A Comparison of Methods for On-Farm Determination of Failure of Passive Transfer of Immunoglobulin to Dairy Calves

The Journal of Undergraduate Research, Dec 2003

Two commercially available, qualitative, on-farm test kits (Midland BioProducts Inc., Boone, lA), utilizing either serum or whole blood to evaluate failure of passive transfer (FPT) of immunoglobulins, were evaluated using 38 Holstein calves. Results from the kits were compared with refractometry determination of serum proteins and radial immunodiffusion determinations specific for IgG (RID; Triple J Farms, Bellingham,WA). Blood samples were collected immediately following birth before first colostrum feeding and at 48 h. At birth, serum protein concentrations averaged 4.52 g/dl and IgG averaged 8.6 mg/dl, respectively, for refractometer and RID. Forty eight hours after feeding colostrum, serum protein concentrations averaged 6.02 g/dl and IgG concentrations were 2129.3 mg/dl. Feeding colostrum increased serum protein and IgG concentrations at 48 h (P < 0.01). Serum protein concentrations determined by refractometry and serum IgG determined by RID were positively and significantly correlated (r^ = 0.78, P < 0.01) and the relationship is characterized as: serum protein, g/dl = 0.0007 mglg5/dl + 4.5726. Adequate immune transfer was assumed when serum IgG concentrations were greater than 1,000 mg/dl or FPT with IgG less than 1,000 mg/dl. Using samples of blood from calves collected prior to feeding colostrum, the accuracy of the on-farm plasma kits for adequate passive transfer was 100% (n = 29). The accuracyof the whole blood kits for assessing adequate passive transfer of IgG on samples from newborn calves was 95.5% with 4.5% false positives(n = 22). On blood samples from calves fed colostrum, the whole blood kits presented4.5% false negative readings and 0% false positives (n = 22). On the colostrum-fed calves, the plasma kit predicted passive transfer with 100% accuracy (n = 30).

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A Comparison of Methods for On-Farm Determination of Failure of Passive Transfer of Immunoglobulin to Dairy Calves

e Journal of Undergraduate Research A Comparison of Methods for On-Farm Determination of Failure of Passive Transfer of Immunoglobulin to Dair y Calves Ann Harvey South Dakota State University Part of the Dairy Science Commons Recommended Citation - Article 4 Follow this and additional works at: http://openprairie.sdstate.edu/jur A c o m p a r i s o n o f m e t h o d s for o n - f a r m d e t e r m i n a t i o n o f f a i i u r e o f p a s s i v e t r a n s f e r o f i m m u n o g i o b u i i n t o d a i r y c a i v e s Author: Faculty Mentor: Department: Sponsors: Ann M. Harvey Arnold R. Hippen Dairy Science Joseph F. Nelson Undergraduate Research Mentorship, SDSU Midland BioProducts, Boone, lA Triple J Farms, Bellingham, VA ABSTRACT Two commercially available, qualitative, on-farm test kits (Midland BioProducts Inc., Boone, lA), utilizing either serum or whole blood to evaluate failure of passive transfer (FPT) of immunoglobulins, were evaluated using 38 Holstein calves. Results from the kits were compared with refractometry determination of serum proteins and radial immunodiffusion determinations specific for IgG (RID; Triple J Farms, Bellingham, WA). Blood samples were collected immediately following birth before first colostrum feeding and at 48 h. At birth, serum protein concentrations averaged 4.52 g/dl and IgG averaged 8.6 mg/dl, respectively, for refractometer and RID. Forty eight hoiurs after feeding colostrum, serum protein concentrations averaged 6.02 g/dl and IgG concentrations were 2129.3 mg/dl. Feeding colostrum increased serum protein and IgG concentrations at 48 h (P < 0.01). Serum protein concentrations determined by refractometry and serum IgG determined by RID were positively and significantly correlated (r^ = 0.78, P < 0.01) and the relationship is characterized as: serum protein, g/dl = 0.0007 mglg5/dl + 4.5726. Adequate immune transfer was assumed when serum IgG concentrations were greater than 1,000 mg/dl or FPT with IgG less than 1,000 mg/dl. Using samples of blood from