Evaluation of a turbidimetric immunoassay (TIA) for
measuring IgG concentrations in mare colostrum.
Student Research Project
Utrecht University, Faculty of Veterinary Medicine, Department of Equine
Sciences, The Netherlands
June 2008
Drs. S. Sitters
0052000
Supervisors:
Dr. T.A.E. Stout
Dr. P.M. McCue
Utrecht University, Faculty of Veterinary Medicine, Department
of Equine Sciences, The Netherlands
Colorado State University, College of Veterinary Medicine and
Biomedical Sciences, Equine Reproduction Laboratory, CO,
USA
Contents
Summary
3
Introduction
4
Materials and Methods
7
Results
9
Discussion
15
References
18
2
Summary
A commercial turbidimetric immunoassay (ARS Foal-IgG Test; Animal Reproduction
Systems, Chino, CA), designed for measuring IgG concentrations in foal serum, was
evaluated as a means for quantifying IgG concentrations in the colostrum of 43 mares
(28 post-partum/pre-suckle samples and 15 pre-partum samples). This TIA test was
found to be unsuitable for determining IgG concentrations in either untreated or
centrifuged (fat-free) mare colostrum. The IgG concentrations estimated by TIA were
significantly higher than those quantified by the radial immunodiffusion (RID) assay, and
there was no overall correlation between the two tests.
By contrast, the BRIX (sugar) refractometer proved to be an accurate, fast and easy
technique for estimating colostral IgG concentrations. The BRIX percentage score
correlated highly with colostral IgG concentrations measured by RID (pre - and postfoaling samples: r=0.78, serial pre-foaling samples: r=0.88, serial post-foaling samples:
r=0.99).
Pre-partum colostral IgG concentrations were measured daily in 3 mares during the final
14 days of gestation. During this period, 2 mares maintained relatively constant IgG
concentrations whereas the third mare showed a rise in colostral IgG starting around 12
days before parturition.
Colostral IgG concentrations were also evaluated in 2 mares during the first 24 hours
post-foaling. IgG concentrations dropped by 78% in the first 4-6 hours after birth, and
then reached a plateau that persisted for the next 10 hours.
3
Introduction
Equine neonates are born immunologically naïve because there is no significant in utero
transplacental transfer of macromolecules such as maternal circulating antibodies. The
absence of transplacental maternal macromolecule transfer in the horse is primarily a
factor of the multilayered epitheliochorial type of placenta in which 6 cell layers impede
the transfer of large molecules (Blackmer et al., 2002; Hines, 2003; Jeffcott, 1974;
McGuire & Crawford, 1973; Steven & Samuel, 1975). On the other hand, newborn foals
are immunocompetent and, indeed, an equine fetus is capable of responding to
immunological stimulation from relatively early in its development. Nevertheless, because
no antigenic stimulation takes place in utero, foals are born virtually agammaglobulinemic
(Blackmer et al., 2002; Erhard, 2001; Hines, 2003; Jeffcott, 1974; Massey et al., 1991;
McGuire & Crawford, 1973). As a result, adequate uptake of maternal immunoglobulins
(primarily IgG) from the dam’s colostrum (passive transfer of immunity) is essential for
the initial protection of newborn foals against infectious disease.
During the last 2 weeks of pregnancy, antibodies (primarily IgG with some IgM class
antibodies) from the mare’s blood are selectively concentrated in the colostrum (Blackmer
et al., 2002; Genco et al., 1969; Hines, 2003; Knottenbelt et al., 2004). The average IgG
concentration of equine colostrum at foaling is approximately 7,000 mg/dl (70 g/L), with a
normal range of approximately 3000 to 12,000 mg/dl (30 to 120 g/L: Curadi et al., 2001; Erhard
et al., 2001; Knottenbelt et al., 2004; Kohn et al., 1989; Lavoie et al., 1989; LeBlanc et al.,
1992; Massey et al., 1991; Morris et al., 1985; Pearson et al., 1984; Perryman, 1980;
Rouse & Ingram, 1970; Shideler et al., 1986; Townsend et al., 1983). Besides providing
the newborn foal with antibodies, colostrum is an important source of nutrients,
complement and lactoferrin. In addition, colostrum promotes cellular immunity and may
have a variety of other physiological activities such as the activation of granulocytes and
the provision of a local protective effect in the gastrointestinal tract. (Blackmer et al.,
2002; Hines et al., 2003; Knottenbelt et al., 2004). In multiparous mares, the average rate of
colostrum secretion is approximately 300 ml/h and the total volume of colostrum produced is 2-5
liters (Knottenbelt at al., 2004; Lavoie et al., 1989; Massey et al., 1991).
