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A modified zinc sulfate turbidity test for the detection of immune status in newlyborn foals
Refereed
A MODIFIEDZINC SULFATETURBIDITYTEST FOR THE DETECTION OF IMMUNE STATUSIN NEWLYBORNFOALS M.M. LeBlanc, DVM, ACT; 1 J.P. Hurtgen, DVM, PhD, ACT; 2 and S. Lyle, DVM 1
ter. Serum containing <400 mg of IgG/dl had no precipitate or slight precipitate and transmitted >80% light. SUMMARY
The accuracy of a modified zinc sulfate turbidity test, in estimating a serum IgG concentration of 400 mg/dl, was evaluated. Sera from 161 foals <18 hours of age were incubated for 15 and for 60 seconds with zinc sulfate. Turbidity was assessed both visually, in a veterinary practice and a laboratory, and with a spectrophotometer. Serum IgG concentration was determined by single radial immunodiffusion assay. Twenty five of the 161 foals (15.5%) had <400 mg of IgG/dl of serum. Failure of passive transfer (serum IgG concentration <400mg/dl) was correctly identified visually in 21 of the sera (84%) in the laboratory, and in 20 of the sera (80%) in the veterinary practice. Spectrophotometric values (light transmittance 550 nm) from zinc sulfate assays were inversely proportional to the serum IgG content (r= -0.88; p <0.0001). Serum containing <400 mg/dl transmitted >80% light. Evidence of adequate passive transfer was detected visually in 100% of the samples (136 serum samples) and96% of these samples transmitted <80% light (131 of 136). Seventy four percent of those foals sampled by 10 hours after foaling had >400 mg of IgG/dl. The modified zinc sulfate turbidity test detected accurately all foals with failure of passive transfer when sera was assessed visually and by spectrophotomeAuthors' address: 1 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610. 2120 Freedom Avenue, New Freedom, PA 17349. Acknowledgements: This study was completed with the financial support of the Equine Neonatal Study Group, University of Florida. The assistance of E. L. Pritchard is gratefully acknowledged.
36
INTRODUCTION
Foals rely on the adequate intake and absorption of colostral immunoglobulins (Ig) for protection against infectious diseases during the first few weeks of life.6,7,12,13'16IgG concentrations >400 mg/dl provide adequate humoral immunity to foals thus minimizing their susceptibility of developing an infectious disease, tz13,16Passive transfer status (serum IgG concentration) in the foal needs to be evaluated early to enable prophylactic management of the foal with inadequate IgG .1,s,10,13,16Foals are routinely tested at 18 to 36 hours of age to determine maximal absorption of colostral IgG. 1,6,7,9,12,13'16 Evaluating IgG status prior to 18 hours gives the veterinarian the option of supplementing hypogammaglobulinemic foals orally with either colostrum or an Ig product prior to gut closure. Timely identification of foals with failure of passive transfer requires an accurate test with rapidly available results. Single radial immunodiffusion assay is accurate but time consuming (48 hours).~,~5Also, it is expensive to perform and requires greater technical skill than other test kits. The zinc sulfate precipitation test is a semiquantitative assay of the globulin fraction in serum samples. 4,9,1sWhen combined with 0.025% zinc sulfate solution, serum Ig will precipitate at 400500 mg/dl. 4,9.15The test is rapid, technically simple, inexpensive, and will yield qualitative results in field situations. 9.~5 However, the one hour incubation required has made it less convenient to use than other tests. EQUINE VETERINARY SCIENCE
Objectives of the present study were to decrease the incubation time of the zinc sulfate turbidity test and determine if the modified test estimated accurately a serum IgG concentration of 400 mg/dl in foals <18 hours of age.
spectrophotometer~set at a wavelength of 550 nm. Test serum IgG concentrations were estimated by comparing the amount of light the serum transmitted to a standard curve calibrated for IgG content. The regression line for the standard curve was plotted by measuring the serum IgG concentrations of the first 21 foals studied in 1987 by SRID.
