Uninfected Calves Research Articles

Effects of acute bovine trypanosomosis ( Trypanosoma vivax) on plasma kinetics of intravenously administered lactose

Four calves infected with Trypanosoma vivax and four uninfected control calves were each injected intravenously with repeated doses of 0.5 g lactose kg −1 body weight, thrice daily at intervals of 4 h. Plasma samples were collected at specified time intervals and analysed for lactose. Pharmacokinetic parameters were calculated from the data. T. vivax infection delayed excretion of lactose from the body, thus leading to significantly ( P<0.001) increased biological half life ( t1/2) and a significantly ( P<0.001) reduced elimination rate constant for lactose in the body. The apparent volume of distribution and total clearance of lactose were not affected by the infection. T. vivax infection also appeared to cause accumulation of lactose in the plasma after repeated intravenous administration.

Detection of Proviral DNA of Bovine Immunodeficiency Virus in Bovine Tissues by Polymerase Chain Reaction (PCR) and PCRin SituHybridization

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCRin situhybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conservedpolgene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIVpolgene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed byin situamplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected toin situhybridization with an internal biotinylated probe and detected with streptavidin–gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissuesin vivoand has a broad cell tropism.

Isolation and phenotypic characterization of abomasal mucosal lymphocytes in the course of a primary Ostertagia ostertagi infection in calves.

Isolation and characterization of surface marker phenotypes of abomasal intraepithelial (IEL), lamina propria (LPL) and abomasal lymph node lymphocytes (ABLN) from uninfected calves were conducted, and the dynamics of change in these populations during the course of a primary Ostertagia ostertagi infection were defined. To obtain viable IEL and LPL from the abomasal mucosa of cattle, a modified isolation method was developed. The phenotypic characterization of abomasal lymphocytes was accomplished by indirect immunofluorescence staining. In uninfected animals, numbers of T cells exceeded the number of immunoglobulin-bearing cells in IEL, LPL and ABLN. The predominant T cell type in IEL and LPL was CD8+ cells, while the CD4+ T cell predominated in ABLN. Levels of activated cells and T cell receptor-1 gamma delta T cells were higher in IEL and LPL compared to ABLN. Within 3 weeks of infection, the number of lymphocytes recovered from the abomasal lamina propira and the mass of the ABLN was dramatically increased when compared to uninfected animals. Laser flow cytometric analysis demonstrated increased levels of immunoglobulin-bearing cells, gamma delta T cells, and activated T cells in IEL, LPL and ABLN in the infected animals. The greatest changes in LPL and ABLN took place during the first days of infection, and these changes were apparent throughout the 28 days covered by the experiment.

Activation of intestinal intraepithelial T lymphocytes in calves infected with Cryptosporidium parvum.

The objective of this study was to identify disease-related changes in lymphocyte populations within ileal mucosae of calves with cryptosporidiosis. Groups of five neonatal calves were orally infected at 3 days of age with 10(8) oocysts and maintained in enteric-pathogen-free conditions until clinical disease was established or until the animals had recovered from disease. Age-matched uninfected calves were used for comparison. Ileal mucosal lymphocytes were collected, quantitated, and phenotyped to determine whether changes in lymphocyte composition occurred in infected animals. We observed significantly larger numbers of intraepithelial CD8+ T lymphocytes in ileal mucosae from acutely infected calves compared with those from control animals. In addition, a proportion of intraepithelial CD4+ T cells from acutely infected calves coexpressed CD25, whereas there was an absence of coexpressed CD25 on CD4+ T cells from control calves. Ex vivo reverse transcriptase PCR of RNA from intraepithelial lymphocytes from control calves showed a cytokine expression pattern consisting of tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma), while intraepithelial lymphocytes from calves with cryptosporidiosis expressed IFN-gamma but not TNF-alpha. Together, the results indicate that changes occur in the ileal intraepithelial lymphocyte population coincidently with Cryptosporidium parvum-induced enteric disease.

