Uninfected Calves Research Articles

Detection of group B rotavirus in fecal specimens by dot hybridization with a cloned cDNA probe

Cloned cDNA copies were synthesized from the genomic RNA of the IDIR strain of group B rotavirus (GBR) isolated in Baltimore, Md. These clones were screened for hybridization with heterologous GBR to identify cDNA for use in dot hybridization experiments. In multiple screening experiments, cDNA clones derived from gene segment 3 provided the most intense hybridization signals. 32P-labeled probes were produced from one of the gene 3 clones, and these were employed in dot hybridization assays. Purified preparations of bovine GBR were detected in concentrations of greater than or equal to 0.5 ng, and GBR was detected in fecal specimens obtained from five of six infected calves. Four of six human fecal specimens containing the Baltimore strain of GBR were also positive in the hybridization assay, while GBR was identified in only one of the six specimens by means of immunoelectron microscopy. A fecal specimen obtained from a patient infected with the adult diarrhea rotavirus strain of GBR was also positive in the dot hybridization assay. Fecal specimens from uninfected humans, calves, and rats, as well as specimens containing group A rotaviruses, did not hybridize with the cloned cDNA probe.

In vitro Proliferative and Cytotoxic Responses of PBL from Theileria annulata‐immune Cattle*

Peripheral blood lymphocytes were isolated from healthy calves and were subsequently infected with sporozoites of Theileria annulata in vitro. The infected cells were passaged for 50 times and thereafter inoculated into animals from which they were previously isolated. Within 4-5 days, schizont-containing cells were demonstrable in the lymph nodes of all animals. Few days later, merozoites were detected in erythrocytes. A slight decrease in the counts of lymphocytes and leucocytes was also found. After 2 months these animals and a group of uninfected calves were heavily infected by tick-infestation and showed severe symptoms of theileriosis with 60% schizont-containing cells in the lymph nodes and a parasitaemia of about 35%. Because of the severity of the infection, all control calves were treated with Halofuginone. In contrast, the initially immunized cattle (by inoculation of culture cells), survived the infection without chemotherapy. Less than 10% of their lymph node cells contained schizonts, whereas less than 1% of their erythrocytes were found to be infected with merozoites. In all immunized animals, specific cytotoxic PBL, with the capacity to lyse autologous but not allogeneic infected cells, were demonstrated. In addition, a population of PBL were found to be able to inhibit the growth of T.annulata-infected culture cells in vitro. However, in comparison to PBL of immune animals, PBL of acute infected calves were superior in their capacity to inhibit the proliferation of schizont-containing cells. In mixed lymphocyte reactions, T. annulata-infected cells could induce a more pronounced proliferative response in PBL from immune than in PBL of uninfected animals.

Protection from parasite-induced weight loss by the vaccination of calves with excretory-secretory products of larval Oesophagostomum radiatum

Excretory-secretory products (ESP) were collected from in vitro maintained Oesophagostomum radiatum larvae during the period in which the larvae molt from the third to fourth larval stage. The ESP were used to immunize 13 uninfected calves which were subsequently challenged with 1.7×10 4 infective O. radiatum larvae. Worm recoveries from immunized calves were reduced 23% compared to 12 unimmunized controls, while the number of intestinal nodules was 72% greater compared to unimmunized controls; however, neither difference was statistically significant. Immunized calves had enhanced serum IgG and IgA anti-ESP antibody responses upon challenge. No differences in serum IgG 2 or IgM antibody or cellular immune responsiveness, as determined by in vitro antigen-induced lymphocyte proliferation, were seen. Vaccination with ESP did significantly protect calves from the weight loss seen in non-immunized calves. Unimmunized calves ceased gaining weight ∼5 weeks after challenge and by 10 weeks after challenge had lost an average of 4 lbs. per calf. Over the same time interval (i.e., 5–10 weeks after challenge), the immunized calves gained an average of 23 lbs. per calf. These results strongly suggest that vaccination with ESP conferred an advantage to calves that is not correlated solely with the number of worms developing from challenge infection.

