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
South Dakota State University
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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
Ann M. Harvey
Arnold R. Hippen
Joseph F. Nelson Undergraduate Research
Midland BioProducts, Boone, lA
Triple J Farms, Bellingham, VA
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
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
, 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
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
. TheIgG2 makes up approximately two-thirds of the
IgG in serum
. All species examined produce IgG and IgM, but may vary
in their ability to produce the remaining classes
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"
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,
. "Intestinal epithelial cells lose theirability to absorb intactmacromolecules after
about 24 h because of the maturation of the cells and development of the intracellular
. "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,
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
. 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 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,
(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
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.
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.
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