Effect of acute Trypanosoma vivax infection on cattle erythrocyte glutathione and susceptibility to in vitro peroxidation

During acute Trypanosoma vivax infection of calves, produced by intravenous inoculation, the mean packed cell volume and red blood cell counts of the infected animals decreased significantly ( P < 0.05) between Days 6 and 13 post-infection (pi). The moderately severe normocytic anaemia started to develop during the first wave of parasitaemia which occurred from Day 2 pi and peaked between Days 4 and 5 pi. The mean erythrocyte glutathione (GSH) concentration of the infected calves decreased significantly ( P < 0.05) from 58 ± 11.4 mg 100ml −1 red blood cells (RBC) on Day 0 pi to 44.5 ± 12.8 mg 100ml −1 RBC on Day 5 pi. As the GSH values recovered on Day 6 pi and increased thereafter, another slight decrease ( P > 0.05) in GSH concentration occurred on Day 12 pi at the second peak of parasitaemia followed by a significant ( P < 0.05) increase to 79.1 ± 14.6 mg 100ml −1 RBC on Day 13 pi. In the uninfected calves, the mean GSH values ranged from 47.7 ± 7.0 to 60.8 ± 6.8 mg 100ml −1 RBC. When washed, erythrocytes of the infected and uninfected calves were separately challenged with hydrogen peroxide. They produced comparable amounts of thiobarbituric acid reactive substances as a measure of by-products of lipid peroxidation. This suggested that the ability of the erythrocytes to prevent peroxidative injury was not reduced, because GSH regeneration was probably enhanced and the antioxidant capacity of the erythrocytes was maintained.

Nitric oxide inhibits establishment of macroschizont‐infected cell lines and is produced by macrophages of calves undergoing bovine tropical theileriosis or East Coast fever

Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN-gamma). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont-infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont-infected cell lines was suppressed either by incubating sporozoites with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite-infected cells with SNAP. Proliferation of established macroschizont-infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neutrotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.

A study on the pathogenesis of Trypanosoma evansi in buffaloes, Holstein Friesian and Ongole cattle

A study on the pathogenesis of Trypanosoma evawi was carried out in 5 buffalo calves and 5 buffalo adults, 6 Holstein-Friesian calves and 6 Holstein-Friesian adults, and 6 Ongole calves and 6 Ongole adults, each ofwhich was divided into 3 infected and 2 uninfected buffalo calves and adults, and 3 infected and 3 uninfected calves and adults of Holstein Friesians and Ongoles. None of infected animals showed acute clinical signs along the course of the observation period, however roughness of the hair and skin, emaciation, weakness and loss of weight gains were the common clinical signs. Clinical signs of calves were more severe than adults, and those of buffaloes were more severe than cattle . Gross pathological changes were not specific . The mortality rate was 2/3 in buffalo calves, 1/3 in Holstein-Friesian calves and 1/3 in Ongole calves . None of infected adults died of infection. Buffaloes had longer and higher parasitemia than Holstein-Friesians or Ongoles. Erythrocyte counts of infected animals decreased to lower levels than controls, however they fluctuated in the normal values . Haemoglobin and PCV values of infected animals were significantly lower than those of non-infected controls, and those of calves were more severe than adults, and those of buffaloes were more severe than cattle . Infections resulted in loss of weight gains which was the greatest in buffaloes then followed by Holstein-Frisians and finally Ongoles.

Experimental bovine schistosomiasis in zebu calves

Five calves were each experimentally infected with 30,000 cercariae of Schistosoma bovis and three calves were kept as controls. S. bovis eggs first appeared in feces of the infected animals by week 5 post infection and all animals were shedding by week 6 post infection. Between week 7 and 9 post infection, where fecal egg counts were highest, the infected animals developed mucoid and then hemorrhagic diarrhoea and they became dull and depressed. Packed cell volumes and hemoglobin concentrations of the infected animals showed progressive reductions compared to the uninfected control calves. The animals were necropsied and perfused at week 12 post infection and tissue egg densities and worm burden were determined.

Effect of administration of serum thymic factor (FTS) in calves and rabbits infected with bovine immunodeficiency-like virus.

The effect of serum thymic factor (FTS) administration in bovine immunodeficiency-like virus (BIV)-infected calves and rabbits was examined. We previously found that some of the macrophage functions were depressed and humoral immune responses against foreign proteins were delayed in BIV-infected calves compared to uninfected calves. After FTS administration, however, no delay of antibody responses against foreign proteins was observed in BIV-infected calves. Though the chemiluminescence (CL) responses of macrophages in BIV-infected calves were significantly depressed (p < 0.05), FTS administration resulted in the recovery of the CL responses in the BIV-infected calves comparable to those in the control calves. Antibody responses against foreign proteins in BIV-infected rabbits were significantly depressed (p < 0.025) as compared with those in uninfected rabbits, though the depression became no significant after FTS administration.