Dermal cellular responses of helminth-free and Ostertagia ostertagi-infected calves to intradermal injections of soluble extracts from O. ostertagi L 3 larvae

Twelve calves were raised helminth-free until 9 weeks of age when six were orally inoculated with 100 000 Ostertagia ostertagi infective stage larvae (L 3). Three uninfected and three experimentally infected calves received intradermal injections of sterile saline and soluble larval extract (SLE) from O. ostertagi L 3 with a protein concentration ranging from 1 to 200 μg ml −1. Biopsies were performed 48 h post-injection. A kinetic study was performed on the remaining six calves, three infected and three uninfected, using a 100 μg ml −1 concentration of SLE and taking biopsies 1, 4, 8, 12, 24, and 72 h post-injection at both the saline and SLE-injected sites. All calves had an immediate wheal and increase in skin thickness at the SLE-injected sites. The numbers of eosinophils infiltrating SLE-injected sites as comparred to saline-injected sites were significant in both uninfected and infected calves, but the infected calves had significant numbers to a wider range of SLE concentrations and had significantly higher numbers than uninfected calves in the kinetic study. Infected calves also had significant numbers of basophils in the dose response study at concentrations of 5 and 100 μg ml −1 SLE. Neutrophil infiltration was similar in both groups and was significant at SLE-injected sites early in the kinetic study. Detectable mast cells were decreased in SLE-injected sites of infected animals and perivascular accumulation of mononuclear and some polymorphonuclear cells was observed in the deep dermis of infected animals.

Lymphocyte blastogenic responses of calves experimentally infected with Ostertagia ostertagi

Twelve 9-week-old calves were divided into four groups; two groups were maintained helminth-free as controls and the other groups were given Ostertagia osttertagi infective-stage larvae (L 3) orally. One group received 100 000 L 3 as a single inoculum and the other group received L 3 in increasing dosages at weekly intervals for 8 consecutive weeks. The blastogenic responses to concanavalin A (Con A), phytohemagglutinin (PHA), and a soluble larval antigen from O. ostertagi (SLA) of peripheral blood lymphocytes were evaluated using tritiated thymidine incorporation into DNA as a measure of blastogenesis. The responses to Con A of all infected calves were significantly depressed while the responses to PHA were not. SLA, at concentrations of 4 μg ml −1 and above, caused blastogenic activity in lymphocytes from uninfected calves. Using SLA at 1 μg ml −1 in lymphocyte cultures supplemented with autologous serum, an antigen-induced blastogenic response was detected in calves receiving serial inoculations of L 3.

Suppressor activity of bovine FCy positive cells during a persistent infection with Mycobacterium avium

The cell mediated immune response (CMI) was measured in calves after experimental infection with Mycobacterium avium. Using the tuberculin skin test a CMI response could be measured from four to 14 weeks after infection, and with a lymphocyte stimulation (LS) test from six to 40 weeks. One year after infection no CMI response was detected by either of the tests, in spite of the fact that in such calves M avium bacteria could be found in the intestinal lymph nodes at autopsy. After removal of mononuclear cells bearing receptors for the Fc part of IgG, the peripheral blood lymphocytes obtained from a calf infected one year earlier responded to M avium pure protein derivative in the LS test in contrast to lymphocytes obtained from uninfected calves.

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.

THE USE OF A MODIFIED BROMOSULPHTHALEIN EXCRETION TEST FOR THE MEASUREMENT OF HEPATIC BLOOD FLOW IN CALVES

Single intravenous injections of bromosulphthalein (BSP) were given to calves before and at intervals after the oral administration of 1000 metacercariae of Fasciola hepatica. The change in BSP concentration with time was analysed by computer to obtain the proportion of BSP extracted from plasma by hepatic cells (E) and the rate of hepatic blood flow (F). The increasing age of the normal calves during the 34 weeks of experiment had no significant effect on E or F. There was no significant effect of the duration of the fasciola lesion on E or F. When all tests on infected calves were compared as a group with tests on uninfected calves, there was a significant reduction in E(P less than 0.05) and in F(P less than 0.001) in the calves infected with F. hepatica. A survey of previously reported results suggests that sheep and cattle have a wide range of F values and that F is probably lower in 3-month-old calves than in adult cattle.

Salmonella typhimurium infection in calves: delayed specific skin reactions directed against the O-antigenic polysaccharide chain.