After a neonatal foal has drunk colostrum, specialized enterocytes in the small intestine
non-selectively absorb the colostral immunoglobulins by pinocytosis. In fact, the
enterocytes engulf droplets of colostrum from the intestinal lumen, transfer the droplets in small
vacuoles across the cell and then discharge the contents into lymphatic vessels. The antibodies
are subsequently transported via the lymphatic system to the blood stream (Blackmer et al.,
2002; Hines, 2003; Knottenbelt et al., 2004). The absorption of colostral antibodies via
the enterocytes is greatest during the first 6-8 hours after birth, and has essentially
stopped by 24-36 hours of age because the specialized enterocytes have been replaced
by epithelial cells incapable of pinocytosis (‘gut closure’: Blackmer, 2002; Hines, 2003;
Jeffcott, 1971, 1974; Knottenbelt et al., 2004; Pearson, 1984). To acquire sufficient
antibodies to offer reasonable protection against infectious disease, the average foal needs to
ingest approximately 2-4 liters of good quality colostrum within the first few hours of life
(Blackmer et al., 2002; Hines, 2003; Knottenbelt et al., 2004; Lavoie et al., 1989; LeBlanc et al.,
1992; Massey et al., 1991; Perryman, 1981).
4
In short, a newborn foal is entirely dependent on the maternal antibodies absorbed
following the ingestion of its dam’s colostrum during the first few hours of life for immune
protection. Failure of this passive transfer of maternal antibodies significantly increases
the risk that a foal will succumb to infectious disease (Crawford et al., 1977; Jeffcott,
1974; Koterba et al., 1984; McGuire et al, 1975; Raidal, 1996; Robinson et al, 1993),
although the degree of risk also depends on other factors such as management and level
of exposure to pathogens. Nevertheless, in foals developing septicemia, levels of
immunoglobulins (IgG) have been shown to be significantly lower than normal (Carter et
al, 1986; Koterba et al., 1984).
Despite, or maybe because, of its importance, there is considerable discussion with
regard to the neonatal circulating IgG values that signify either failure of passive transfer
of maternal antibodies (FPT) or partial failure of passive transfer (PFPT). This is partly
because it has not been possible to precisely determine the concentration of foal serum
IgG required for adequate protection of a healthy vigorous neonate. Nevertheless, it is
generally accepted that a foal has suffered complete failure of passive transfer (FPT) if
plasma IgG levels are < 200 mg/dl at 18-24 hours after birth. Partial failure of passive
transfer (PFPT) has variously been defined as a plasma IgG concentration of 200 -400
mg/dl or 200-800 mg/dl (Crawford et al., 1977; Hines, 2003; Madigan, 1997; McGuire et
al, 1977). As a result, it is generally advised that plasma IgG levels ≥400 mg/dl should
be adequate for a healthy foal in a clean environment with minimal exposure to
pathogens (Kohn et al., 1989; McGuire et al, 1977; Perryman, 1980; Rumbaugh et al,
1979). On the other hand, an IgG concentration ≥ 800 mg/dl is considered optimal
because it is rarely possible to ensure ‘minimum exposure to pathogens’ (Blackmer et al.,
2002; Hines, 2003; Koterba et al, 1984).
Reported incidences of FPT and PFPT range from 10-24 % and 14-19 %, respectively
(Crawford et al, 1977; Clabough et al., 1991; Erhard et al., 2001; Kohn et al, 1989;
LeBlanc et al., 1986, 1990, 1992; McGuire et al., 1973, 1977; Pemberton et al., 1 980;
Perryman & McGuire, 1980; Raidal, 1996; Stoneham et al., 1991; Tyler-McGowan et al.,
1997). Variation in the reported incidences may in part be a factor of the different tests
used to measure IgG concentrations, different reference ranges used to def ine FPT and
PFPT, the age of foal when tested and associated management procedures.
In any case, the most important risk factor for the development of FPT is thought to be
poor colostrum quality (i.e. IgG concentration of < 1,000 - 3,000 mg/dl: LeBlanc et al.,
1986, 1992; Morris et al., 1985; McGuire et al., 1977; Perryman & Crawford, 1979; Tyler McGowan et al., 1997). Other major factors associated with FPT include maternal factors
such as agalactia, premature lactation and rejection of the foal, and foal factors like
inability or lack of desire to nurse, prematurity, dysmaturity, and failure to absorb colostral
immunoglobulins (Blackmer et al., 2002; Crawford et al., 1977; Hines, 2003; Jeffcott,
1971, 1974; LeBlanc et al, 1986; McGuire & Crawford, 1973; Pearson et al., 1984;
Perryman & Crawford, 1979; Perryman 1981; Rumbaugh et al., 1979). Little is known
about the effects of season, climate or maternal factors on neonatal foal colostral IgG
absorption, while reports on the effects of breed, mare age, parit y, gestation length,
dystocia, retained placenta, foal sex, and meteorological factors are contradictory
(Clabough et al., 1991; LeBlanc et al., 1992; Morris et al, 1985; Shideler et al., 1986).
