L.E. Hulbert

Innate immune responses of temperamental and calm cattle after transportation

The objective was to investigate measures of cellular innate immune responses among calm and temperamental Brahman bulls in response to handling and transportation. Sixteen Brahman bulls (344 ± 37 d of age; 271.6 ± 45.5 kg BW) classified as either calm (n=8) or temperamental (n=8) were loaded onto a trailer, transported for 4h to a novel facility, rested 16 h overnight, and then were returned to their original facility after a 4h transport. Blood samples were collected immediately prior to (time 0) and at 24, 48, and 96 h after initial loading for analyses of innate immune and blood parameters. Leukocyte counts did not differ (P>0.05) due to temperament before or after transportation, but neutrophil:mononuclear cell ratios were greater in temperamental bulls compared to calm bulls at 24h. At 24h, expression of peripheral neutrophil β(2)-integrin decreased among all bulls compared with 0 h (P<0.01). Temperamental bulls had greater glucose and cortisol than calm bulls (P<0.01) at 48 h; whereas calm bulls had elevated neutrophil L-selectin expression, and phagocytic and oxidative burst activity compared with temperamental bulls (P<0.10) at 48 h. The supernatant collected from endotoxin-stimulated whole blood cultures had greater TNF-α concentrations at 48 h than at the other time points (P<0.05), but no temperament effect was observed (P>0.05). In contrast, 96 h after initial loading the supernatant TNF-α concentrations were lower (P<0.05) among all cattle. Lastly, transportation increased neutrophil phagocytosis, oxidative burst, and cell adhesion molecule expression 96 h post-transportation and the effect was more pronounced among calm bulls. These data suggest that neutrophils from calm bulls are more likely to resist microbial invasion at 96 h after transportation than neutrophils from temperamental bulls.

Technical note: Development of a self-contained, indwelling rectal temperature probe for cattle research

A device was developed to monitor rectal temperature automatically in cattle for application in research settings. Compared with manual measurement of rectal temperature, this device decreases labor and time requirements and allows data collection without the influence of animal handling or restraint. The device consists of a custom-fabricated aluminum tail harness that supports an indwelling rectal temperature data logger. Materials cost approximately US

Effects of changing milk replacer feedings from twice to once daily on Holstein calf innate immune responses before and after weaning

300 per unit, and units are completely reusable. Use of this device would increase the conditions under which accurate rectal temperature measurements can be obtained in experiments with cattle.

The effects of early weaning on innate immune responses of Holstein calves1

The objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during the fourth week of life (24 ± 2.3 d of age; once-fed n=22; twice-fed n=22) on innate immune responses, and to evaluate whether carry-over effects occurred when the calves were weaned during the seventh week of life. Peripheral blood samples were taken immediately before the change in feeding strategy (24 d of age) and at 27, 31, 45, 48, 52, and 66 d of age and were analyzed for circulating cortisol, haptoglobin, total leukocyte counts, neutrophil:mononuclear cells, and hematocrit percentage. Heparinized whole blood was also stimulated with lipopolysaccharide (LPS) for 24h and the concentration of tumor necrosis factor-α (TNF-α) in the supernatant was analyzed. Neutrophil L-selectin and β(2)-integrin expression were analyzed by flow cytometry. Simultaneous neutrophil phagocytic and oxidative burst responses to a heat-killed Escherichia coli were quantified by dual-color flow-cytometry. Treatment (once-daily or twice daily feeding) had no effect on pre- or postweaning performance. Once-fed calves tended to have more circulating neutrophils at 27 d of age, greater expression of L-selectin on neutrophils at 31 and 45 d of age, and greater intensity of phagocytosis at 45 d of age. Once-fed calves secreted less TNF-α in LPS-stimulated whole blood cultures at 45 d of age compared with twice-fed calves and this tended to persist through the immediate postweaning period. None of the other immune parameters differed after weaning between the preweaning feeding strategies. Consolidating calf milk replacer into one feeding during the fourth week of life was likely a mild and acute stressor, as evidenced by transient neutrophilia in the absence of suppressed functional capacities of neutrophils. Future research should address the mechanism and immunological significance of the persistent decreased TNF-α response in once-fed calves.

