Performing Department
College of Vet Medicine
Non Technical Summary
Mortality in young dairy calves exceeds 10% (USDA 2010), and historical losses are worse in warmer climates such as the Southeast (rref 3). A South Carolina study showed dairy calf mortality from birth to 3 months or birth to 1 year ranged from 6.5 to 22.0% (Jenny, 1981). Heifer health, including sickness and death, was reported as the top challenge rated very important by 92.5% of dairy calf-raisers in a recent study (USDA, 2011). Yet only half of dairy calf-raising operations reported testing heifers for any disease during 2010 (USDA, 2011). Producers need low-cost, on-farm tests that give near immediate results that can be used to dictate treatment (personal interviews). Such tests do not currently exist. The testing used is typically specific to a particular disease and requires submission to central labs that take days or weeks to provide results.The blood leukocyte differential (BLD) could meet producer needs for testing. Blood leukocytes, or white blood cells, play a crucial role in defense against infection (Blumenreich). The total number and percentages of leukocyte types change at the onset of and different stages of disease (Berend 2001). Thus, the leukocyte differential has become widely accepted in human hematology and it is commonly used for screening, case finding, diagnosis, and monitoring of hematologic and nonhematologic disorders (Berend 2001). The number and type of cells not only indicate the presence of infection or disease but also differentiate between bacterial, viral and parasitic infections. Similarly, in companion animals the differential is a useful triage tool that improves the rapid diagnosis and care of pets (Idexx training tool). Such a tool would be useful in calf management to reduce mortality and morbidity, improve overall animal health, improve producer profitability and minimize the opportunity for antibiotic resistance.Technology from Advanced Animal Diagnostics (AAD) is well-suited to provide a rapid BLD test that can identify the presence of a disease or infection and indicate the causative agent (bacteria, virus or parasite) in less than one minute from a single drop of blood. Given the limited number of therapeutics available, accurate treatment decisions can be made by confirming the presence of disease and identifying the causative agent. AAD has developed similar technology to perform a leukocyte differential in milk. The company is preparing for commercial sales of a reader instrument that acts as a rapid robotic cytologist. With different software customized for blood, the company could use the existing system to produce a BLD.The College of Veterinary Medicine, North Carolina State University, and AAD intend to collaborate on a project to develop the framework for using total and differential leukocyte counts from calf blood. The intended field use is the development of protocols to use the technology to effectively improve health management of calves, such as to identify calves with specific disease or to indicate whether antibiotic treatment is warranted. Technical objectives include: Step 1: Longitudinal BLD Values for Healthy Calves: Determine normal values for dairy calves by age (up to 6 months) using AAD's BLD. Differentials change over time as the immune system develops, so normal ranges by age must be determined first to eventually interpret "abnormal." Step 2: BLD Values for Key Calf Diseases: Determine the BLD patterns (totals and percentages) for dairy calves to be used in the detection of three serious calf diseases:Septicemia, the life-threatening presence of bacteria in the blood stream,Pneumonia ,an infection of the calf's lungs, andDiarrhea, the most common health problem for young calves.For pneumonia and diarrhea, patterns for infections caused by both bacteria and viruses will be identified so the producer can understand whether antibiotic treatment is warranted.AAD has the development, manufacturing and dairy sales and customer support infrastructure to quickly leverage the results of this research into a successful commercial product.
Animal Health Component
100%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
Mortality in young dairy calves exceeds 10% (USDA 2010), and historical losses are worse in warmer climates such as the Southeast (rref 3). A South Carolina study showed dairy calf mortality from birth to 3 months or birth to 1 year ranged from 6.5 to 22.0% (Jenny, 1981). Heifer health, including sickness and death, was reported as the top challenge rated very important by 92.5% of dairy calf-raisers in a recent study (USDA, 2011). Yet only half of dairy calf-raising operations reported testing heifers for any disease during 2010 (USDA, 2011). Producers need low-cost, on-farm tests that give near immediate results that can be used to dictate treatment (personal interviews). Such tests do not currently exist. The testing used is typically specific to a particular disease and requires submission to central labs that take days or weeks to provide results.The blood leukocyte differential (BLD) could meet producer needs for testing. Blood leukocytes, or white blood cells, play a crucial role in defense against infection (Blumenreich). The total number and percentages of leukocyte types change at the onset of and different stages of disease (Berend 2001). Thus, the leukocyte differential has become widely accepted in human hematology and it is commonly used for screening, case finding, diagnosis, and monitoring of hematologic and nonhematologic disorders (Berend 2001). The number and type of cells not only indicate the presence of infection or disease but also differentiate between bacterial, viral and parasitic infections. Similarly, in companion animals the differential is a useful triage tool that improves the rapid diagnosis and care of pets (Idexx training tool). Such a tool would be useful in calf management to reduce mortality and morbidity, improve overall animal health, improve producer profitability and minimize the opportunity for antibiotic resistance.
