Performing Department
Medicine & Epidemology
Non Technical Summary
Depending on the source, dairy bull calves delivered to calf ranches have variable passive transfer of immunity status. Failure of passive immunity (FPI) is assumed in delivered bull calves on some calf ranches in California. Major reasons for not feeding sufficient colostrum to dairy bull calves include their relatively lower economic value compared to heifer calves, and shortage of colostrum. Whilst calves with FPI might be raised successfully, they have an increased risk for morbidity and mortality, particularly from diarrhea and bovine respiratory disease (BRD). Consequently, production of calves with FPI incur increased costs through treatments and loss from mortality. In order to encourage producers to feed dairy bull calves sufficient volumes of non-contaminated and or pasteurized colostrum, prior to delivery, calf ranches may offer monetary incentives for calves with adequate immunity, on arrival. However, the incentives are based on test results from serum total protein (STP) determination, an on-farm test, which is variable when calves are dehydrated. Sick calves are more likely to be dehydrated and will have relatively high STP from hemoconcentration, without necessarily having adequate passive transfer of immunity. Thus, calf ranches might end up paying more for sick calves, if monetary incentives to producers are based on STP determination alone. Dairy calves diagnosed with diarrhea or BRD may become septic (presence of bacteria and or endotoxin in blood) thereby negatively altering prognosis for survival. In clinic settings determination of bacteremia through bacteriological culture and Complete Blood Count (CBC) are 2 methods used to evaluate sepsis. Determination of bacteremia and CBC evaluation are impractical and expensive methods for on-farm use to evaluate sepsis in calves. In contrast, qualitative determination of presence of endotoxin in blood is a practical on-farm or clinical settings method to detect calves with endotoxemia. To the investigators' knowledge, qualitative endotoxin determination has not been reported in dairy calves. The purpose of this proposal is to evaluate the on-farm utility of a qualitative test for detection of endotoxin in dairy calves. The test fits the criteria as determined by the FDA for endotoxin detection.Justification: Currently, monetary incentives for delivering dairy bull calves with adequate transfer of immunity to calf ranches are based in part by determination of STP. The STP levels consistent with adequate passive immunity in sick calves is ≥ 5.5 g/dL. Because the correlation between STP and serum immunoglobulin concentrations (reference laboratory method) varies, a significant number of sick calves have high STP (>6.0g/dL) but with serum immunoglobulin concentrations consistent with FPI (< 1000mg/dL). Thus, a more objective, practical, method is required to assess sick calves with higher STP. Because sick neonatal calves are likely to become septic, determination of endotoxin is a practical, on-farm method to assess such calves. Therefore, calf ranches may assess for endotoxemia in sick calves with high STP, which are delivered to their premises. Calves with endotoxemia have a guarded prognosis with or without treatment, thus the endotoxin test results might be useful in making specific treatment decisions. The information would also enable calf ranches to detect patterns in the origin of calves diagnosed with sepsis, and recommend colostrum management practices aimed at reducing the incidence of septicemia. Such recommendations may include feeding sufficient volume of non-contaminated and or pasteurized colostrum. Furthermore, the results of this proposal will be applicable to clinical settings for admitted calf patients.
Animal Health Component
100%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Goals / Objectives
Research hypothesis: Endotoxin determination using an on-farm qualitative test will differentiate sick septic calves from sick non-septic calves during the first 14 days of life.Objectives:a. Determine the presence or absence of blood endotoxin in healthy, sick non-septic (based on low clinical septic scores), and sick septic (based on high clinical septic scores) calves.b. Determine the association between detection of endotoxin and changes on CBC.c. Determine the mortality rates between healthy calves, sick non-septic calves, and sick septic calves.
Project Methods
Research hypothesis: Endotoxin determination using an on-farm qualitative test will differentiate sick septic calves from sick non-septic calves during the first 14 days of life.Objectives:a. Determine the presence or absence of blood endotoxin in healthy, sick non-septic (based on low clinical septic scores), and sick septic (based on high clinical septic scores) calves.b. Determine the association between detection of endotoxin and changes on CBC.c. Determine the mortality rates between healthy calves, sick non-septic calves, and sick septic calves.Project details:a. Design: This is a prospective cohort study, with sample size calculated based on a power of 80%, α = 5 %, an estimated 10 % incidence of endotoxemia in sick calves, and an assumption that sick septic calves are 3 times more likely to test positive for presence of endotoxin compared to sick non-septic calves. The minimal sample required per groups was 59 calves. To account for a 20 % dropout from mortality prior to weaning or missed records, 71 calves/group (213 total calves) will be enrolled. A calf ranch in Kings County, California, has been identified as the farm of study. The ranch processes 400 - 500, 2-3 day old calves, delivered to the farm, per day. The ranch has a calf morbidity prevalence of 15 - 20 % during the first 14 days of life. The farm assesses passive immunity status of calves using serum total protein (STP) determination. Calves will be divided into 3 groups on delivery to the ranch based on clinical examination by one of the investigators into; healthy (control), sick non-septic (negative control) and sick septic. Presence of sepsis on clinical examination will be based on a previously described clinical sepsis scoring system. Serum will be collected for STP and serum immunoglobulin G (IgG) by refractometry and radial immunodiffusion, respectively. Serum endotoxin determination will be performed using a qualitative, commercial gel clot assay kit, with a minimum endotoxin detectable concentration of 0.25 EU/ml, according to the manufacturer's recommendations. Briefly, lyophilized limulus amebocyte lysate (LAL) will be reconstituted with LAL reagent water. Following reconstitution, 0.1ml of test serum sample will be mixed with 0.1 ml of LAL in an endotoxin free vial. The mixture will be incubated at 370C in a water bath or oven for 60 ± 2minutes. The negative control for the test will be the LAL reagent water, whereas the positive control will be an E. coli endotoxin standard, supplied with the kit. Presence of endotoxin will be characterized by formation of a gel clot that is stable when the vial is inverted, whereas absence of endotoxin will be characterized by the absence of the gel clot when the vial is inverted. To assess the relationship between the presence of endotoxin and CBC findings (leukopenia, elevated fibrinogen, presence of bands, and toxic changes) randomly selected samples (35 samples/group) will be submitted for CBC evaluation at UC Davis. Mortality up to weaning (80 days) among the 3 groups will be recorded.b. Data analysis: Prevalence of endotoxemia and FPI among the 3 groups will be calculated. Differences in proportions of calves with endotoxin detected among the 3 groups will be assessed by Chi-square or Fisher's exact tests. Association between sepsis score (risk factor) and subsequent detection of endotoxin (outcome) will determined by calculation of risk ratio. Association between presence of endotoxin (risk) and CBC changes (outcome) will be determined using risk ratios. Conditional logistic regression will be used to determine the probability of a calf having endotoxin detected (present or absent) as a function of group, serum IgG concentration, age, medical treatment, and cause of morbidity at the time of clinical examination. A Cox proportional hazard model will be used for multivariate analysis with age at detection of endotoxin, serum IgG concentrations, group, medical treatment, and cause of morbidity considered as exposures. Entry into the study will be at delivery (2-3 days old), weaning as the exit point, and the outcome of interest will be death. The endotoxin test is not the reference method for detecting endotoxin, therefore the sensitivity and specificity of the test, will be determined using maximum likelihood estimation.