Temperament influences endotoxin-induced changes in rectal temperature, sickness behavior, and plasma epinephrine concentrations in bulls.

The objectives of this study were to compare innate immune responses of calves weaned early (EW; n=23; weaned at 23.7 ± 2.3 d of age) with those of conventionally weaned calves (CW; n=22; weaned at 44.7 ± 2.3 d of age). All calves were fed 3.8L of colostrum within 12h of birth and were subsequently fed milk replacer twice daily. The weaning process began by withdrawal of the afternoon milk-replacer feeding. Milk was fully withdrawn, and the calf was considered completely weaned when it consumed 900 g of calf starter as-fed for 2 consecutive days. Blood samples were collected from all calves at 24, 27, 31, 45, 48, 52, and 66 ± 2.3 d of age. Early weaned calves took a variable amount of time to completely wean from milk replacer; therefore, data were also analyzed by comparing calves grouped by latency to completely weaned (fast=1 to 5 d; intermediate=6 to 8 d; slow=15 to 17 d). Slow-EW calves weighed less than either the fast- or intermediate-EW calves before initiating weaning. At 27 d of age, circulating neutrophils were greater among EW calves than CW calves. Moreover, fast-EW calves had lower neutrophil:mononuclear cell ratios at 45 d of age than other EW calves. Slow-EW calves had lower TNF-α concentrations from whole blood stimulated with endotoxin at 27 and 31 d of age compared with fast- and intermediate-EW calves. All EW calves had decreased neutrophil L-selectin at d 27 and increased neutrophil L-selectin at 31 d of age. At 31 d of age, neutrophil β(2)-integrin was the greatest among the fast-EW calves. All EW calves had decreased neutrophil oxidative burst at 27 and 31 d of age. Three days after CW calves were weaned they had higher neutrophils, hematocrit percentages, and circulating cortisol than EW calves. In addition, 3 d after CW calves were weaned they had decreased neutrophil oxidative burst responses to Escherichia coli. Weaning, irrespective of age, suppressed many innate immune responses. In addition, early weaning transiently suppressed L-selectin expression on neutrophils; however, the immunological significance in the context of the resistance to disease is unknown because EW calves likely had greater protection from passively derived immunoglobulins when they were weaned. Finally, calves with lower BW around 24 d of age may not be suitable for early weaning programs as evident in the suppressed secretion of TNF-α from whole blood cultures during the week following the initiation of weaning.

Administration of anesthetic and analgesic prevent the suppression of many leukocyte responses following surgical castration and physical dehorning.

This study was designed to determine the influence of temperament on endotoxin-induced changes in body temperature, sickness behavior, and stress hormone concentrations in cattle. Brahman bulls were selected based on temperament score measured 28 d prior to weaning. In dwelling recording devices were used to monitor rectal temperature, and jugular catheters were used to collect blood samples to determine cortisol and epinephrine concentrations before and after LPS administration (0.5 μg/kg body weight). Temperamental bulls had the lowest peak rectal temperature and sickness behavior scores relative to the Calm and Intermediate bulls. Prior to the administration of LPS, Temperamental bulls had greater cortisol and epinephrine concentrations than Calm or Intermediate bulls. Cortisol concentrations increased following LPS administration but were not affected by temperament. Epinephrine concentrations peaked 1 h after LPS administration in Calm bulls. Temperamental bulls did not exhibit an epinephrine response to LPS challenge. These data demonstrate that the temperament of calves can modulate the physiological, behavioral, and endocrine responses of pre-pubertal Brahman bulls to endotoxin challenge. Specifically, temperament differentially affected the rectal temperature, sickness behavior and epinephrine, but not cortisol, responses to LPS challenge.