calves collected prior to feeding colostrum, the accuracy of the on-farm plasma kits for adequate passive transfer was 100% (n = 29). The accuracy of the whole blood kits for assessing adequate passive transfer of IgG on samples from newborn calves was 95.5% with 4.5% false positives(n = 22). On blood samples from calves fed colostrum, the whole blood kits presented4.5% false negative readings and 0% false positives (n = 22). On the colostrum-fed calves, the plasma kit predicted passive transfer with 100% accuracy (n = 30). Thus,dairyproducers can use thesequalitative assessment tools to ensurethat calves that test adequate for passive transfer do indeedhaveadequate bloodIgG concentrations and avoid FPT in calf rearing systems. Key words: Calves, Dairy, Immunoglobulin, Passive transfer Abbreviations: FPT, failure of passive transfer; Ig, inununoglobulin; PK, plasma kit; RID, radial inununodiffusion; WBK, whole blood kit INTRODUCTION According to McVicker (2002) , the economic value of passively acquired inununity in bullHolstein calves is $23.04 per calf. One can assume that the value of a healthy immiinp system in heifercalves would be significantly higher. Thus, the ability to rapidly and accurately assessthe immime status of newborn dairy animals is extremely important to the dairyproducer. The only way to assessthe inununestatusis to measure the level of circulating antibodies in the animal's bloodstream. Antibodies are globulins that are synthesized under a directing influence in vertebrate animals. They are responsible for the serological specificity of serumbecause of their ability to react with antigens in someobservable way (Gray, 1970) . Antibodies thatcomprise the majority of the neonate's immune system are called immunoglobulins (Ig). Goldsby, et al (2000) define immunoglobulins as an antibody or a heavy or light polypeptide chain that is partof an antibody molecule. There arethree major classes of Ig in cattle: IgGj andIgG2, IgA, andIgM. The IgM molecule is the firstto appear in response to immunization. Its presence in the bloodstream is transient and limited in amount, failing to exceed 5-8% of the circulating immunoglobulins. Aftera shortinterval in the immune response, IgMis overtaken and replaced by IgG production, possibly by inhibitory antibody feedback to antigen-sensitive IgM lymphocytes. The IgG eventually comprise 75-80% of the circulating antibody (Gray, 1970) . TheIgG2 makes up approximately two-thirds of the IgG in serum (Butler, 1973) . All species examined produce IgG and IgM, but may vary in their ability to produce the remaining classes (Gray, 1970) . Neonates rely on colostrum for passive immunization, or the acquisition of immunity by receipt of preformed antibodies ratherthanby active production of antibodies afterexposure to an antigen (Goldsby, et al, 2000) . "Colostrum is a source of immune components and nutrients to the neonate and contains moreprotein, immunoglobulins, non-protein nitrogen, fat, as, vitamins and minerals thanmilkdoes. Because some vitamins do not cross the placental barrier, colostrum is the primary source of these nutrients for the calf after birth" (Quigley, 1998) . Immediately afterbirth, neonate mammals possess the ability to absorb these large protein molecules through theintestinal epithelial cells. Eventually these cells willlose theirability to absorb inununoglobuUns. Thisis whatis known as closure (Stott, et al, 1979) . "Intestinal epithelial cells lose theirability to absorb intactmacromolecules after about 24 h because of the maturation of the cells and development of the intracellular digestive apparatus" (Quigley, 1998) . "From birth until themaximum concentration of immunoglobulin in serum is reached is the period of absorption. Following the peak concentration, there is a gradual decline in serum content due to discontinuance of absorption and, presumably, to the reflected catabolism of the immunoglobulin in serum and/or transfer to other metabolic pools, making the peak quite evident" (Stott, et al, 1979) . In a study looking at the immunoglobulin transfer in calves, conducted by Stott, et al (1979), "The age at first colostrum