Cellular immune status of coronavirus-infected neonatal calves

A preliminary study was conducted to determine the cellular immune status of neonatal, colostrum-deprived calves following inoculation with either attenuated or virulent bovine coronavirus (BCV). Uninfected calves served as controls. To determine the intestinal and systemic cellular immune status, we performed MHC-restricted cytotoxic lymphocyte (CTL) assay on mesenteric lymphocytes, enumerated T cell subsets in peripheral blood lymphocytes, and examined histopathological alterations in mesenteric lymph nodes and gut-associated lymphoid tissue. Target cells for the CTL assay were autologous testicular cells and effector cells were mesenteric lymphocytes from calves infected with BCV. No appreciable specific lysis was observed in any group of calves indicating the absence of demonstrable CTL responses. The T C/T S population was severely depressed in the calf inoculated with the virulent virus but not in those inoculated with either the attenuated virus or placebo. The mesenteric lymph nodes and Peyer's patches of calves inoculated with the virulent virus showed severe depletion of lymphocytes. These calves developed intestinal antibody responses in the acute phase of infection (1 week after infection) but were immunosuppressed in the later stage of infection.

A field evaluation of the polymerase chain reaction procedure for the detection of bovine leukaemia virus proviral DNA in cattle

A polymerase chain reaction (PCR) procedure that detects proviral bovine leukaemia virus (BLV) in peripheral blood mononuclear cell DNA was evaluated. Blood samples from all animals (164) in a commercial dairy herd with a 30% prevalence of BLV infection, and from 194 animals from BLV free herds were tested. The absence of any positive PCR results in animals from BLV free herds confirmed the specificity of the assay. Initial testing of the infected herd using a single amplification PCR (SA-PCR), detected BLV infection in 62 of 72 adult animals that were seropositive by the agar gel immunodiffusion (AGID) test and in one persistently seronegative cow. Infection in this cow was confirmed by sheep bioassay. Subsequent testing of the SA-PCR negative, seropositive animals using a double amplification PCR (DA-PCR) detected proviral BLV in eight of nine animals that were available for retesting. The PCR assay was also able to distinguish BLV infected calves from uninfected calves that were serologically positive because of the presence of colostral antibody. Lymphocytes from all seropositive animals were cultured for determination of BLV antigen expression. Cultures from 37 of 62 SA-PCR positive animals produced detectable quantities of viral antigens. However, antigen expression was not detected in cultures from seropositive animals that were negative in the SA-PCR. In addition, in experimental transmission tests, inoculation of more than 10 6 lymphocytes from these cows was required for sheep to become seropositive to BLV. These results suggest that the failure of the PCR assays to detect some seropositive animals was due to a low proportion of lymphocytes being infected with BLV in these animals. The DA-PCR detected BLV infection with a sensitivity comparable to that of the AGID test and the sheep bioassay. PCR assays may be an alternative to the sheep bioassay as an adjunct to serological testing for use in situations where it is essential to detect all infected cattle. However, the stringent precautions found to be essential to prevent false positive results due to contamination of samples with PCR product are likely to preclude the routine use of PCR for diagnosis of BLV infection.

Enterotoxic effect of stool supernatant of Cryptosporidium-infected calves on human jejunum

Background/Aims:The clinical pattern of cryptosporidial diarrhea suggests an enterotoxic mechanism. No evidence for this mechanism has been reported thus far. This study aimed to look for enterotoxic effect elaborated by Cryptosporidium.Methods:The effects on human intestinal transport of stool supernatant of diarrheal calves infected with Cryptosporidium parvum were examined. Aliquots of centrifuged and filtered stools were added to the mucosal or serosal side of human jejunum obtained from patients undergoing surgery and mounted in Ussing chambers. Electrical parameters were recorded. Stool supernatants of uninfected calves served as a control.Results:The mucosal addition of 2.5 mg protein of fecal supernatant from diarrheal calves induced a prompt and significant increase in short circuit current with no effects on tissue conductance. The serosal addition of this material and the addition of control supernatant to either side did not induce modifications of electrical parameters. The enterotoxic effect was dose-dependent and saturable. It was reversible by withdrawing the supernatant from the incubation medium. The electrical effect was chloride- and calcium-dependent and was sensitive to heating.Conclusions:An enterotoxic activity is present in the stools of Cryptosporidium-infected calves. This activity may be responsible for secretory diarrhea in humans.