Project Methods
TASK 1: AAD System ModificationsThe AAD system is currently configured to perform a milk leukocyte differential. To develop the BLD, the following modifications must be made:Optimize stain formulation for calf blood. AAD staff have already completed significant research with various stain formulations in human and cow blood.To finalize selection of a stain, calf blood will be collected from 10 animals determined to be healthy by physical examination and tested at AAD in combination with a variety of stain formulations.AAD cytologists and software engineers will evaluate images collected from each sample to select the stain best suited to work for blood in the reader instrument marketed by AAD.Modify reader software to confirm the five-part differential on calf blood reported by the AAD reader correlates well with manual differentials.Calf blood will be collected from 30 animals determined to be healthy by physical examination and of varying ages. Images will be captured using the AAD reader and stain identified in A2 above.Cells will be manually identified by type, and the resulting images will be used to developing a cell recognition and identification algorithm for the five cell types in blood.To test the accuracy of the software vs. manual differentials, calf blood will be collected from an additional 30 animals of varying ages. Each sample will be run through the AAD reader, which will provide a five-part differential count. In addition, a manual wet stain smear will be made from each sample. Manual cytology will be performed on the smears and results will be compared to the AAD differential.TASK 2: Establish normal ranges of leukocyte differential patterns for different ages of calves from newborn to six months.Calf blood will be collected from the jugular vein using standard techniques from 10 different calves all determined to be healthy by physical examination at each of 5 farms in 3 regions of the country recommended by their veterinarian and judged to have an adequate calf management system. Samples will be collected every day for the first week of life. (10 X 5 X 3 = 150 samples collected daily).Calf blood will be collected from 10 different calves all determined to be healthy by physical examination at each of 5 farms in 3 regions of the country recommended by their veterinarian and judged to have an adequate calf management system. Samples will be collected every week for the first month of life.Calf blood will be collected from 10 different calves all determined to be healthy by physical examination at each of 5 farms in 3 regions of the country recommended by their veterinarian and judged to have an adequate calf management system. Samples will be collected monthly for the first 5 months of life.Farm health and testing records and the above will be analyzed. Any calves determined to be infected by routine testing will be evaluated separately. BLDs from healthy animals will be evaluated to establish the blood leukocyte differential values for healthy calves.TASK 3: Determine the specific leukocyte differential patterns associated with septicemia.Fifty calves suspected of septicemia will be identified, examined by a veterinarian, and scored for clinical sepsis based on hydration, fecal appearance, attitude, appearance of scleral vessels and umbilical abnormality (Fecteau). Blood will be aseptically collected from the clipped jugular vein of calves into blood culture tubes tube that contain sodium polyanetholesulfonate (SPS) and then subjected to routine blood culture. Isolating a pure culture of bacteria and Fecteau's clinical sepsis scoring system will confirm the diagnosis of sepsis.BLDs will be compared with blood culture results to determine the BLD ranges indicative of septicemia.TASK 4: Determine the ability of leukocyte differential patterns to differentiate between bacterial and viral pneumoniaOne hundred calves suspected of pneumonia will be identified, examined by a veterinarian, blood will be drawn from the jugular vein using standard techniques, an earnotch sample will be collected and methods will be used to isolate organisms from the lower respiratory tract (e.g., transtracheal wash procedure). Samples will be submitted for bacterial and viral testing and the BLD will be performed on AAD reader.BLDs will be evaluated from calves confirmed to have pneumonia and compared to any not diagnosed with pneumonia. Further, those with viral pneumonia will be evaluated to see if the BLD differs from those with bacteria pneumonia. BLD patterns indicative of each will be developed.TASK 5: Determine the ability of leukocyte differential patterns to identify the organism associated with diarrhea (scours).From at least 5 farms, a total of 100 calves with diarrhea will be identified. Diarrhea will be defined as feces of a fluid consistency not considered normal. Pictures of feces categorized as "diarrheic" will be provided and the feces of the calf at trial entrance will be photographed and kept on file. Each calf will be examined by a veterinarian, and standard physical examination data will be recorded. Blood will be drawn for the BLD from the jugular vein using standard techniques. Feces will be collected for culture (Escherichia coli, Salmonella spp.), analysis of Cryptosporidium spp. by appropriate methodology, tested for viral causes of diarrhea (Rotavirus, Coronavirus), and tested for the presence of coccidian and other parasites. The cause of scours will be classified as bacterial, cryptosporidial, viral, and coccidiosis or other parasites.BLDs will be evaluated from calves confirmed to have viral infection and compared to any not diagnosed with viral infection. BLDs will be evaluated from calves confirmed to have bacterial l infection and compared to any not diagnosed with bacterial infection. Further, the BLD will be evaluated for each population to see if the pattern differs for viral vs. bacterial infection. BLD patterns indicative of each will be developed.