feeding influenced closure as indicated by differences in the mean closure time among the age groups for each immunoglobulin class. The trend appears linear, with closure time earlier with calves fed at 0 h and the period of absorption increasing with each increment of age up to 24 h when the final colostrum feeding was initiated. The data indicate that as feeding of colostrum is delayed, the estimated time for closure is also delayed. However, since the coefficient is less than 1.0, the length of time that the calves absorbed the immunoglobulin decreased as time to the initial feeding was delayed. Hence, in calves fed initially at birth, (0 h), closure occurs at approximately 21 h for IgG, 23 h for IgM, and 23 h for IgA. However, if feeding is delayed until 24 h after birth, then closure occurs at 33, 31 and 32 h for IgG, IgM and IgA, respectively. Thus, the length of time the calf is actually absorbing colostrum is reduced from about 21 h to about 8 h.." Serum IgG concentrations of less than 10 g/L are termed failure of passive transfer (FPT). In a study conducted by the National Animal Health Monitoring System, 40% of all calves sampled between 24 and 48 h had IgG concentrations below the recommended level of 10 g/L and over 25% had less than 6.2 g/L. That study indicated that over half of the deaths of calves with serum IgG concentrations less than 10 g/L were attributed to lack of IgG intake (Quigley, 1998) . Things that influence the amount of IgG absorbed are: sex of the calf, age at first feeding, body weight, amoimt of IgG consumed, and colostrum quality (Quigley, 1998) . "Colostrum must have a minimum IgGj concentrationof 35.2 mg/ml to provide 100 g of IgGj in 2.84 L. Similarly, for feedings of 1.89L or 3.78 L, the minimumIgGj concentrations were 52.9 and 26.5 mg/ml, respectively" (Pritchett, et al, 1994) . The development of a healthy immune system is important for neonate dairy heifers to survive to maturity. Knowing this, a method to assess the immune status of heifers on the farm is needed. Midland BioProducts, Boone, lA, developed two on-farm test kits that qualitatively assess whether the animal has FPT or adequate immune transfer. One of these kits uses whole blood and the other uses serum. The objective of this project was to determine the accuracy of these kits for diagnosing FPT in newborn calves. The hypothesis was that producers who use these kits will be able to confidently rely on the results of these kits, thus allowing proper steps to be taken to care for animals with FPT. MATERIALS AND METHODS Thirty-eight Holstein and Brown Swiss calves bom at the South Dakota State University Dairy Research and Training Facility, Brookings, SD, were used for this trial. Information collected, in regards to the general status of the calves, was calving difficulty score, birth weight, amount of colostrum fed, method of colostmm feeding, and source of colostrum. Hematocrit readings, to assess the clinicaldehydration levelof the calves, were also obtained. The kit results were compared to reffactometry and a radial immunodiffiision assay (RID) that was donated by Triple J Farms, Bellingham, WA. The RID results were considered the "gold standard" for comparison and that is howfinal accuracy determinations were made. Jugular vein blood samples were drawn following birth before first colostrum was fed, and again 48 h later using an 18-gauge needle and 20-ml syringe. Forplasma collection, 10ml of blood were placed in a K3 EDTA vacutainer andinverted several times to allow mixing of theanti-coagulant materials and blood andthen immediately placed in a cooler with icepacks until the sample could be tested immediately at the lab. For serumcollection, another10-mlof the samplewere placedin a vacutainer without additives andallowed to clotat room temperature for 30-90 min. After sufficient clotting, serum collections were centrifuged at 1200 x g for 10 min. The serum was then frozen at -20°C until analyses could beperformed. Prior to testing with theMidland Quick Test Kit Plasma Calf IgG (PK), samples were placed in a test tube rack and left at room temperature for approximately 30 min. Aftercompletion