5
It is, however, clear that serum IgG concentrations in a newborn foal correlate well with
the IgG concentrations in its mother’s colostrum (Curadi et al., 2001; Erhard et al., 2001;
Kohn et al., 1989; Lavoie et al., 1989; LeBlanc et al., 1986; Morris et al., 1985). And since
poor quality colostrum is one of the most important risk factors for the development of
FPT, a logical first step in the prevention of FPT is to determine colostrum quality in the
immediate postpartum period, prior to nursing. If the colostrum quality is poor, the foal
can be supplemented with frozen-thawed colostrum, or a colostrum substitute. An
optimal screening test for colostrum quality should therefore be rapid, accurate,
repeatable, inexpensive and quantitative.
Common qualitative techniques used to assess colostrum quality include the
colostrometer (LeBlanc et al, 1986) and the BRIX (or sugar) refractometer (Cash, 1999).
Other less widely used techniques include gluteraldehyde precipitation (Blackmer et al.,
2002; Knottenbelt et al., 2004), latex agglutination (Knottenbelt et al., 2004 ; Waelchli et
al., 1990) and an enzyme-linked immunosorbent assay (ELISA) (Erhard et al., 2001). The
colostrometer measures the density or specific gravity of colostrum (Knottenbelt et al.,
2004; LeBlanc et al, 1986; Massey et al., 1991; Waelchi et al, 1990). Colostrum with high
IgG levels has a higher density and, therefore, specific gravity. However, determination
of the specific gravity of equine colostrum with a colostrometer depends on accurate
measurement of an exact volume (15 ml) of colostrum; even small errors in volume
measurement can lead to significant inaccuracy in specific gravity determination
(Chavatte et al., 1998).
Refractometry measures the concentration of dissolved solids in a solution. In the case of
a BRIX refractometer, a small amount of colostrum is placed on the prism and the light
plate is closed so that colostrum is spread evenly across the prism. The refractometer is
then pointed at a light source and the deviation or refraction of light is evaluated as a
percentage score. Colostrum with a low concentration of dissolved solids (i.e. low IgG
level) will scatter less light and therefore have a lower percentage score. Colostrum with
high amounts of dissolved solids (i.e. high IgG levels) will cause more light scatter and
thereby achieve a higher percentage score. BRIX refractometer evaluation of equine
colostrum quality has been shown to be both repeatable (r= 0.98) and highly correlated
with foal plasma IgG levels (r=0.85) as measured by the radial immunodiffusion assay
(Cash, 1999; Chavatte et al., 1998). Similar correlations have been reported for BRIX
refractometer evaluation of bovine colostrum (Molla, 1980).
Direct quantification of IgG levels in colostrum can be performed using the radial
immunodiffusion (RID) assay (Fleenor et al., 1981; Knottenbelt et al., 2004; Mancini et
al., 1965). Unfortunately, RID cannot be performed easily on–farm, and it takes
approximately 24 hours to obtain a result. Furthermore, the test is expensive.
Recently, a commercial turbidimetric immunoassay (TIA) was validated for measuring
IgG levels in the plasma of neonatal foals, to detect failure of passive transfer (McCue,
unpublished data). The assay (ARS Foal-IgG Test; Animal Reproduction Systems, Chino,
CA) is rapid, repeatable and correlates well with RID measurements of plasma IgG
concentration. The primary objective of this study was to evaluate the commercial TIA
test, designed for measuring IgG concentrations in foal serum, as a means for quantifying
IgG concentrations in the colostrum of mares. A second objective was to evaluate
colostrum IgG levels during the last days of pregnancy and the first 24 hours post -foaling.
6
Materials and Methods
Colostrum samples (2 ml) were collected from 28 mares (various breeds and parities)
immediately postpartum and prior to nursing (i.e. pre-suckle). Colostrum samples (2 ml) from
another 15 mares (also various breeds and parities) had been previously collected on
randomly selected days shortly prior to foaling. All 43 samples were frozen in plastic
cryovials (-20 C) until evaluation. Colostrum IgG concentrations were subsequently
evaluated using the commercial TIA test for quantifying serum IgG concentrations.
Objective I:
To evaluate a commercial TIA test for quantifying colostral IgG
concentrations.
Study 1: Comparing colostral IgG concentrations as measured by TIA with those measured
using the BRIX refractometer or by RID.
Frozen colostrum samples were thawed in a water bath at room temperature, and thereafter
immediately evaluated using a BRIX refractometer, the TIA test and by RID.
