B divergens-infected Ixodes ricinus ticks for two months. Between 24 and 41 days after exposure all the control animals contracted babesiosis and six of them reacted severely. None of the immunised animals suffered clinical babesiosis although 14 had detectable low-level infections. The relative severity of the reactions of the groups of calves was reflected in their haematology.

Bacteriophage contamination in calf sera collected by different methods from various sources

A systematic study was carried out in order to compare the presence and the frequency of different phage types in foetal calf sera collected aseptically in slaughterhouses, in sera collected aseptically from young healthy donor calves and in calf sera obtained under clean but not sterile conditions in slaughterhouses. Twenty-five batches of aseptic calf sera collected from 300 donor calves bred in closed herds were consistently phage-free while 14 out of 16 batches of foetal calf sera showed phage contamination. Calf sera were positive to a lesser extent (8 out of 16 batches). E. coli B and E. coli K 12 proved to the most sensitive strains for detecting phages and their presence can give information about the frequency and the abundance of other enterobacteria phages containing sera. The advantages of using sera collected aseptically from donor calves are discussed.

Isolation and Partial Characterization of Exo- and Endoantigens of Cooperia punctata Cultured In vitro

Cooperia punctata were cultured in vitro in Ae medium for 21 days, washed in BSSA (Balanced Salt Solution-Antibiotics), and incubated for 72 hr in fresh BSSA. Exoantigens (metabolic antigens of mol wt > 10,000) harvested from incubation fluids and partially purified by ultrafiltration were shown to contain protein. An endoantigen (somatic extract) was prepared of the same worms from which exoantigens were harvested. Immunodiffusion tests made with serum of calves immune to C. punctata and exoor endoantigens gave 2 precipitin bands. Similar tests with mucosal extracts (of a calf immune to C. punctata) showed no activity against the exoantigens; one precipitin band developed using endoantigens. A pattern of complete identity was demonstrated between one component of exoantigens and the endoantigens; a pattern of partial identity developed between another component of the exoantigens and the endoantigens. Disc electrophoresis of the exoantigens resolved 3 distinct and 3 to 4 faint protein bands; the endoantigens were resolved into 10 to 12 reproducible protein bands. Incorporation of the disc gels in immunodiffusion tests indicated two distinct areas of activity when diffused against immune serum in the case of exoantigens and endoantigens whereas the mucosal extract had activity against one of the same areas of protein bands as the immune serum. Both antigens were fractionated by Sephadex G-200 into 3 peaks, and in each case, the first 2 peaks were serologically active; only the first peak of the endoantigens was active against the mucosal extract. Products of metabolism have been suspected as a major source of protective antigens in immunity to parasitic worms. The inability to harvest metabolic antigens of parasitic worms in vivo stimulated interest in in vitro cultivation as a means by which large numbers of parasitic worms and products of their metabolism (of antigenic potential) could be harvested. The in vitro cultivation of parasitic stages of Cooperia punctata was reported by Leland (1961). Later, C. punctata was cultured in vitro from egg to egg (Leland, 1962). Douvres and Alicata (1962) were able to culture artificially ensheathed L3 of C. punctata to sexually immature adults in a vitamin-supplemented medium. The development of C. punctata from L3 to egg-laying or sperm-containing adults in a modification of Ae medium was reported by Eckert (1967). Leland (1967) indicated the final step to completion of the life cycle of C. punctata in culture was in vitro fertilization. In 1971, Zimmerman and Received for publication 12 November 1973. * Contribution No. 178, Department of Infectious Diseases, College of Veterinary Medicine and the Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506. Supported by NSF Research Grant GB 7532 and contributing to Regional Project W-102 Biological Methods of Control for Internal Parasites. Leland reported the completion of the life cycle of C. punctata in vitro. At least the minimum nutritional and physicochemical conditions comparable to those in vivo had been approximated. It could be assumed that in vivo and in vitro metabolisms were comparable and therefore the products of metabolism in both conditions should be similar. The purpose of this study was to isolate and partially characterize antigens of C. punctata cultured in vitro. The term exoantigen is used for products of worm metabolism (i.e., metabolites, secretions, excretions); endoantigen refers to soluble somatic worm material (i.e., extract of worm tissue). MATERIALS AND METHODS A schematic of the procedure for antigen harvest and characterization is given in Figure 1. Cultivation techniques Two thousand infective larvae (L3) were isolated from 14-day-old fecal-vermiculite cultures (made with feces of a donor calf containing a monospecific infection of C. punctata) by the Baermann technique, processed, and inoculated into a culture tube containing 2 ml Ae medium a cording to the procedures of Leland (1963). The number of tubes inoculated at any one time varied from about 50 to over 300, depending on the availability of L3. Incubation was at 38.5 C in roller drums ( 1/ rpm). Worms were washed in BSSA (Balanced Salt Solution-Antibiotics, Leland, 1963) and transferred to tubes of fresh medium weekly except that the first transfer pro-