Effects of intravenous Escherichia coli dose on the pathophysiological response of colostrum-fed Jersey calves

The objectives of the current research were to determine the physiological effects and responses of many leukocytes following surgical castration and/(or) physical dehorning and the influence of anesthetics and analgesics in 3-month-old calves. Eighty 3-month-old Holstein bull calves were completely randomized to treatments in a 2 × 2 × 2 factorial arrangement with castration, dehorning, and anesthetic/analgesic as the main effects. Peripheral blood samples were collected just before (0) and 0.5, 1.5, 2.5, 4, 6, 24, and 72 h after the respective procedure(s) and analyzed for total leukocyte and differential counts, as well as plasma cortisol and haptoglobin concentrations. Blood from the 0, 0.5 and 24h collections were analyzed for many ex vivo leukocyte responses. Data were analyzed using a repeated measures analysis of variance with the fixed effects of treatment, time, and the interaction of treatment × time. Pre-planned contrasts were performed to determine the effect of (1) management procedure (castration and/(or) dehorning), (2) anesthetic/analgesic, and (3) were the management procedures additive. There were treatment × time interactions (P<0.05) on plasma cortisol and haptoglobin concentrations as well as for total leukocyte and neutrophil concentrations in blood. Castration and dehorning increased cortisol concentrations and the effect of the procedures was additive (P<0.02). Dehorning alone elicited a greater (P<0.05) cortisol response than castration alone. In contrast, the leukocytosis and neutrophilia was greater (P<0.01) among castrated calves. In addition, haptoglobin concentrations at 24h after castration were elevated (P<0.01) in calves that were castrated. Both castration and dehorning suppressed (P=0.04) many leukocyte responses including the secretion of tumor necrosis factor-α when whole blood cultures were stimulated with lipopolysaccharide, surface expression of L-selectin on peripheral blood neutrophils, and the oxidative burst intensity of peripheral blood neutrophils when co-cultured with an Escherichia coli. The effects of castration and dehorning on blood leukocyte counts or any of the leukocyte responses were not additive (P>0.23). Castration and dehorning effects of plasma haptoglobin concentrations tended (P=0.10) to be additive at 72 h after the procedure(s). Prior administration of local anesthetic and a systemic analgesic attenuated (P<0.001) the cortisol response and prevented (P=0.03) the observed leukocytosis, neutrophilia, and leukocyte suppression. These data suggest that calves should be castrated and dehorned on the same day rather than spreading them out across two days and calves should be administered pain relief prior to performing either procedure.

Sexually dimorphic stress and pro-inflammatory cytokine responses to an intravenous corticotropin-releasing hormone challenge of Brahman cattle following transportation

Abstract Objectives of the present study were to characterize the dose dependency of an intravenous Escherichia coli O111:H8 challenge in colostrum-fed Jersey calves and to identify any biochemical markers indicative of septicemia. Eighteen 3-week old colostrum-fed Jersey calves were completely randomized to 1 of 6 doses of E. coli O111:H8. The challenge doses included 0, 1.5×105, 1.5×106, 1.5×107, 1.5×108, and 1.5×109 colony-forming units (CFU) given intravenously as a bolus in 5mL of sterile isotonic saline. Peripheral blood samples were collected at 0, 2, 4, 8, 12, 24, and 48h relative to the challenge for biochemical, total leukocyte count, and differential analyses. Rectal temperatures were collected via indwelling rectal temperature probes at 5-min intervals, and hourly averages calculated from 2 d prior to the challenge till 2 d after the challenge. All calves survived the 48h observation period following the challenge. Calves given 1.5×108 and 1.5×109 CFU displayed sickness behaviors (P <0.01) beginning 0.5h after the challenge and returned to that of the control calves by 6 and 32h for calves challenged with 1.5×108 and 1.5×109 CFU, respectively. There were treatment×time interactions (P <0.01) on total leukocyte counts and plasma glucose and zinc concentrations. Calves administered 1.5×108 and 1.5×109 CFU had leucopenia beginning 2h after the challenge and returning to counts similar to the control calves within 24h. Additionally, those calves were hypoglycemic from 4 to 12h after the challenge with the degree of hypoglycemia inversely related to the dose of the E. coli. All calves challenged with E. coli had decreased plasma zinc concentrations, and the magnitude was inversely proportional to the challenge dose. There were treatment×time interactions (P <0.001) on rectal temperatures following the challenge. All calves challenged with E. coli developed a febrile response, but the intensity and duration of the response were dependent on the challenge dose. These data indicate that calves intravenously challenged with 1.5×108 and 1.5×109 CFU of the E. coli O111:H8 showed immediate clinical and biochemical signs indicative of septicemia. However, calves administered 1.5×107 or less of the E. coli had febrile responses, but did not develop septicemia. Blood glucose and zinc concentrations may be dose responsive indicators that could potentially differentiate between a septicemic versus non-septicemic calf.