A commercial refractometer (Equine Colostrum Refractometer, Animal Reproduction
Systems, Chino, CA) was used to obtain the BRIX percentage score. The relationship
between colostrum quality and the BRIX % refractometer reading, as reported by Cash
(1999), is shown in the table below.
BRIX (%)
<15
15-20
20-30
>30
IgG concentration (g/L)
0-28
28-50
50-80
>80
Colostrum quality
Poor
Fair
Good
Very Good
Table 1: Equine Colostrum Refractometer Guide, Animal Reproduction Systems, Chino, CA.
The TIA test was performed using a commercial kit (ARS Foal-IgG Test; Animal
Reproduction Systems, Chino, CA), as follows. All materials were first allowed to equilibrate
to room temperature. A colostrum sample was then diluted 1:19 with buffer (diluent
containing < 0.1% sodium azide; Midland BioProducts Corporation, Boone, IA), which was
expected to ensure that the IgG concentration fell within the working range of the assay.
Next, 25 l of the diluted colostrum was added to 1.5 ml of dilution buffer in a cuvette and
mixed gently; 180 l of the second dilution was then added to 1.3 ml of a polymer buffer in a
second cuvette and again mixed. The second cuvette was then inserted into a
spectrophotometer (Densimeter Model 590a, Animal Reproduction Systems, Chino, CA) and
the system was zeroed. Next, 180 l of goat anti-equine IgG serum (Midland BioProducts
Corporation, Boone, IA) was added and the solution was mixed gently. The cuvette was then
replaced in the spectrophotometer and the percentage light transmission (% T) was
measured. The spectrophotometer automatically converted the % T score into an estimated
IgG concentration in mg/dl. This value had to be multiplied by 20 (initial dilution factor) to
obtain the IgG concentration of the original colostrum sample.
7
RID was performed at the Veterinary Diagnostic Laboratory of Colorado State University,
using a commercially available kit (Immunocheck, VMRD, Inc., Pullman, WA), as described
by Mancini et al (1965). As for the TIA test, colostrum samples were first diluted 20-fold with
sterile water to ensure that concentrations would be within the measurable range. The RID
assay was then performed using 3 l of the diluted colostrum. Sample precipitation rings
were evaluated after approximately 18 hours of incubation at room temperature. Again the
value had to be multiplied by 20 to obtain the IgG concentration of the original colostrum
sample.
Correlation between the IgG concentrations measured using the various tests were
examined using SPSS and Microsoft Excel software.
Study 2: Validating the TIA test by serial dilution of colostrum samples.
Colostrum samples from 4 mares were subjected to halving dilutions using a buffer (diluent
containing < 0.1% Sodium azide, Midland BioProducts Corporation, Boone, IA).
As previously, colostrum samples were first diluted 20-fold. The resulting solution was then
diluted 1:1 with buffer to obtain a 1:40 dilution. Depending on the IgG concentrations
measured in these solutions, either a less (1:80) or a more (1:10) concentrated solution was
prepared to create a series of 3 dilutions per sample within the working range of the assay;
1:80 dilutions were prepared by 1:1 dilution of the 1:40 solution with buffer, while 1:10
dilutions were made by diluting the original colostrum sample. The IgG concentration of all
solutions was then evaluated by both TIA and RID. Correlation analysis was performed.
Objective II:
To Evaluate pre- and post-partum colostral IgG concentrations.
Study 1: Evaluating colostral IgG concentrations during the immediate pre-partum period.
Colostrum samples were collected from 3 mares daily for 22 (n=1) or 13 (n=2) days prior to
foaling, to monitor colostral antibody concentrations during the pre-foaling period. The IgG
levels of these samples were determined using the BRIX (sugar) refractometer and, in the
case of one mare, by RID.
Study 2: Evaluating post-partum colostral IgG concentrations.
Colostrum samples were collected from 2 mares every 2 h after foaling for 22 and 24 h,
respectively (i.e. 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h), to monitor the decline in
colostral antibody concentrations post-foaling. IgG concentrations were measured using both
the BRIX refractometer and by RID. Correlation analysis was performed.
8
Results
The utility of the commercial TIA test for quantifying colostral IgG concentrations.
Pilot study:
Comparing colostral IgG concentrations measured by TIA and RID.
The TIA test was first performed on 1:20 and 1:40 dilutions of 5 randomly selected colostrum
samples, to determine which dilution factor was most appropriate for bringing IgG
concentrations into the working range of the assay. It was immediately clear that the IgG
concentrations measured by using TIA were not accurate (Table 2) since, for example, the
doubling dilution from 1:20 to 1:40 did not result in the expected halving of the measured IgG
concentration. Since the measured IgG concentrations were also unfeasibly high, it was
concluded that the TIA test is unsuitable for measuring IgG concentrations in untreated
equine colostrum.