of the test kits, the samples were refrozen. TheMidland Quick Test KitWhole Blood CalfIgGtest (WBK) was performed upon arrival at the lab. This test used the whole blood from the K3 EDTA sample. Upon completion of the testkit, the blood was thencentrifuged at 1200 x g for 25 min. The plasma portion was collected and frozen at -20°C. Figure 1 shows contents of thekits: a testcartridge, dilution vial and pipettes. The kits aresimple to use. All the producer hasto dois obtain a blood sample and, depending on whether the kit is for wholebloodkit or plasma, the serumis separated. Afterthe serumor wholebloodis obtained, a filled pipetteof the sample is placedinto the dilution vial andthepipette is flushed several times to mix the sample and ensure quantitative transfer. The same pipette is then used to transfer the diluted sample onto the test cartridge. After waiting at least 20, and not more than 40 min, results can be obtained. These kits are a qualitative test. They specify adequate or inadequate passive transfer. Inside the cartridge there is a complexing agent specific for IgG molecules. If there are inadequate amounts of IgG in the sample (>10 mg/ml), the IgG does not complex completely with the complexing agent and reacts with the immobilized "T" and "C" lines. Two lines became visible; however, if there were adequate amoimts of IgG (< 10 mg/ml), the IgG complexes with the agent, the complex migrates through the'T" indicator, and only one line is visible. The kits have with a built-in control mechanism. Regardless of adequate transfer or FPT, the "C" line should develop. If it fails to develop, it means the test was erroneous. Figure 2 shows what the cartridges look like after completion. The top cartridge shows adequate immime transfer and the bottom indicates Colostrum was tested for IgG concentrations, with Quick Test Colostrum Kit (Midland BioProducts, Boone, lA), and a colostrometer to insure that calves were receiving adequate amounts of IgG in the colostrum they received. If frozen colostrum was fed, the method and temperature of thawing was recorded, the temperature of the colostrum was obtained before sampling and before testing with the kit, and a colostrometer reading was obtained. To adjust the colostrometer reading to achieve a corrected reading at 20°C, a formula developed by Mechor et al, (1991 ), of (uncorrected reading -13.2 + (0.8 temperature (°C)) was used. — ^ — To determine the accuracy of the whole blood and plasma kits, both kits as well as reffactometer readings, were compared with a radial immunodiffusion (RID) assay, which is quantitatively specific for IgG. The refractometer (Figure 3) measures total serum proteins in g/dl. It assumes that IgG is the largest portion of the total protein. By refractometry, serum protein concentrations of greater than 5 g/dl are considered indicative of adequate immune transfer. To use the reffactometer, a droplet of serum is placed on the glass and the slipcover is placed on top. To obtain the serum protein concentration, one must look through the ocular piece and read the appropriate scale. The RID assay (Figure 4) worked on the basis of antibodies reacting to a particular antigen. The wells on the plate were each filled to capacity with plasma or serum and were allowed to incubate for 24 h. During that time, the antigen diffused through the agarose gel that had a specific antiserum for IgG. A circle developed until equilibrium between the antigen and antibody was reached. The diameter of the ring was a direct function of the IgG concentration. RESULTS AND DISCUSSION Serum protein concentrations, as determined by refractometry, at birth and at 48 h are shown in Figure 5. The increase in proteins at 48 h can be attributed to the IgG that were absorbed from colostrum before gut closure occiured. The IgG concentrations, as determined by RID, are shown in Figure 6. At birth, there are virtually no IgG in the calf, which is to be expected since IgG must be obtained through passive transfer from the dam. After colostrum was fed, IgG increased to 2100 mg/dl of serum. Figure 7 shows the relationship between serum protein levels and IgG