Specific delayed skin reactions developed after intradermal injection of Salmonella typhimurium lipopolysaccharide in 12-week-old calves which had been orally infected with S. typhimurium 5 weeks earlier. Uninfected calves showed no delayed skin reactions. Skin biopsies from skin swellings showed a massive infiltration of mononuclear cells in the skin of infected calves but not in those of uninfected calves. Persistence of infection in infected calves was confirmed by isolation of S. typhimurium from fecal specimens. The delayed skin reactions could be shown to be specific and directed against the O-polysaccharide chain of the lipopolysaccharide since none of the lipopolysaccharide chain of the lipopolysaccharide since none of the lipopolysaccharide preparations from a rough mutant of S. typhimurium, two strains with different O-polysaccharide chains, or lipid A elicited skin reactivity. To cause a reaction, the O-polysaccharide had to be in a macromolecular complex, since skin swellings were seen only after injections of either O-polysaccharide chains cross-linked by 1,2,3,4-diepoxybutane or octasaccharides from the O chain covalently linked to a straight 12-carbon aliphatic chain forming an artificial glycolipid. Injection of the pure octasaccharides of O-polysaccharide chains failed to elicit delayed skin reactions.

Plasma pepsinogen, inhibited larval development, and abomasal lesions in experimental infections of calves with Ostertagia ostertagi

Single doses of Ostertagia ostertagi, followed in 42 days by multiple increasing doses in calves, were monitored by fecal egg counts and plasma pepsinogen. The level of plasma pepsinogen increase was related to the increase in graded levels of inoculation and to the fecal egg counts. Plasma pepsinogen in uninfected controls remained below 1 IU/L. Plasma pepsinogen in all calves reached levels greater than 5 IU/L at 48 days of the extended inoculation period, although fecal egg counts remained low in the previously inoculated calves. The previously infected calves had a higher proportion of early fourth-stage larvae, while larger adult worm burdens were found in the previously uninfected calves. Early fourth-stage larvae were observed in extra-glandular sites, notably between the glandular epithelium and basement membrane or within the lamina propria. An immunological response of the host was suggested by the lymphoid cell infiltration in the mucosa. This host immune response may account for the greater larval inhibition exhibited by the previously infected calves.

The Repellent Effect of Gastric Contents from Uninfected Calves for Sheathed and Exsheathed Larvae of Ostertagia ostertagi

Stringfellow (1974, J. Parasitol. 60: 293297; 1977, Proc. Helminthol. Soc. Wash. 44: 76-81) studied the histochemistry of abomasa from uninfected calves and those infected with Ostertagia ostertagi. Chief cells were denuded of their pepsinogen granules. Carbonic anhydrase activity was not detected histochemically in parietal cells from infected calves. These observations, as well as biochemical evidence, suggested a mechanism whereby pepsinogen was released directly into the blood from chief cells. They also provided a mechanism for the rise in pH of the ,gastric contents during ostertagiasis. (Stringfellow and Madden, 1979, Proc. Helminthol. Soc. Wash. 46: 233-239). Those studies described the worm's environment at both the tissue and biochemical level in abomasa from both uninfected and infected calves. The present paper reports further analyses of selected components in the gastric contents of abomasa from uninfected calves. It compares the response of both sheathed and exsheathed larvae of 0. ostertagi to components of both calf origin and from commercial sources. Six 3-mo-old, helminth-free calves were taken off feed 12 hr before they were stunned with a captive bolt gun. Their throats were then slit. The anterior part of the fundus and the pylorus were tightly tied with string which retained the gastric contents and prevented the leaking of omasal and duodenal contents. The abomasum was slit and its contents were drained into a bucket with little or no bleeding. The contents were filtered through gauze, measured, and centrifuged. The clear supernatant was recovered and stored in vials on dry ice. The pH of the gastric contents was measured electrometrically immediately with standard buffers. Sodium and potassium ions were measured with a Corning 450 flame photometer with suitable standards. Chloride ion was measured electrometrically with Markson specific ion electrodes (liquid membrane type) and compared with a suitable calibration curve. Albumin and globulins were determined with Harleco kits on a Clinicard spectrophotometer. Pepsin (E.C. 3.4.4.1) was measured according to Edwards et al. (1960, Br. Med. J. 1: 30-32). Sigma Fraction V albumin was incubated at pH 2 for 24 hr at 37 C. The products were estimated with FolinCiocalteau reagent, and the samples were read in a Spectronic 20 spectrophotometer at 540 nm. Standard tyrosine (0.2 mmol) and a water blank served as references. Results are expressed as milliunits in Table I (Reid et al., 1967, Vet. Rec. 80: 1-4). Lipase (E.C. 3.1.1.3) was measured with the procedure of Cherry and Crandall (1965, In The practical manual for clinical laboratory procedures, Chemical Rubber Company [ed.], pp. 30-31). The response of 0. ostertagi larvae to test agar blocks was bioassayed on agar petri plates. Each petri plate had a background pH of either 2.3 or 7. The agar was adjusted to pH 2.3 with concentrated HC1. It was allowed to cool without adjustment to attain pH 7. A value of 2.3 represented the pH of the gastric contents from an uninfected calf. The pH 7 represented gastric contents from severe, advanced infections with this abomasal worm (Stringfellow and Madden, 1979, loc. cit.). Selected data at background pH 7 are presented in Figure 1. Only data with a common background pH were compared statistically. The test components were incorporated into 2% agar at the concentrations shown in Table I. Defined gradients were established as explained by Ward (1973, Proc. Nat. Acad. Sci. 70: 817-821). The agar background was adjusted with an appropriate compound such that only the substance in the agar block could be detected. Less defined components were prepared as follows: Fundic tissue (wet weight: 0.1-0.2 g) was homogenized with 10 ml of deionized, distilled water. Three milli-