Efficacy of Commercial Bovine Serum as a Component of Media Used to Grow Cooperia punctata In vitro

Several commercial serums-agamma calf, bovine, fetal bovine, from calves 10 days to 6 months old, and from calves younger than 10 days-were incorporated into Ae medium (Leland, 1963, 1965). Each serum's efficacy in promoting in vitro growth and development of Cooperia punctata, a nematode parasite of ruminants, was compared with medium Ae containing from a donor calf. Culture tubes inoculated with exsheathed, 3rd-stage C. punctata larvae were maintained in a roller drum at 38.5 C for 35 or 56 days; some cultures were transferred weekly into new media and some were not. At termination, worms were preserved, measured, and counted, and data for each medium were compared and evaluated. All serums tested, when used as a component of Ae, promoted growth and development of C. punctata to the adult stage. Bovine was least effective as a substitute for obtained from a donor calf. Although medium containing fetal bovine was unsatisfactory because it tended to precipitate after thawing, it resulted in good growth and development during the first 3 weeks of culture. A minus gamma globulin (agamma serum) is our choice as a substitute for serum; this resulted in a clear medium and L, from eggs laid in vitro were observed after 28 days. Living worms were observed in all media in which worms were grown for 56 days without transfer, but weekly transfers into new media produced better cultures in tubes maintained that long. After 35 days in culture, a negative correlation between size of inoculum and percentage of worms reaching the adult stage was noted, although no correlation was observed between size of inoculum and size of adults. Largest adults were produced in cultures maintained for 56 days but transferred weekly into new media. Largest females were produced in media containing either from a donor calf (Ae) or commercial fetal (AeF); largest males were produced in AeF. The parasitic stages of Cooperia punctata grow well in medium Ae which normally contains from calves; omitting the results in fewer and smaller adult worms, and females so grown produce no eggs (Leland, 1963). In contrast, nonparasitic stages of C. punctata (L1 and L2) do not develop in medium containing (Leland, 1967b). If a readily available commercial could be substituted for helminth-free serum it would facilitate experimentation using the in vitro culture system, and also might simplify comparisons of studies from different laboratories. Thus we tested the efficacy of Ae medium in which various commercially available bovine serums were substituted for obtained from