Innate immune responses and health of individually reared Holstein calves after placement into transition-pens 23 d after weaning.

This study was designed to characterize potential sexually dimorphic stress and immunological responses following a corticotropin-releasing hormone (CRH) challenge in beef cattle. Six female (heifers) and six male (bulls) Brahman calves (264 ± 12 d of age) were administered CRH intravenously (0.5 µg of CRH/kg body mass) after which serum concentrations of cortisol increased from 0.5 h to 4 h. From 1 h to 4 h after CRH administration, serum cortisol concentrations were greater in heifers than in bulls. In all cattle, increased serum concentrations of TNF-α, IL-6 and IFN-γ were observed from 2.5 h to 3 h after CRH, with greater concentrations of IFN-γ and IL-6 in heifers than bulls. Heifer total leukocyte counts decreased 1 h after CRH administration, while bull leukocyte counts and percent neutrophils decreased 2 h after CRH administration. Heifers had greater rectal temperatures than bulls, yet rectal temperatures did not change following administration of CRH. There was no effect of CRH administration on heart rate. However, bulls tended to have increased heart rate 2 h after CRH administration than before CRH. Heifer heart rate was greater than bulls throughout the study. These data demonstrate that acute CRH administration can elicit a pro-inflammatory response, and cattle exhibit a sexually dimorphic pro-inflammatory cytokine and cortisol response to acute CRH administration.

Technical note: Determination of preanalysis storage temperature and time allowances for ex vivo innate immune responses

Sixty-four Holstein dairy bull calves were all reared in individual calf-hutches and pens until they were randomly assigned to treatments of Grouped (pens of 3, n=36 calves) or Control (left in home hutch, n=8 calves) at age 68±2·3 d (body weight 74·9±1·5 kg). Blood was drawn at age 66, 70, 74 and 88 d for ex-vivo immunological and biochemical analyses. Calf starter intake was measured daily and individual body weights were measured at age 68, 78, and 89 (±2·3 sd) d. Grouped-calves consumed less starter (P<0·05), and weighed 6·4±1·99 kg less (P<0·05) than Control-calves by age 89 d. Group housing was a mild stressor, as evident by a transient suppression in neutrophil oxidative burst at age 70 d, but there was a lack of difference in the neutrophil:lymphocyte ratio of peripheral leucocytes and neutrophil expression of L-selectin at age 70 and 74 d. However, grouped-calves had elevated total peripheral leukocyte counts at age 70 d (P<0·05) and tended (P<0·10) to be greater at age 74 and 88 d. In addition, neutrophil phagocytosis of Escherichia coli increased (P<0·05) at age 74 d in Grouped-calves. These data indicate that moving calves into transition-pens with 3 calves per group decreases performance, but this may not be due primarily to extreme stress or disease. These data do indicate that it is important that calves have a competent immune system and any potential stressors are limited when they are moved into transition-pens because they are exposed to a wider diversity and (or) load of pathogens.