Comparison of cytokine mRNA expression in peripheral CD4+, CD8+ and γδ T cells between healthy Holstein and Japanese Black calves

Japanese Black (JB) calves are more susceptible to infectious diseases compared to Holstein (Hol) calves. To clarify the immunological differences between JB and Hol calves, expression of cytokine messenger RNA (mRNA) was examined using peripheral CD4⁺, CD8⁺ and γδ T cells. Healthy calves, 24 from each species, were examined. Blood samples were obtained from calves at 1 week, 1 month and 3 months old, eight calves for each age of each species. Peripheral blood mononuclear cells were stimulated with phytohemagglutin (PHA), and T cell subsets were isolated by positive selection using magnetic cell sorting (MACS). Levels of interlekin (IL)‐2, IL‐4, IL‐10 and interferon (IFN)‐γ mRNA in three T cell subsets were analyzed. WC1‐N1⁺ γδ T cell percentages were significantly lower in JB calves at 1 week and 1 month of age compared to Hol calves. In addition JB calves had significantly lower IL‐2, IL‐10 and IFN‐γ mRNA in WC1‐N1⁺ γδ T cells at 1 and 3 months of age, whereas there were no significant differences in cytokine mRNA of CD4⁺ and CD8⁺ cells between the two groups. Decreased cytokine mRNA and cell number of peripheral γδ T cells may affect negatively on the immune system of JB calves.     

Relationship between diarrhea and peripheral leukocyte population in neonatal Japanese black calves

Neonatal Japanese Black (JB) calves show a high incidence of diarrhea. The objective of this study was to analyze the immune cell populations of neonatal JB calves in detail and examine its correlation with the incidence of diarrhea immediately after birth. Understanding the immune cell populations is helpful in clinics in order to determine the condition of the immune system for prevention of diseases. Blood samples were obtained from JB calves on the day of birth. The peripheral leukocyte populations were analyzed separately for calves that had diarrhea within 2 weeks after birth (diarrhea group; n = 26) and for calves without diarrhea (control group; n = 74). The numbers of the peripheral blood CD3(+)TcR1-N12(+) and CD8(+) T cells were significantly lower in the diarrhea group compared with the control group. These findings suggest that the congenital lower peripheral γδ and CD8(+) T cells results in a high risk of diarrhea in neonatal JB calves.     

Transient detection of proinflammatory cytokines in sera of colostrum-fed newborn calves

To obtain basic information on the state of proinflammatory cytokines in newborn calves, we determined the kinetics of 5 cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist) in sera of newborns during the first 4 weeks of life. At birth, none of the 5 cytokines were detected in almost all serum samples, but the cytokines became detectable within 12 hr after being fed colostram. The mean concentrations of the cytokines reached peak levels by 24 hr and then gradually decreased and became undetectable by 4 weeks after birth. Cytokine mRNA expressions in peripheral blood mononuclear cells of newborns were observed without reference to the cytokine concentrations in sera. Serum cytokines detected in newborn calves are probably colostral origin.     

Age-related alterations in trimethoprim-sulfadiazine disposition following oral or parenteral administration in calves.

Age-related changes in the absorption and distribution patterns of trimethoprim/sulfadiazine were studied following oral or subcutaneous administration of 15 mg/kg of the drug combination in calves. Following oral administration, the time course of trimethoprim/sulfadiazine appearance and dissipation in serum, synovial fluid and urine was followed for periods up to 48 hours in calves one day, one week and six weeks of age. The profiles of drug appearance-disappearance in these body fluids were also determined after subcutaneous administration in seven week old calves. The peak serum and synovial fluid levels of trimethoprim/sulfadiazine achieved following oral administration were substantially lower with increasing maturation. In ruminating (six and seven week old) calves, subcutaneous or oral administration of the combination led to high serum levels of sulfadiazine but little or no serum trimethoprim was detected in animals at this age. The data indicate that, while therapeutic concentrations and optimum ratios of the drugs may be achieved for extended time periods in neonatal life, this dosage is unable to produce optimum serum and synovial fluid concentrations as the calves mature.     