M A T E R I A L S AND METHODS
Serum Collection. One postsuckling serum sample was collected (age range, 5 to 18 hours) from 161 foals located on 10 farms. The blood tube was stored upright and 0.1 ml of serum was subjected to a zinc sulfate turbidity test within 30 minutes of collection. The remaining serum was decanted in 6 to 10 hours. Hemolysis, if present, was recorded. At that time, a second zinc sulfate turbidity test was performed by 1 of 3 individuals in the New Freedom Veterinary Hospital at 15 seconds and a spec.trophotometric reading was taken at one minute. The serum was then stored at -20C in labelled, one ml aliquots until shipped to the Equine Reproduction Laboratory, University of Florida. At the University of Florida, a third zinc sulfate turbidity test was performed at 60 seconds and serum IgG concentrations were quantified by radial immunodiffusion assays (SRID). Zinc Sulfate Turbidity Test. Zinc sulfate turbidity tests were prepared as described previously? Briefly, 250mg of zinc sulfate" were added to one L of boiled distilled water and stored in an air tight container in the dark. New zinc sulfate solution was made every four months at the veterinary hospital and was made in two batches, of one L each, at the University of Florida. For each assay one tenth ml of test serum was added to six ml of zinc sulfate solution. The tube was rotated three to five times. Turbidity was rated subjectively from 0 (no precipitate) to 3 (heavy precipitate). The turbidity rating given to each serum in the veterinary practice by one of three individuals at 15 seconds was compared to a rating determined by one individual at the Equine Reproduction Laboratory at 60 seconds. These time intervals were compared because turbidity was interpreted between 15 and 60 seconds on the farms. Results from zinc sulfate turbidity tests performed on farms were not included in this study due to incomplete farm records.
IgG quantification. Foal serum IgGconcentrations were determined by SRID, as described previously,u Samples were evaluated in duplicate. Statistical analysis. Mean values + standard deviation are given throughout. Least squares linear regression and correlation analysis (p<0.05) were used to analyze the relationship between light transmittance and serum IgG concentrations. 17
RESULTS
Light transmitted through serum incubated with zinc sulfate was inversely proportional to the IgG content of the serum (r= -0.88; p < 0.0001; Fig 1). Twenty five of the 161 foals (15.5%) had <400 mg IgG/dl of serum. Sera from these foals transmitted >80% light. A turbidity rating of 0 was given to 21 of the 25 sera (84 %) when evaluated in the laboratory and to 20 (80%) of the sera when evaluated in the veterinary hospital (Table 1). Investigators in the veterinary hospital agreed on a turbidity rating of 0 in 19 of the 21 samples (86%) rated as 0 in the laboratory. Three of the 25 sera consistently had precipitate, however these sera were not hemolysed. Agreement on turbidity ratings of 1-3 was less consistent. Serum containing >400 mg of IgG/dl had visible precipitate and transmitted 10-80% light except for 5 sera. These 5 contained 400-460 mg of IgG and transmitted >80% light. Seventy four percent of those foals sampled by 10 hours had >400 mg of IgG/dl whereas 80% of those foals sampled by 14 hours had >400 mg IgG/dl (Table 2). Mean time from foaling to blood collection was 12 + 2 hours with a range of 5 to 18 hours. Mean serum IgG concentration of those foals evaluated by 12 hours was 1270 mg/dl. Mean time from foaling to nursing was 2 + 1 hours.
Spectrophotometric Determination. Optical density was measured by absorbance and light transmittance. Our results are expressed in light transmittance because the regression line generated for light transmittance had a higher correlation coefficient than that for absorbance (light transmittance r2 = 0.79; absorbance r 2 = 0.72). Correlation coefficients may have differed because the two parameters differ mathematically. Absorbance is a function of the log equation A=2 - log % transmittance whereas light transmittance is a linear function. Light transmittance was recorded at one minute on a
Accurate, rapid assessment of the immune status of a newlyborn foal is desirable for the detection of inadequate passive transfer of IgG. In this and previous studies, a serum IgG concentration of 400 mg/dl or less is considered to be indicative of a failure of passive transfer. 2'5's'1°This condition has been detected in 2.9-24% of foals evaluated and increases their susceptibility to infectious disease. 12'14'16There are
"Sigma Chemical Co, St. Louis, Missouri
bBaush and Lomb, Chicago, III.