1
2
3
4
5
Colostrum sample
Date of collection
A
B
C
D
E
29/01/2006
21/02/2006
03/03/2006
20/02/2006
23/02/2006
1:20 dilution
IgG (mg/dl)
1871.0
1303.3
1249.8
1294.2
408.2
1:40 dilution
IgG (mg/dl)
1402.7
664.2
1412.7
1665.5
-
Table 2. Effect of serial dilution of (untreated) colostrum on IgG levels, as determined by TIA.
However, because it was possible that the discrepancies in the measured IgG concentrations
were due to interference, e.g. from contained fat, the samples were reanalyzed after
centrifugation at 1000 g for 15 min to remove cellular debris and fat. The fat-free colostrum
located between the superficial fat layer and the underlying precipitate was harvested and
examined by TIA (Table 3; Fig. 1). A full 3 sample serial dilution was not performed on
sample D, because it was anticipated that other dilutions would result in IgG levels outside
the range of the TIA. Serial dilution of fat-free colostrum did result in the desired linear
decline in measured IgG concentrations (mg/dl). However, the absolute IgG concentrations
recorded were still unrealistically high.
Colostrum sample
1
2
3
4
5
A
B
C
D
E
1:10 dilution
IgG (mg/dl)
1383.4
1530
1:20 dilution
IgG (mg/dl)
776.5
750.6
1970.2
1072.8
810.8
1:40 dilution
IgG (mg/dl)
367.7
344.2
1109.5
330
541.5
Table 3. IgG concentrations in serial dilutions of fat-free colostrum, as determined by TIA.
9
1:80 dilution
IgG (mg/dl)
235.9
546.9
-
2500
IgG (mg/dl)
2000
sample A
1500
sample B
sample C
1000
sample E
500
0
Serial dilutions
Figure 1. IgG concentrations in serial dilutions of fat-free colostrum, as determined by TIA.
To determine whether the IgG concentrations measured by TIA were accurate, 3 dilutions of
3 fat-free colostrum samples were measured by RID and TIA in parallel. In summary, the IgG
concentrations measured by RID were significantly lower than those suggested by TIA, and
although there was often an obvious relationship between the two tests for a given sample,
this relationship varied between samples such that here was no overall correlation
(correlation coefficient (r) = 0.43, P = 0.25, Fig. 2).
It was concluded that the TIA test is not suitable for determining IgG concentrations in either
untreated or centrifuged (fat-free) colostrum. As a result, experiments to examine changes in
colostral IgG concentrations during the pre and post-partum periods used alternative tests.
IgG concentrations (m g/dl) by TIA and RID
1000
IgG by RID
800
600
400
200
0
0
500
1000
1500
2000
2500
IgG by TIA
Figure 2: IgG concentrations in fat-free colostrum as determined by TIA versus RID (n= 3 serial dilutions of
3 samples).
Next, the accuracy of BRIX refractometry for estimating IgG concentrations was examined by
comparing BRIX scores with RID test scores, because the former is a simple way of
estimating colostral IgG concentrations.
10
Evaluating the BRIX (sugar) refractometer as an estimator of colostral IgG concentrations.
Study 1: Comparing BRIX percentage scores with IgG concentrations measured by RID.
BRIX scores (%) were compared with RID IgG concentrations (mg/dl) (Figs. 3 a, b). Because
the maximum value measurable using the BRIX refractometer was 32 %, samples with a
score > 32 % were omitted from comparisons. This left 35 pre- and post-foaling colostrum
samples with an accurate BRIX score (Fig. 3a). Of these 35 samples, 21 were postfoaling/pre-suckle (Fig. 3b). The mean IgG concentration of the 28 post-foaling/pre-suckle
colostrum samples was 11,384 mg/dl (range; 4,660 to 19,520 mg/dl) as measured by RID.
A correlation coefficient of 0.78 (P<0.001) was obtained for the relationship between the IgG
levels as measured by BRIX and RID in the pre- and post-foaling colostrum samples. For the
post-foaling samples a correlation coefficient of 0.72 (P<0.001) was found.
Pre- and postfoaling colostrum sam ples
25000
IgG (mg/dl)
20000
15000
10000
5000
0
0
5
10
15
20
25
30
35
Brix score (%)
Figure 3a. Comparison of pre- and post-foaling colostrum IgG concentrations using BRIX (sugar) refractometry
versus RID.
Postfoaling colostrum sam ples
25000
IgG (mg/dl)
20000
15000
10000
5000
0
0
5
10
15
20
25
30
35
Brix score (%)
Figure 3b. Comparison of post-foaling colostrum IgG concentrations using BRIX (sugar) refractometry versus
RID.
11
Evaluating pre- and postpartum colostral IgG concentrations.