Experimental infection of calves with two strains of bovine virus diarrhoea virus: Certain immunological reactions

Certain immunological responses of 4–6 month old calves experimentally inoculated with either cytopathic or non-cytopathic bovine virus diarrhoea virus (BVDV) were compared with those of uninfected control calves. The tests used to demonstrate the immunological responses were the transformation of lymphocytes by PHA mitogen, the percentage of lymphocytes with surface immunoglobulin, and the antibody titres induced by an intravenous inoculation of killed Brucella abortus . There were no significant differences between the two groups of calves and therefore, the mild experimental disease produced by BVDV did not appear to affect adversely the immunological response.

Pathological Changes in the Small Intestine of Neonatal Calves with Enteric Colibacillosis

Three neonatal calves, protected from colisepticaemia by intravenously administered immunoglobulin M, were infected orally with Escherichia coli type 0101 K?(A). All calves developed severe enteric colibacillosis. When they were 4 days old stunting and fusion of villi were seen in the distal half of the small intestine and were associated with adhesion of the challenge organism to the mucosa. Two uninfected control calves kept under similar conditions, did not develop diarrhoea and had no lesions.

Hypotension in acute Babesia bovis (= B. Argentina) infections of splenectomized calves

Systemic blood pressure changes were measured in groups of 4 splenectomized calves on days 3, 6, 7, 8 and 9 after acute infection with Babesia bovis. In addition, changes in five uninfected calves were measured on day 0. Carotid arterial, right atrial, right ventricular pulmonary arterial and ascending vena cava pressures were all lower on day 3 and had fallen dramatically by days 8 to 9. All animals died on days 8 and 9. The PCV began to fall and kallikrein activation started during days 2 to 3. Parasites were first detected on days 2 to 3. These results are discussed in relation to other protozoan diseases of man and animals and to anaphylaxis in cattle. It is concluded that many of the changes observed in acute B. argentina infections are due to kallikrein and kinins, possibly released by the action of parasite secretions during infection.

Biochemical indicators of liver injury in calves with experimental fascioliasis.

Twenty calves were infected with 1000 metacercariae of Fasciola hepatica, the activities of 10 enzymes in plasma or serum were assayed and concentrations in serum of proteins, urea and bilirubin were determined. These values were compared with control data obtained from 14 uninfected calves. Aspartate aminotransferase, lactate dehydrogenase, sorbitol dehydrogenase, glutamate dehydrogenase, ornithine carbamoyl transferase and gamma-glutamyl transpeptidase activities increased in infected calves. Total serum protein increased, albumin decreased, globulin increased and the albumin/globulin ratio was decreased in infected calves. Plasma alanine aminotransferase, leucine aminopeptidase, alkaline phosphatase and cholinesterase activities and serum concentration of urea and bilirubin were unaffected. It was concluded that glutamate dehydrogenase and gamma-glutamyl transpeptidase were the most sensitive indicators of liver cell damage in fascioliasis.

The possible effects of gastro-intestinal nematodes upon intestinal enzymes of calves

Observations were made on the effect of combined infections of gastro-intestinal nematodes on small intestinal disaccharidases and alkaline and acid phosphatases in 4–5-month old Friesian calves. The tests were carried out 3 weeks following exposure of these calves to infected pasture in a wet tropical environment. Sucrase activity, at a very low level, was detected in all parts of the small intestine of infected and uninfected calves. There was no significant difference in maltase, trehalase, cellobiase, sucrase, lactase, acid β-galactosidase, acid and alkaline phosphatase activities between uninfected control calves and all infected calves in which Cooperia species predominated. In heavily infected calves with worm burdens greater than 20 000 Cooperia species significantly elevated ( P < 0.05) duodenal maltase and acid phosphatase levels were recorded.