Completion of the Life Cycle of Cooperia punctata In vitro

L3 Cooperia punctata were cultivated in vitro to adult worms that laid large numbers of eggs; many of these eggs hatched to L1 and L2 and a few of the latter developed to L.. Parasitic stages were cultivated in Ae medium (38.5 C); free-living stages were cultivated in A-s medium at 27 C. Embryonation, hatching, and development initially were observed only in cultures with a population density of fewer than 1,742 worms/2 ml medium. Embryonation was not observed in cultures where 15-day-old worms were combined with worms 29 days old. Embryonation and hatching were detected in 28 of 271 subsequent cultures inoculated with 1,500 larvae. The cultivation of the parasitic stages of Cooperia punctata in vitro, including the production of egg-laying or sperm-containing adults in Ae medium, was reported by Leland (1961). Leland (1962) reported the cultivation of C. punctata from egg to egg in vitro. Douvres and Alicata (1962) cultured C. punctata from artificially exsheathed L3 to sexually immature fifth-stage adults in vitamin-supplemented medium. Eckert (1967) also developed C. punctata from L3 to egg-laying or spermcontaining adults in a modification of Ae medium. Leland (1967) reported the cultivation of C. punctata in vitro from egg to egg (infertile). Nonparasitic stages were cultivated in A-s medium (serum component of Ae replaced with BSSA); parasitic stages were cultivated in Ae medium. Infertile eggs were produced by virgin females and females in male-containing cultures. Leland (1970) reported the development of C. punctata from L3 to egg-laying adults in Ae medium in as few as 21 days. The final portion of the life cycle of C. punctata to be accomplished in vitro was hatching eggs produced by adults reared in vitro. The purpose of this study was to determine if hatching of eggs was not occurring because (1) males and females were maturing at Received for publication 17 September 1970. Contribution VIII No. 149, Department of Infectious Diseases, College of Veterinary Medicine, Kansas Agricultural Experiment Station, Manhattan, 66502. Supported in part by NSF Research Grant GB 7532, the National Defense Education Act program, and contributing to Regional Project W-102-Biological Methods of Control for Internal Parasites. different times or (2) the nematode-population density in culture was not suitable. In the course of this work the hatching of eggs was detected. MATERIALS AND METHODS L3 isolated from 14-day-old vermiculite cultures (made with feces taken by rectal collection from a donor calf containing a monospecific infection with C. punctata) were exsheathed and inoculated according to the procedures of Leland (1963). Larval suspensions used as inoculum contained approximately 200 to 2,000 larvae per 0.2 ml. Media used were Ae (Leland, 1963, 1965) or A-s (Leland, 1963, 1967). A-s was a modification of Ae in which the serum component was replaced with BSSA (Balanced Salt Solution-Antibiotics, Leland, 1963). Incubation was in roller drums (1/5 rpm) at 38.5 C or 27 C. Either weekly or semiweekly, worms were washed in BSSA and transferred to tubes of fresh medium. To combine different aged cultures, worms 15 days old (tubes 190, 191, 192, 193, 194, and 195) were placed respectively in tubes containing worms 29 days old (tubes 200, 201, 202, 203, 204, and 205). This procedure provided an optimum density of approximately 1,500 larvae/2 ml medium (Leland, 1970) in each tube during the precombination period. After combining, the population density was doubled (3,000 worms/2 ml). This was necessary because the in vitro entangling characteristic of the nematode prevented division of the culture without injury to the worms. Worms were cultured until no further worm movement was detected (day 72 after combining cultures; refer to Table I for termination ages of population density cultures). Subsequent culture manipulations were as described above, except for the numbers of larvae inoculated into each of tubes 170, 182 to 189, 196, and 206 to 208; the level of inoculum in them ranged from approximately 200 to 2,100 larvae per culture (Table I). Many L1 and L2 produced by in vitro adults were transferred either to Ae medium at 38.5 C, or to BSSA, Ae, or A-s at 27 C. C. punctata larvae were isolated from calf feces by the Baermann technique after 24 hr (L1), 48 hr

Attempts to transmit bovine anaplasmosis with Anopheles quadrimaculatus Say (Diptera: Culicidae) and Dermacentor andersoni Stiles (Acarina: Ixodidae).

Females of Anopheles quadrimaculatus were fed on donor calves acutely infected with Anaplasma marginale. In 1 trial, the mosquitoes were interrupted in their feeding and immediately transferred to a splenectomized recipient calf and completed engorgement; in other trials, mosquitoes were held for 3, 7, 8, or 14 days before receiving their second blood meals on splenectomized recipient calves. The mosquitoes did not transmit anaplasmosis to any of the 8 recipient calves. In contrast, adult Dermacentor andersoni, which engorged as nymphs on the same donor calves while the mosquitoes were feeding, later transmitted anaplasmosis to other splenectomized recipient calves in 5 of 7 trials. These results suggest that Anopheles quadrimaculatus plays little or no role in either the mechanical or biological transmission of Anaplasma marginale.

The part played by the extracellular components (collagen and albumin) in the decrease in reactivity of adipose tissue in old rats

The age of the albumin donor (calf or adult ox) has no effect on the basal lipolysis of fatty tissue in vitro or in lipolysis induced by adrenaline. The amount of collagen in adipose tissue is increased in old rats when related to nucleic acid content. Adipose tissue collagen from older rats (6 and 15 months) was found to be significantly less digestible by collagenase than that of 3-month-old rats as measured by the released hydroxyproline value. The significance of the qualitative changes in adipose tissue collagen is discussed in relation to previous findings of a decrease in lipolysis with age.