Study 1: Evaluating colostral IgG concentrations during the immediate pre-partum period.
In the mare from which colostrum samples were collected daily for 22 days pre-foaling,
colostral IgG concentrations tended to rise as foaling approached, as measured by both
BRIX refractometry and RID (Fig. 4a). In fact, BRIX readings showed a fairly consistent
increase from a baseline of approximately 5 % at >12 days prior to foaling to 27 % on the
day of foaling. RID measurements gave a broadly similar trend, with an increase from a
baseline of approximately 2500 mg/dl at >12 days before foaling to a maximum of 12.000
mg/dl 3 days before foaling, although the increase was less consistently progressive.
Nevertheless, the correlation coefficient for the relationship between IgG concentrations
measured by TIA and RID was 0.88 (P<0.001) (Fig. 4b).
14000
35
12000
30
10000
25
8000
20
6000
15
4000
10
2000
5
0
Brix score (%)
IgG (mg/dl)
Mare A
RID
BRIX
0
1
3
5
7
9
11
13
15
17
19
21
23
Time (days), day 23=day of foaling
Figure 4a. Pre-foaling colostral IgG concentrations determined by BRIX (sugar) refractometry and RID: mare
A.
IgG concentrations by refractom etry and RID
14000
IgG (mg/dl)
12000
10000
8000
6000
4000
2000
0
0
5
10
15
20
25
30
Brix score (%)
Figure 4b. Comparison of IgG concentrations determined in pre-foaling colostrum samples using BRIX
(sugar) refractometry versus RID: mare A.
12
In the two other mares from which pre-foaling colostrum samples were collected and
analyzed by BRIX refractometry (Fig. 5), BRIX values were high (mean = 27 and 19 % for
mares B and C, respectively) and relatively constant throughout the period examined (from
13 days pre-foaling).
Brix refractom eter
35
Brix score (%)
30
25
20
Mare B
15
Mare C
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time (days), day 14=day of foaling
Figure 5. Pre-foaling colostral IgG concentrations estimated by BRIX (sugar) refractometry: mares B & C.
Study 2: Evaluating post-partum colostral IgG concentrations.
For the 2 mares examined serially, IgG concentrations in colostrum declined during the
post-partum period (Figs. 6 a, b). From an initial IgG concentration of around 9000 mg/dl
(BRIX scores of 26 % and 31 %), concentrations fell rapidly during the first 4 -6 hours
post-partum to a plateau of approximately 2000 mg/dl (BRIX score of 10%), which was
then maintained over the subsequent 10 hours.
The correlation coefficient for IgG concentrations measured by TIA and RID was 0.99
(P<0.001) (Fig. 6c).
Mare A
10000
35
IgG (mg/dl)
25
6000
20
4000
15
10
2000
Brix score (%)
30
8000
RID
BRIX
5
0
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (hours)
Figure 6a. Post-foaling colostral decline in IgG concentrations determined by BRIX (sugar) refractometry
and RID: Mare A.
13
Mare B
10000
35
IgG (mg/dl)
25
6000
20
4000
15
10
2000
Brix score (%)
30
8000
RID
BRIX
5
0
0
0
2
4
6
8
10
12
14
16
18
20
22
Time (hours)
Figure 6b. Post-foaling colostral decline in IgG concentrations determined by BRIX (sugar) refractometry
and RID: Mare B.
IgG concentrations by refractom etry and RID
12000
IgG (mg/dl)
10000
8000
6000
4000
2000
0
0
5
10
15
20
25
30
35
Brix score (%)
Figure 6c. Comparison of IgG concentrations in post-foaling colostrum samples using BRIX (sugar)
refractometry versus RID (n=2 mares).
14
Discussion
Although a newborn foal possesses a functional immune system, it is immunologically
naïve because there is no significant transplacental transfer of maternal antibodies. As a
result, initial immune protection of the newborn foal is critically dependent on adequate
passive transfer of immunity (primarily IgG class antibodies) via the dam’s colostrum.
Failure of passive transfer therefore significantly increases the risk of a neonatal foa l
succumbing to infectious disease.
Failure of passive transfer can occur for several reasons, some of which are easily
identified and can therefore be managed effectively; examples include maternal
agalactia, failure to drink due to neonatal weakness, delayed development of the sucking
reflex, maternal non-cooperation, and loss of colostrum due to premature lactation. By
contrast, failure of the dam to produce colostrum of sufficient quality is more difficult to
identify and tends, therefore, to be less effectively managed (Morris et al., 1985).