The serological response of three month old calves to infection withTaenia saginata (Cysticercus bovis) and their resistance to reinfection

Three calves, to which had been given100,000 eggs ofTaenia saginata at3 months of age, were significantly more resistant to a challenge infection10 months later than two previously uninfected control calves, although their resistance to reinfection was not complete.

Nonenzymic Histochemistry of Bovine Abomasal Tissue Infected with Ostertagia ostertagi

Abomasa from calves infected with 0. ostertagi and 0. radiatum, 0. radiatum only, and an uninfected calf were studied histochemically at 2, 5, 7, 14, and 22 days after infection (DAI). Collagen, accompanied by fibroblasts, was deposited in the tissue about the dilated gastric glands beginning at 7 DAI. Structural fibers staining with reticulum stains were abundant about the necks of the infected gastric glands. Compared to uninfected tissue and tissue adjacent to the dilated gastric glands, fewer chief cell granules stained for tryptophan, SS bonds, and SH groups immediately about the dilated gastric glands from 2 through 22 DAI. The cytoplasm of chief cells, agranular leukocytes, and fibroblasts stained intensely for RNA in uninfected and adjacent tissue, whereas degenerating chief cells immediately about the dilated gastric glands stained less intensely beginning at 7 DAI. Hemoand lipofuchsin methods stained a PAS-positive iron-negative pigment in macrophages and degenerating cells in infected abomasa. Frandsen (1965) studied the nonenzymic histochemistry of oesophagostomiasis at 8 and 10 days and ostertagiasis at 8 days postinoculation. He observed no significant changes in the histochemistry of abomasal tissue infected with 0. ostertagi probably because of an absence of intimacy between the larvae of 0. ostertagi and abomasal tissue. Because intimacy of the parasite with host tissues might influence the time and degree of host response, it became pertinent to extend the period of infection and reinvestigate the nonenzymic histochemistry of bovine ostertagiasis emphasizing ground substance, structural proteins, and fibers. The histochemical data were interpreted relative to the observed pathology which was similar to that described by Ritchie etal. (1966). MATERIALS AND METHODS Abomasa from 5 calves, infected with 350, 350, 150, 75, and 150 thousand larvae of 0. ostertagi, were removed at 2, 5, 7, 14, and 22 days after inoculation (DAI), respectively. All calves were 7 months old, except the third calf which was 9 months old. At necropsy, tissues were excised from proximal and distal parts of the abomasum and fixed in (1) neutral buffered formalin, (2) formol-calcium, (3) Bouin's, (4) Carnoy's, and (5) Zenker's fixatives. Hereafter, the histochemical method will be followed by a number designating the kind of fixation used. Additional tissues were quenched at -70 C in isopentane, cooled with dry ice, and stored in screw-cap vials on dry ice. Received for publication 17 May 1973. *Animal Parasitology Institute, ARS, Beltsville, Maryland 20705. Because these calves were also infected with Oesophagostomum radiatum for other studies, control tissues consisted of abomasal tissue from a calf infected with 20,000 0. radiatum larvae only and from an uninfected calf. Where appropriate, calf tissue positive controls were processed and studied concurrently. To verify the appropriate larval stage in tissue, whole mounts of 0. ostertagi were cleared in dimethylsulfoxide (DMSO) and compared with stages described and illustrated by Douvres (1956).

Babesia argentina: The Infection of Splenectomizcd Calves with Extracts of Larval Ticks (Boophilus microplus)

Larval ticks (Boophilus microplus) infected with Babesia argentina were fed on uninfected calves until forms of the organism infective for cattle were produced in the ticks, 3-5 days after attachment. Subcutaneous and intravenous inoculations of homogenates of the whole larvae plucked from the host at this stage transmitted B. argentina to other uninfected calves. These extracts were considered to be a possible source of erythro-cyte-free parasites for the vaccination of cattle against B. argentina infection and for the study of parasite antigens.

The preferential invasion of young erythrocytes in acute Babesia bigemina infections of splenectomised calves.

The mean corpuscular diameters (MCD) of both parasitised and unparasitised erythrocytes in B. bigemina-infected splenectomised calves, and erythrocytes in uninfected splenectomised control calves were measured.