Effect of nutritional conditions on changes in leukocyte populations in Japanese black calves

To clarify the effect of nutritive conditions on changes in immune cells in Japanese Black (JB) calves during the growth period, leukocyte populations were analyzed in ten healthy JB calves managed in one herd. The calves were divided into two groups: five calves in Group 1 were given insufficient nutrition, and the other five calves in Group 2 received adequate nutrition. The levels of serum total cholesterol and glucose were significantly lower in Group 1 than in Group 2 at 1 month. The numbers of CD3+, CD4+ and CD8+ cells tended to be lower in Group 1 than in Group 2 at months 1 and 2, and the difference in CD4+ was significant at month 2. The number of MHC class-II(+high) cells was significantly lower in Group 1 than in Group 2 at months 1 and 2. These results suggest that adequate nutrition might stimulate an increase in immune cells in calves during the growth period.     

Differential cytokine gene expression in CD4+ and CD8+ T cell subsets of calves

The distinct patterns of cytokine expression in CD4+ and CD8+ T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4+ and CD8+ T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4+ and CD8+ T cells of calves with or without Concanavalin A (Con A) stimulation. CD4+ and CD8+ T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4+ T cells spontaneously expressed the mRNAs of interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta, and augmented the mRNA expression of IL-10, IFN-gamma and TNF-beta after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4+ T cells. CD8+ T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-alpha, TNF-beta and TGF-beta, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-gamma) and GM-CSF after Con A stimulation. It was found that CD4+ T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8+ T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4+ and CD8+ T cells of calves, indicating that CD4+ T cells can be distinguished from CD8+ T cells by the cytokine gene expression of IL-1alpha, IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4+ and CD8+ T cells serve to interpret an individual function of T cell subsets in the immune system of calves.     

Changes in peripheral leukocyte populations of weak calf syndrome of Japanese black calves

To clarify the cellular immune condition in Japanese Black (JB) calves with a weak syndrome, peripheral leukocyte populations were analyzed by flow cytometry. Twenty JB calves were divided into two groups based on clinical observations; one group of calves was weak, because they had experienced an onset of diarrhea within 3 days after birth and needed treatment (Group 1 ;n=10), and the other group of calves was healthy (Group 2; n=10). With regard to leukocyte populations, CD8(+) cells and gamma delta T cells in Group 1 were markedly lower than those in Group 2 during the experimental periods. It is possible that immune-insufficiency might be based on T lymphocyte function in weak syndrome JB calves during the growth process.     

Comparison of the peripheral blood leukocyte population between Japanese Black and Holstein calves

Japanese black (JB) calves have greater susceptibility to infectious diseases compared to Holstein (Hol) calves. In order to clarify the differences in cellular immune status between JB and Hol calves, the leukocyte population and lymphocyte proliferative ability were analyzed. In total 200 healthy calves, 1 day to 14 weeks of age, were examined: 105 JB and 95 Hol calves. Lower numbers in peripheral blood and percentage in peripheral blood mononuclear cells of CD3⁺TcR1‐N12⁺ T cells and major histocompatibility complex class‐II⁺CD14^‐ B cells were observed in the JB compared to the Hol. The percentage of TcR1‐N12⁺CD25⁺ T cell in the JB was significantly lower than that of the Hol at 4–6, and 8–10 weeks. Interleukin (IL)‐2 sensitivity in the JB was lower than that in the Hol, and significant differences were observed in age groups of 6–8 weeks and 10–14 weeks. These findings indicated that the lower numbers of γδ T cells and B cells in the JB compared to the Hol might be associated with the specificity of the immune systems in JB calves.     

Phenotypical characterization of peripheral blood leucocytes in the newborn calf

The present study was undertaken to establish reference values for the composition of blood leucocyte populations in neonatal calves by differential leucocyte counts and immunophenotyping. Neonatal calves 1 h post partum (p.p.) were found to have a very high absolute number of granulocytes while the number of peripheral blood mononuclear cells was lower than in calves aged 3-9 weeks. The relative numbers of T cell subpopulations were similar in newborn and older calves, but newborn calves had lower percentages of B cells and MHC class II positive cells. Within the first 4 h of life the relative numbers of CD2+, CD6+, and CD8+ T cells declined in colostrum-fed as well as in colostrum-deprived calves. In contrast, the percentage of MHC class II positive cells and monocytes increased from 1 h to 4 h p.p. particularly in colostrum-fed calves. Although there is some evidence for immaturity of lymphocytes in neonatal calves, the immune system of these animals seems to be fully present at birth.     