Moreover, poor colostrum quality is thought to be the most important risk factor for failure
of passive transfer (Crawford et al., 1977; McGuire et al., 1977; Perryman, 1979). On the
other hand, the reasons for low absolute colostral IgG concentrations are largely
unknown, although a genetic defect in a mare’s ability to concentrate IgG from serum into
the colostrum in late gestation (Perryman, 1979) has been proposed, and increasing
maternal age has been associated with reduced colostral IgG concentration (LeBlanc).
And while premature lactation might be an important factor in the total amount of colostral
IgG available (Jeffcott, 1974; Morris et al., 1985), other factors may be more important
determinants of initial colostrum quality.
The postpartum/pre-suckle colostral IgG concentrations recorded in the present study fall
within the range of mean values reported previously for mares immediately post -partum
(Table 3).
Breed
Various breeds
Various breeds
Various breeds
Not specified
Crossbred
Thoroughbred
Thoroughbred
Thoroughbred
Arabian
Arabian
Arabian
Quarter Horse
Quarter Horse
IgG (mg/dl)
11,384
5,450
11,324
16,583
8,867
5,036
2,354
4,608
9,691
10,847
6,394
5,843
1,080
Sample size
28
139
18
6
10
39
32
22
14
12
25
21
1
Standardbred
Standardbred
Standardbred
Shetland pony
Heavy draft mares
3,932
5,128
8,912
4,600
6,438
15
136
36
9
11
Reference
Present study
Erhard et al., 2001
Massey et al., 1991
Lavoie et al., 1989
Curadi et al., 2001
LeBlanc et al., 1992
McGuire et al., 1977
Pearson et al, 1984
Pearson et al, 1984
Perryman, 1980
LeBlanc et al., 1992
LeBlanc et al., 1992
McGuire & Crawford,
1973
LeBlanc et al., 1992
Morris et al, 1985
Kohn et al., 1989
Rouse & Ingram, 1970
Townsend et al., 1983
Table 4: Average IgG concentrations in colostrum of mares at the time of parturition.
15
The large differences in mean colostral IgG concentrations between studies may in part
be breed-related (Kohn et al., 1989; LeBlanc et al., 1992; Pearson et al., 1984), although
there is also considerable within-breed variation. Another important factor is the time of
colostrum collection post-partum, because colostral IgG levels decrease rapidly after
foaling, as will be discussed later. Pre-suckle colostrum samples collected immediately
after foaling will therefore have significantly higher IgG concentrations than samples
collected several hours post-partum, when the foal has had the opportunity to nurse.
Factors affecting individual colostral IgG concentrations (Lavoie et al., 1989; LeBlanc et
al., 1992; Morris et al., 1985; Pearson et al., 1984; Shideler et al., 1986) may include
repeated vaccination, particularly in the last month before foaling (Pearson et al., 1984;
Perryman, 1981; Shideler et al., 1986). Again, however, it is unlikely that vaccination
history alone will account for all of the between-mare difference. Age and health of the
dam, number of lactations, total colostrum yield and management are other factors that
have been suggested to influence colostral IgG content (Knottenbelt et al, 2004).
Whatever its determinants, the importance of colostral quality to foal morbidity makes
determination in the immediate postpartum period, prior to nursing, highly desirable.
Initial postpartum colostral IgG content appears to be a good estimator of total colostral
IgG available to a foal (Lavoie et al., 1989), and correlates well with subsequent foal
serum IgG concentrations (Curadi et al., 2001; Erhard et al., 2001; Kohn et al., 1989;
LeBlanc et al., 1986; Morris et al., 1985). If colostrum quality is found to be poor, a foal
can be supplemented with frozen-thawed colostrum, or a colostrum substitute. Oral
supplementation is the easiest, safest and most effective way and is possible until the
moment of ‘gut closure’, but preferably within 12 hours after birth (LeBlanc et al., 1992;
Raidal et al., 2005). An optimal screening test for colostrum quality must therefore be
rapid, accurate, repeatable, inexpensive, and quantitative.
In the current study, a commercial turbidimetric immunoassay (Foal-IgG Test; Animal
Reproduction Systems, Chino, CA) was evaluated for its ability to measure IgG
concentrations in mare colostrum. This TIA test had recently been validated for
measuring IgG concentrations in the plasma of neonatal foals. Unfortunately, the current
study demonstrated that the commercial TIA test is not suitable for determining IgG
concentrations in either untreated or centrifuged (fat-free) mare colostrum. Not only were the
IgG concentrations estimated by TIA significantly higher than those measured by RID, but
there was no overall correlation between the two tests. Although the reason for the
differences between the two tests is unclear, they could arise at any stage of the TIA
procedure. For example, it is possible that the goat anti-equine IgG-antibodies reacted with
colostral substances other than IgG, and that the increased non-specific coagulation was
misinterpreted as a raised IgG concentration. In addition, even after centrifugation, colostrum
is a thick emulsion containing approximately 25 % fat, 16 % total protein and 22 % dry matter
(Csapó-Kiss et al., 1995; Csapó et al., 1995). This would presumably mean that initial light
transmission would be much lower than in a serum sample, and thereby reduce the
proportional change in transmission after addition of the goat anti-equine IgG in a
sample/individual specific fashion. In this respect, there was an apparent correlation between
the results of TIA and RID within individual samples (Fig. 2).