Effect of the Antioxidant 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline on the Development of the Free-Living Stages of Some Nematode Parasites of Cattle

The antioxidant used in commercial animal feeds, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin, Santoquin, or EMQ) had an inhibitory effect on larval nematode development. Increasingly fewer larvae were recovered from fecal cultures as the quantity of EMQ fed to infected calves, or mixed with feces from infected calves, was increased from the amount commonly used as a feed preservative (1/4 lb/ton) up to 100 times that amount. For example, the number of Cooperia pectinata larvae recovered from fecal cultures from a calf fed untreated chopped alfalfa hay was 40.4% as compared to 9.5% when 100 X EMQ was mixed with the hay. Similarly, 25 X EMQ resulted in reductions as follows: Trichostrongylus colubriformis from 33.2 to 13.5%, T. axei from 28.3 to 10.8%, and Oesophagostomum radiatum from 45.3 to 4.8%. Comparing commercial pelleted alfalfa hay with and without EMQ showed reductions, varying from 17% for C. punctata to 64% for T. colubriformis. Larval development in feces-vermiculite cultures treated with various concentrations of EMQ showed reductions as follows: T. axei from 27.9 to 0.0% with 500 X, C. pectinata from 22.8 to 0.8% with 30 X, and T. colubriformis from 24.6 to 15.3% with 4 x. Studies on the effect of host diet on the bionomics of the free-living stages of some cattle nematodes showed a reduction in the number of larvae recovered from cultures when the diet was changed from alfalfa hay to dehydrated alfalfa pellets. Further investigations indicated that inhibition of larval development could be due to the chemical preservative or antioxidant used in the preparation of commercial animal feeds. Of the antioxidants used in the food industry, 6-ethoxy1,2-dihydro-2,2,4-trimethylquinoline (known as ethoxyquin, Santoquin, or EMQ) is probably the most prevalent because of its high activity and desirable physical properies (Bickoff et al., 1954). According to Machlin et al., 1957, and Wilson et al., 1959, this chemical is rapidly absorbed and its metabolites are readily and nearly completely excreted in urine and feces. This paper considers the effect of EMQ on larval development when mixed with various feeds, and when added to feces obtained from donor calves monospecifically infected with various cattle nematodes. Received for publication 28 September 1966. * Resigned 13 August 1966. t In cooperation with the Georgia Agricultural Experiment Station. Station Journal Series Paper No. 546. MATERIALS AND METHODS Commercial dehydrated alfalfa pellets (16 or 17% crude protein) and alfalfa meal (17% crude protein) were compared with alfalfa hay and with chopped alfalfa hay, to which various amounts of EMQ (Santoquin-Mixture 6, Monsanto Company, St. Louis) had been added. The chopped hay was passed through a hammer mill with a 1/2-inch screen. The standard concentration of EMQ, designated as 1 X, was based on the amount commonly used in the manufacture of commercial pellets and other animal feeds (1/4 lb/ton, or 125 Mig/g). Higher dosages were in multiples of the standard dosage, and were designated, for the sake of brevity, from 1 X to 100 X. Dairy calves, monospecifically infected with various cattle nematodes, were used as experimental hosts. The species present were Cooperia punctata, C. pectinata, Trichostrongylus axei, T. colubriformis, and Oesophagostomum radiatum. Feces were collected from the hosts' rectums hourly during the day (8 AM to 4 PM) for various days while the particular host calf ate, ad lib., one of the rations. No collections were made 3 or 4 days following a change in diet in order to allow the elimination from the calf of all waste products of the previous diet and for the rumen flora to become adjusted to the new diet. The total daily output was mixed thoroughly, samples were taken for nematode egg counts, and the remainder was cultured in plastic pans. The cultures, each containing 1,000 g of feces, 166 g vermiculite, and any necessary water, were incubated for 9 days in constant temperature cabinets set at 25 C. All cultures were weighed daily and opened to aerate and to replace any water lost through evaporation. At the end of the incubation period, the cultured material was placed in Baermann funnels, left over-

Serological and Immunological Responses of Calves to Infection with Trichostrongylus axei

Serological and Immunological Responses of Calves to Infection with Trichostrongylus axei