Comparison of distal forelimb conformations between Japanese Black and Holstein-Friesian newborn calves

Flexural and hyperextension deformities are congenital problems in calves. We, therefore, aimed to investigate the distal limb conformation in 1 day- and 28-day-old female Holstein-Friesian (HF) calves (n=21), male Japanese Black (JB) calves (n=15), and female JB calves (n=15). The claw angle of the forelimb dorsal claw wall in a standing position and recorded other parameters, including body weight, withers height, circumference of forelimbs, and flexor tendon thickness in the forelimbs, were measured and compared these between the three groups. At 1 day old, the mean claw angles were 51.1° in female HF calves, 47.0° in male JB calves, and 41.8° in female JB calves; the 95% confidence intervals (CIs) of the claw angles showed large distributions in all three groups. One female HF and one male JB calves showed mild flexural deformity, whereas four JB calves showed hyperextension deformity. At 28 days old, the mean claw angles were 51.7° in female HF calves, 51.2° in male JB calves, and 48.4° in female JB calves; the 95% CIs of the claw angles showed smaller distributions than those at 1 day old in all groups. For all groups, the limb deformities had improved without treatment at 28 days old. As a feature of the breed, female JB calves were apt to show hyperextended deformities inversely proportional to the body weight. These limb deformities healed spontaneously and were thought to be physiological.     

Toll-like receptor, MHC II, B7 and cytokine expression by porcine monocytes and monocyte-derived dendritic cells in response to microbial pathogen-associated molecular patterns

To evaluate effects of treatment with pathogen-associated molecular patterns (PAMPs) on toll-like receptor (TLR), MHC II, B7 and cytokine expression, pig monocytes and monocyte-derived DCs (moDCs) were treated with LPS, CpG, lipoteichoic acid (LTA), poly IC or peptidoglycan (Pep). Monocytes and moDCs treated with LPS, CpG, LTA, poly IC or Pep altered expression of at least one TLR (4, 5 and 9) and up-regulated MHC II and/or B7. The mRNA for IL-4 was not detected after any treatment. Treatment with LPS or LTA tended to up-regulate mRNA for TLR 4, Th-1 (IFN-gamma and IL-12p35) and Th-2 cytokines (IL-10 and IL-13). Poly IC or CpG tended to up-regulate TLR 9 and Th-1 cytokines. Porcine monocytes and moDCs like those of humans and mice responded to microbial PAMPs by altering TLR expression, up-regulating MHC II and B7 and altering cytokine expression toward Th-1 and/or Th-2, which may steer immune response. Hence, porcine moDCs and monocytes are likely able to discriminate between microorganisms using TLRs which determine cytokine expression and immune response bias.     

The effect of intermittent treatment with sulphadimidine on coccidiosis in preruminant calves

Preruminant calves excreted coccidia oocysts in their faeces after 3 weeks of group housing. Two weeks of oral sulphadimidine (SDM) administration, 50 mg/kg on the first day of treatment followed by daily administration of 37.5 mg/kg, under the same housing conditions kept the faeces free of oocysts. Three weeks later, these calves excreted oocysts again. Repetition of the same treatment for 2 weeks controlled the infection again, but a second treatment for 5 days did not suffice. The repeated long treatment affected immunoglobulin levels adversely. SDM given repeatedly at a lower dose rate (30 mg/kg) for 1-week periods with medication-free intervals of 1 week controlled the infection and no adverse effects were noted. In comparison with controls, weight gains were greater in treated calves.     

Variations in the immune response to natural Schistosoma mattheei infections in calves born to infected mothers

During previous work Schistosoma antibodies and circulating antigens were detected at birth in the serum from some calves born to Schistosoma mattheei infected mothers. The objectives of the present survey were: (1) to investigate the proportion of calves, born to cows infected with S. mattheei, which have specific antibodies and circulating schistosome antigens present in their serum at birth and (2) to investigate whether the presence or absence of these specific antibodies and/or circulating antigens at birth may affect the pattern of a natural S. mattheei infection in calves from 4 to 5 months of age, when the colostral antibodies are thought to be of negligible importance. A total of 28 calves born to infected mothers were randomly selected. Faeces, serum and colostrum samples were collected from the cows at calving, serum samples were collected from the calves at birth (day 0), after intake of colostrum (day 1) and monthly thereafter up to the age of 10 months. Both serum and colostrum samples were analysed for IgG(H+L) against SWAP mattheei and schistosome circulating anodic antigen (CAA) levels. The calves were exposed to a natural challenge from the age of 4-5 months. Faecal samples were collected from the calves monthly, starting at an age of 5 months up to 10 months, and were examined for faecal egg counts. Nine (group 1) out of the 28 calves were found to have specific antibodies in their serum at birth, in 5 of them CAA levels were also detected. In the other 19 calves (group 2) no IgG(H+L) or CAA were detected. At the end of the study faecal egg counts and CAA levels were significantly lower in calves from group 1 compared to group 2. Results confirm earlier work that specific antibodies and circulating antigens may be present in serum from calves at birth, and show that these calves have lower faecal egg counts and CAA levels after exposure to a natural challenge.     