Another technique for estimating colostral IgG concentrations is the BRIX (sugar)
refractometer. Previously, it has been shown that the equine colostrum quality as estimated
using the BRIX refractometer correlates highly with foal plasma IgG concentrations (r=0.85),
as measured by RID (Cash, 1999; Chavatte et al., 1998).
In the current study, we found that, as expected, the BRIX percentage score correlated
highly with colostral IgG concentrations measured by RID (pre- and post-foaling samples:
r=0.78, post-foaling samples: r=0.72, serial pre-foaling samples: r=0.88, serial post-foaling
samples: r=0.99). A similar relationship (r=0.89) has been reported for bovine colostrum
(Molla, 1980).
16
The exact relationship between the BRIX score and IgG concentration (mg/dl) estimated in
the current study differed slightly to that provided by the manufacturers of the refractometer
(Table 1), with BRIX values in the current study reflecting higher IgG concentrations. The
relationship provided by the manufacturers is based on the study by Cash (1999). In that
study an immunoturbidimetric assay (Hitachi 717 Automated Biochemistry Analyser) was
used to determine colostral IgG (mg/dl) whereas a radial immunodiffusion assay was used in
the current study. The difference in techniques most probably explains the difference in the
exact relationship between BRIX score and IgG concentration. For this reason, the
regression (IgG (mg/dl) = 472.48 * BRIX (%) – 1013.50) found in this study (Fig. 3a) is used
in Table 5.
BRIX (%)
<15
15-20
20-30
>30
IgG Concentration (g/L)
0-60
60-80
80-130
>130
Colostrum quality
Poor
Fair
Good
Very Good
Table 5. Equine Colostrum Refractometer Guide, as measured in the current study.
In any case, BRIX refractometry proved to be an accurate, fast and easy method for
estimating colostrum IgG concentrations and was subsequently used to evaluate pre- and
post-partum colostral IgG concentrations. In this respect, it has been reported that IgG
antibodies from the mare’s blood are selectively concentrated in the colostrum during
approximately the last 2 weeks prior to parturition. However, in the current study, mares B
and C maintained relatively constant colostral IgG concentrations during the final 14 days of
gestation (Fig. 5), and it is assumed that the rate of IgG accumulation was matched by the
rate of fluid accumulation in the mammary gland. By contrast, mare A showed a clear rise in
colostral IgG concentration starting at around 12 days before parturition (Fig. 4a), and it is
assumed that transport of IgG exceeded the rate of fluid accumulation; Pearson et al. (1984)
reported similar individual differences in IgG sequestration. The greater variability in IgG
concentrations measured by RID than refractometry during the period shortly before foaling
in mare A (Fig. 4a) may be explained by greater human error during RID or may reflect real
differences in the rate of accumulation of IgG versus sugars. The variability in the timecourse and absolute levels of pre-partum colostral IgG concentrations in mares, means that it
would be difficult to accurately predict final colostrum IgG concentration before the foal is
born.
In the current study, colostral IgG concentrations dropped by 78% in the first 4-6 hours after
birth, and then reached a plateau that persisted for at least 10 hours (Fig. 6a, b). Similar
results have been reported previously (Knottenbelt et al., 2004; Lavoie et al., 1989; Pearson
et al., 1984; Rouse & Ingram, 1970; Erhard et al., 2001; Curadi et al, 2001), and speed of the
fall in colostral IgG concentrations emphasize the significance of the first colostrum in the
passive transfer of immunity, and explains why the initial colostral IgG concentration
correlates well with total available IgG (Lavoie et al., 1989).
It is concluded that measuring the IgG concentration of colostrum post-partum, preferably
pre-suckle, is of great value for the prevention of failure of passive transfer (Kohn et al.,
1989; Morris et al., 1985; Raidal, 1996; Stoneham et al., 1991; Tyler-McGowan et al., 1997).
Unfortunately, the large (individual) variation in the rate of increase in colostral IgG
concentration in the period leading up to foaling means that estimating colostrum quality in
advance is likely to be difficult. Moreover, because it is not clear what factors influence IgG
accumulation it is not clear whether it is possible to manipulate (improve) colostral quality.
Although, the commercial TIA test proved unsuitable for quantifying colostral IgG
concentration, the BRIX (sugar) refractometer proved to be an easy, accurate, rapid,
repeatable and inexpensive way of measuring this parameter.
17
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21