Characterisation of Ostertagia ostertagi antigens by the different bovine immunoglobulin isotypes

Antigenic differences between the developmental stages of Ostertagia ostertagi were studied by SDS-PAGE and Western blotting. Gel electrophoresis showed a complex protein pattern different for every stage with the O ostertagi fourth stage larvae ( L4) showing an intermediate protein pattern between the third stage larvae ( L3) and the adult stage. Immunoblotting showed that IgG 1, IgG 2 and IgM immunoglobulins present in serum from uninfected calves identified several O ostertagi antigens at every stage. When using serum from O ostertagi infected calves, O ostertagi specific IgG 1 was the predominant bovine immunoglobulin. Specific IgG 2 and IgM responses were also observed, while specific IgA antibodies were hardly detectable. Severe IgG 1 cross reactivity was demonstrated when using anti- Cooperia oncophora serum.

Detection of stable diagnostic antigen from bile and feces of Fasciola hepatica infected cattle

Diagnostic antigens in bile and feces from Fasciola hepatica infected cattle were detected and characterized by enzyme-linked immunotransfer blot (EITB) techniques. As sources of antigen, samples of bile, intestinal contents and feces were collected from five uninfected calves and from 10 calves with known Fasciola hepatica burdens. A band detected by EITB using a densitometer in the area corresponding to 26 kDa reacted with rabbit anti-fresh fluke antigen and infected cattle sera but not with fluke-negative rabbit sera, rabbit anti- Fasciola hepatica egg sera, Fascioloides magna positive or negative cattle sera. This band was not detected by Coomassie blue in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or by Ponceau-S stained nitrocellulose strips. Band groups located at 104-66, 66-42, 42-26 and 25-16 kDa reacted inconsistently with the above sera. Sera from mice hyperimmunized with Fasciola hepatica excretory-secretory (ES) products detected only the 26 kDa band by EITB, without cross-reactivity with bands in the other molecular weight (MW) ranges. The results suggest that the 26 kDa antigen may consist of a stable component of ES products and/ or tegument-related worm antigen. Diagnosis of Fasciola hepatica through detection of specific, stable antigens in feces of infected animals offers potential advantages over serum-based tests of better sample accessibility, discrimination between previous and current infections, and possible semi-quantitation of fluke burdens.

The efficacy of ivermectin against the eyeworm, Thelazia skrjabini, in experimentally infected cattle

The anthelmintic efficacy of ivermectin (administered subcutaneously at 200 μg kg −1 body weight) was assessed for control of Thelazia skrjabini in experimentally infected calves. Twenty-four uninfected male Holstein calves, 1–2 weeks old, were artificially infected with Thelazia skrjabini by placing 15 third-stage larvae under the third eyelid of calves. The challenge larvae were recovered from naturally infected face flies, Musca autumnalis. The exposed calves were randomly assigned to either an ivermectin treatment group or a control within pairs ranked by weight. Equal numbers of calves were housed in each of four indoor fly-free rooms. The calves were treated with ivermectin or saline 35 days post-infection, then slaughtered 14 days later to determine eyeworm numbers. All eyes and associated tissues (including the lacrimal glands and ducts) were removed and examined for total numbers, species and viability of eyeworms. Thelazia skrjabini was found in the control group of calves only. The efficacy of ivermectin against Thelazia skrjabini was thus shown to be 100%.

Immune modulation by Ostertagia ostertagi and the effects of diet

IgG 1 antibody responses to Ostertagia ostertagi third stage larvae (L 3) and the third party antigen, keyhole limpet haemocyanin (KLH), and faecal egg counts were determined in calves infected with a single dose of O. ostertagi and in uninfected, pair-fed calves. The infected and uninfected calves were given diets either high (H) or low (L) in protein and energy. The diets were within the normal range of husbandry practice in the UK. IgG 1 antibody responses to L 3 antigen were significantly greater from 6 weeks post-infection in infected calves given the L diet than in infected calves given the H diet ( P < 0.05). The effects of diet and infection on anti-KLH IgG 1 responses were independent of each other. IgG 1 responses to KLH were decreased by infection and by the L diet compared with the H diet.