Blood picture of calves from primiparae kept under large-scale production conditions in the early postnatal period]

Twelve calves of the Red Spotted breed and its crossbreds born by first-calvers, kept in a large-capacity stable of a dairy farm, were subject to the study of the ontogenetic development of the values of the blood picture from birth up to the 10th day of age. The erythrocyte and leucocyte count, the packed cell volume and the relative and absolute number of neutrophils reached the significantly highest levels in calves before drinking the colostrum. The amount of haemoglobin, MCV and MCHC reached the highest values on the first day after birth. The values of the red blood picture decreased in the first week of life and reached the minimum on the tenth day. The leucocyte count decreased during the first to second day after birth, the decrease being due to a drop of the absolute number of neutrophils, and on the tenth day the leucocyte count returned to its initial value. Neutrophils prevailed in the peripheral blood of new-born calves whereas lymphocytes prevailed in the peripheral blood from the fourth day of life on. The amount of cosinophiles in the peripheral blood of the calves was about 0.5% up to the fourth day; on the seventh day it exceeded 1%. The relative number of monocytes was at its minimum level just after birth and showed a significant increase during the second to fourth day.     

Oral administration of IL-1 beta enhanced the proliferation of lymphocytes and the O(2)(-) production of neutrophil in newborn calf

Recently, we demonstrated the presence of IL-1 beta in the colostral whey from dairy cows. Here, authors examined oral transmission of colostral IL-1 beta and its immunological effects on the neonatal calves. Biotin-labeled recombinant bovine (rb) IL-1 beta was administered orally to newborn calves and monitored in the serum. The results disclosed the passive transfer of colostral cytokines via the oral route, and a potent increase in white blood cell (WBC) count was observed in all calves administered with rbIL-1 beta. Oral administration of IL-1 beta significantly increased the proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A, and the O(2)(-) production of stimulates neutrophils in newborn calves. These results suggest that the oral administration of IL-1 beta has an immunostimulatory activity in the newborn calf.     

The influence of colostral leukocytes on the immune system of the neonatal calf. II. Effects on passive and active immunization

The influence of colostral leukocytes on the concentration of immunoglobulins and antibodies against an enterotoxigenic strain of E. coli in the sera of newborn calves was investigated for four weeks using four experimental groups. The calves received either complete colostrum (COL-, n = 16), cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during the first three days of life. The cows were not specifically immunized. The sera of the COL+ calves had significantly higher concentrations of antibodies against E. coli mainly of IgG1 specificity on the second day of life as compared to those of the COL-. The sera of the COL+ calves contained significantly more IgM on days 2 and 5 and slightly more IgA during the first week. Both COL groups had equal concentrations of serum IgG. It appears that colostral leukocytes which are an integral part of the colostrum enhance the passive immunity of the neonatal calf, especially in regard of antibodies and immunoglobulin classes which are essential for intestinal immunity. The concentration of IgM in the sera of the MS+ calves was reduced, that of IgG did not rise to appreciable amounts; the IgA synthesis started one week later as compared to the MS- group. The administration of isolated colostral cells led to an impairment of the natural active immunization.     

Effect of bovine lactoferrin feeding on lipopolysaccharide-induced metabolic and hormonal disturbances in preruminant calves