concentrations as a regression model. This project suggests that there is a moderate linear relationship between total protein and IgG levels in the serum, as r2 = 0.7807. The equation for the slope of the line is y = 0.0007x + 4.5726 where y = serum protein in gl/dl and x = IgG in mg/dl. To test the accuracy of the kits, results from the kit determinations were compared with RID. Additionally, reffactometry and RID were compared with each other to test the accuracy of the refractometry. In all comparisons, RID was considered to be the "gold standard." Results were categorized as either false positives or false negatives. False positive was defined as when either the kits or refractometer reported adequate immune transfer and RID reported FPT. False negatives were defined as when either the kits or refractometer suggested FPT and RID reported adequate immune transfer. For comparing the accuracy of refractometry to RID there were 27 samples obtained at birth. Refractometry yielded one false positive for an accuracy rate of 96.3%. At 48 h of age, there were 31 samples obtained and testing with refractometry resulted in 0 false negatives and 1 false positive for an overall accuracy rate of 96.8%. Comparison of the WBK to RID with 22 samples collected at birth resulted in one false positive reading. Accuracy was 95.5%. Analysis by PK of the 29 samples collected at birth resulted in no false negatives or false positives, for an accuracy rate of 100%. Comparing samples collected at 48 h after feeding colostrum, 22 of the WBK analysis resulted in one false negative and 0% false positives, for an overall accuracy rate of 95.5%. Of 30 samples collected at 48 h that were tested with PK, there were no false negatives or false positives, a 100% accuracy rate. 1000 2000 3000 4000 5000 IgG, mg/dl CONCLUSIONS AND IMPLICATIONS The plasma kits tested in this study were 100% accurate, and the whole blood kits were 95.5% accurate when compared with RID, a quantitative measure of IgG. Reffactometry was 96.6% accurate when compared with RID. Use of the plasma kits provides producers with a highly acciu'atetool for assessment of adequate immune transfer in young calve. Use of the whole blood kit provides an assessment tool that is as accurate as refractometry, yet does not require separation of plasma or serum from blood. Based upon these results, both dairy and beef producers can confidently rely on the results of these kits when assessing the immune status of their newborn calves. These new kits provide producers with another tool in their production toolbox and allow them to take the appropriate steps to successfully mzmagethose calves with FPT. Butler , J.E. 1973 . Synthesis and distribution of immunoglobulins . J. Am. Vet Med Assoc . Vol. 163 . 3: 795 - 798 . Goldsby , R.A. , T.J. Kindt , B.A. Osbome. 2000. Kuby Immunology . W.H. Freeman & Co., New York. Pages 97 , 616 . Gray , D.F. 1970 . Immunology: 2nd Edition . An outline of basic principles, problems and theories concerning the immunological behavior of man and animals . American Elsevier Publishing Company, Inc. New York. Pages 2 , 66 - 67 , 71 - 72 , 80 - 83 Mechor , G.D. , and Y.T. Grohn . 1991 . Effect of temperature on colostrometer readings for estimation of inmumoglobulin concentration in bovine colostrum . J. Dairy Sci . 74 : 3940 - 3943 . Pritchett , L.C. , C.C. Gay , D.D Hancock , and T.E. Besser . 1994 . Evaluation of the hydrometer for testing immunoglobulin G1 concentrations in Holstein colostrum . J. Dairy Sci . 77 : 1761 - 1767 . Quigley 111, J.D. , and J.J. Drewry . 1998 . Nutrient and immimity transfer from cow to calf pre- and postcalving . J. Dairy Sci . 81 : 2779 - 2790 . Stott , G.H. , D.B. Marx , B.E. Menefee and G.T. Nightengale . 1979 . Colostral immunoglobulin transfer in calves 1. period of absorption . J. Dairy Sci . 62 : 1632 - 1638 . McVicker , J. 2002 . American Journal of Veterinary Research . Vol 63 : 247 - 250 .


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Ann Harvey. A Comparison of Methods for On-Farm Determination of Failure of Passive Transfer of Immunoglobulin to Dairy Calves, The Journal of Undergraduate Research, 2003,