Pathophysiology of Haemonchus placei infection in calves

This study was conducted to investigate the pathophysiology of Haemonchus placei infection in Friesian calves. Seven calves were divided into two groups, three uninfected calves (control group) and four infected animals. The latter group were infected orally with 500 H. placei larvae kg −1 body weight. Five weeks after infection they were all housed in metabolic crates and infected with 125I-bovine albumin, 51Cr-red cells and 59Fe-transferrin, to study albumin metabolism, erythrokinetics and ferrokinetics. The results showed that there was a significant reduction in the mean haematocrit values and reduced weight gains in the infected calves compared with the controls. There was also a change in the distribution of albumin from the extravascular to the intravascular pool and a significant increase in the plasma and blood volumes of infected calves although the blood and albumin loss via the gastrointestinal tract recorded in this study was similar in both groups.

The vaccination and challenge with bovine viral diarrhea virus (BVDV) of calves previously infected with a non-cytopathic BVDV

Four calves were infected with noncytopathic (NCP) New York-1 strain of bovine viral diarrhea virus (BVDV). During the observation period of one month the calves remained clinically normal but the virus was repeatedly recovered from their pharyngeal swabbings and blood. Thirty days following infection the four calves were vaccinated, together with two uninfected calves, with a modified-live vaccine containing cytopathic (CP) BVDV, infectious bovine rhinotracheitis virus and parainfluenza-3 virus. No detrimental effects were observed after vaccination. Forty-three days after vaccination the calves were challenged by exposure either with the CP TVM-2 strain or the NCP New York-1 strain of BVDV. The vaccinated calves remained healthy throughout the 60-day observation period.

Decrease in Erythrocyte Survival in Theileria sergenti-Infected Calves Determined by Non-Radioactive Chromium Labelling Method.

Pathogenesis of anemia in the calves infected with Theileria sergenti was investigated from the viewpoint of erythrocyte survival decrease in the circulating blood. For investigation of erythrocyte survival a method of erythrocyte labelling with non-radioactive chromium (50Cr) was utilized. It was found that (1) the erythrocyte survival decreased markedly in the T. sergenti-infected calves compared with that in the uninfected calves; the survival rate of 25.7% for infected calves and 86.0% for uninfected ones on the fourth day after re-introduction of the labelled erythrocytes into the original donors, and that (2) the survival of non-parasitized erythrocytes in the infected calves was also decreased, which indicates no obvious relationship between parasitism and decrease in survival of erythrocytes.

Induction of protective immunity in calves immunized with adult Oesophagostomum radiatum somatic antigens

Oesophagostomum radiatum, the nodular worm of cattle, is a reverse pathogen in previously uninfected calves. However, cattle develop a strong protective immune response upon exposure to the parasite. In order to evaluate whether soluble parasite antigens could induce protective immunity, a soluble fraction was obtained from disrupted adult worms, and this fraction was used to vaccinate calves. The vaccination protocol involved two immunizations. The first was administered intramuscularly with complete Freund's adjuvant, the second was given intraperitoneally with antigen plus alum. This immunization reduced the number of worms developing from a subsequent challenge infection by 85% and also reduced clinical signs associated with infection with adult worms. However, vaccination resulted in decreased weight gains during the larval phase of the infection. Analysis of the immune response generated in the vaccinated calves indicated that protection from infection was significantly correlated with the levels of: (1) circulating parasite-specific IgG 2 antibody; (2) cellular immune reactivity as determined in a conventional parasite-specific lymphocyte proliferation assay. Serum anti- O. radiatum IgG 2 antibodies from vaccinated calves were used in immunoblots to identify the major immunogens. There were five major immunogens with molecular weights ranging from 70 to 150 kDa. Fractions separated by high-pressure liquid chromatography contained immunogens that were used to immunize calves. Vaccination with these fractions was found to impart the same level of protective immunity and induced similar IgG 2 antibody and cellular immune responses as the crude whole worm extract even with 100-fold less protein.