One of the biological functions of bovine lactoferrin (LF) is modulation of the host defense system, including cytokine production and immune response. The aim of the present study was to investigate the effect of oral administration of LF in calves on lipopolysaccharide (LPS)‐induced metabolic and hormonal changes in inflammatory response. Thirty Holstein calves at 4 day of age were given one of three oral doses of LF (0, 1, 3 g/day) for 10 days (?10 day to ?1 day). They were injected i.v. with LPS (50 ng/kg bodyweight) the day (day 0) after the end of LF treatment. Plasma samples were obtained on ?10, 0 day (immediately before LPS injection), and at 2, 6, 12, 24, 48, 72, and 96 h after LPS injection. Plasma tumor necrosis factor‐α concentrations at 2 h after LPS treatment were lower (P < 0.05) in LF 1 g/day‐fed claves compared with LF 0 g/day (control) calves. On day 0 there were no significant group differences in plasma LF concentration. Plasma concentration of haptoglobin in control calves was elevated by LPS injection. In LF groups, plasma haptoglobin concentrations slightly increased after LPS injection, but those levels at 6–24 h were lower (P < 0.05) than in the control group. The LF treatment inhibited (P < 0.05) the reduction of plasma ferrin concentration in calves following LPS challenge. The concentration of plasma aspartate aminotransferase in calves treated with LF was lower (P < 0.05) than in control calves at 24–96 h after LPS treatment. The concentration of plasma insulin‐like growth factor‐1 (IGF‐1) in all groups was decreased by LPS treatment, while in the LF groups the IGF‐1 level was higher (P < 0.05) than in the control group. Plasma adrenocorticotropic hormone and insulin concentrations in LF groups were lower (P < 0.05) than in control calves at 2 h after LPS injection. These data suggest that LF has a substantial anti‐inflammatory effect on the modulation of the host defense system in preruminant calves.     

Pathological and immunological changes after challenge infection with Pasteurella multocida in naive and immunized calves

Challenge infections of calves with Pasteurella multocida were established to characterize the local inflammatory response and determine the effect of previous exposure to live bacteria on the post-challenge immune response. Experimental infections were established by intratracheal inoculation of P. multocida in both naive calves and calves that had been previously vaccinated with two subcutaneous (s.c.) injections of live bacteria. Histological, immunohistological and cytokine expression studies were performed on bronchoalveolar lavage (BAL) samples, lung parenchymal tissues and lung lymph nodes (LN). In comparison to uninfected control animals in which no lung lesions were observed, a patchy to confluent bronchopneumonia was observed following infection of naive calves, characterized by abscess formation, haemorrhage, oedema and suppurative consolidation. Cellular analysis following infection of naive animals was characterized by an influx of neutrophils in the BAL, with macrophages and dendritic cells observed in the lesion perimeter. A significant increase in the number of CD8(+) blasts expressing MHC (major histocompatibility) II was also observed in the BAL of infected calves. Decreased expression of interleukin (IL)-1 beta and increased expression of IL-8 compared to naive unchallenged controls was apparent in lung LN. In comparison, a more limited pathology was observed in vaccinated animals post-challenge, indicating partial protection conferred by the s.c. immunization with live bacteria. Studies of vaccinated animals showed the presence of bronchial-associated lymphoid tissue (BALT) in the lung tissue and an increase in the number of B-cells and CD4(+) T-cells expressing MHCII in the lung LN after challenge. In contrast to primary infection, there was no significant influx of neutrophils in the BAL. Instead, a population of newly recruited monocytes/macrophages was observed. Increased IL-2 expression and decreased IL-8 expression was observed in the LN, while IL-1 beta expression was not detected. The reduced neutrophil and increase monocyte response in the vaccinated calves may be associated with significant changes in the gamma delta T lymphocyte population in the BAL.     

Treatment of Neonatal Calf Diarrhea with an Oral Electrolyte Solution Supplemented with Psyllium Mucilloid

Dairy calves under 14 days of age with naturally occurring, uncomplicated diarrhea were treated for 3 days with a hypertonic oral electrolyte solution with (n = 15) or without (n = 12) psyllium. Clinical response and clinical pathology data were compared between the 2 groups. Glucose absorption was evaluated on days 1 and 3 by measurement of plasma glucose and lactate and serum insulin concentrations for 4 hours after formula administration. On day 1, glucose, lactate, and insulin concentrations were lower in psyllium-fed calves than in control calves, with significant differences noted in glucose and lactate concentrations at several time points ( P < 0.05). Plasma lactate concentrations were higher at several times in both treatment groups on day 3 than on day 1 ( P < 0.05). Fecal consistency was markedly different in psyllium-fed calves as compared with control calves within 24 hours of psyllium supplementation. Fecal percent dry matter content was lower in psyllium-fed calves than in control calves at least once a day during supplementation and on day 3 compared with day 0 in the psyllium-fed calves ( P < 0.05). There were no significant differences in clinical performance scores, hydration status, arterial blood gas, serum anion gap, electrolyte, or total CO, concentrations. Addition of psyllium to an oral electrolyte solution resulted in immediate alterations in glucose absorption without impairing rehydration in diarrheic calves, but differences were transient and did not affect clinical outcome.