Volume 10, Number 1, 1990
DISCUSSION
37
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Figure 1. Relationship between light transmittance and serum IgG concentrations of 161 foals. (y=2308 + (-23.7)x; r= -.0.88). numerous methods available for measuring serum IgG in the foal; each has its disadvantages. I.z4.9.11.14,15Although the zinc sulfate turbidity test is semiquantitative, it is an inexpensive, practical method for estimating serum Ig concentrations? .14.15 Its disadvantage is the one hour incubation recommended by previous workers. In the present study, serum IgG concentration was evaluated between 15-60 seconds with the zinc sulfate turbidity test. If less than 400 mg IgG/dl was present, there was no visible precipitate (turbidity rating of zero) in 80% of the cases. Sixteen percent of the samples evaluated visually in the laboratory and 20% of those evaluated in the veterinary practice overestimated IgG in sera containing <400mg IgG/dl. Howe~/er, if a test serum showing slight precipitation (turbidity ~ating of 1) was evaluated on the spectrophotometer and it transmitted >80% light, the serum contained <460mg of IgG/dl. Using this combined interpretation all sera containing <400rag IgG were correctly identified. Overestimating IgG in serum containing <400 mg/dl with the zinc sulfate turbidity test and one hour incubation has been reported previously. 8.14Possible reasons include differences in operators, field conditions, reagent quality, and the presence of hemolysis in the sample. Why the modified zinc sulfate turbidity test was as accurate as the original test at determining a IgG concentration of 400-500 mg is not clearly understood. The 15-60 second incubation time was chosen because we noticed when performing the original test that turbidity occurred within 15 seconds. Also, the number of false positives (test result indicating that a sample had >400 mg IgG/dl when in had <400 mg/dl) were lower when samples were incubated for 15 seconds versus 1 hour. This may be because precipitation continues during the 1 hour incubation and globulins, other than IgG, precipitate. Individuals did not consistently agree on turbidity ratings of 1 to 3. Unless individuals assess numerous precipitated samples together, differences between readings will occur. Therefore, the modified zinc sulfate turbidity test is probably most accurate if turbidity is recorded as present (+) or absent
(-).
A serum IgG concentration of 400 mg/dl may not ade38
Table 1. Comparison of turbidity ratings performed at a veterinary hospital and at a laboratory.
Field ratings
Laboratory ratings
0
1
0
19"
2
1
1
20
14
8
47
23
78
3
24
27
64
47
2 3 20
30
2
3 21 35
Total for each laboratory rating
Total for each field rating aNumberof observations
Table 2. Foals with failure of passive transfer at 5 to 18 hours of age. Time of blood collection (hrs) 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Serum IgG (mg/dl) < 400 2= 1 2 2 1 3 6 3 4 1 0 0 0 0
> 400 0 1 3 4 9 14 2O 25 14 18 10 7 7 5
Cumulative no. of samples 2 4 9 15 25 42 68 96 113 132 142 149 156 161
anumberof foals
EQUINE VETERINARY SCIENCE
quately protect those foals at high risk from the development of infection.3'4~'1°In foals admitted to an intensive care facility for treatment of neonatal disease, there was a relationship between survival rate and the level of IgG in the foal' s serum.5 Fifty-three percent of the foals having <200 mg/dl survived whereas 68% and 76% of the foals survived if serum IgG concentrations were 200-400 mg/dl or400-800 mg/dl, respectively. Furthermore, in foals with <400 mg/dl of IgG/dl, the opsonizing activity of serum is significantly lower than the opsonizing activity of that from adults or from foals with >600 mg/dl. 1° If such foals become ill they do not have adequate concentrations of circulating IgG for opsonizing invading bacteria effectivelyJ° Therefore, a serum IgG concentration of 400 mg/dl or less indicates a need for therapeutic intervention. In the present study, the modified zinc sulfate test allowed a way for rapid assessment of failure of passive transfer to be made. Since the critical level of 400 mg IgG/dl was never underestimated no foal would have been treated when therapy was unnecessary. Of those foals requiring treatment, 84% were identified by simple visual evaluation of serum turbidity. The remaining 16% of foals that required treatment could be identified if serum with a turbidity rating of one was also assessed with a spectrophotometer. Those sera that transmitted >80% light contained <460 mg IgG/dl. Therefore, there is the chance that a small percentage of foals with IgG levels slightly above 400 mg/dl may be treated unnecessarily. Passive transfer status of foals is routinely assessed when foals are 18 to 36 hours old. 2.4.9,12"14,16The minimum age for such tests has not been defined. Because absorption of immunoglobulins from the gastrointestinal tract begins to decrease rapidly beyond 12 hours after birth,6 the best time to evaluate passive transfer status based on a small amount of data collected in out laboratory may be, in our opinion, between 9 and 12 hours. This would give the foal time to absorb Ig but would also allow sufficient time for the veterinarian to treat the foal orally. In the present study, 9 of the 10 foals evaluated at 9 hours had > 400 mg IgG/dl. The modified zinc sulfate turbidity test should be a useful assay for rapidly determining failure of passive transfer in the foal. It is inexpensive (approximately $0.50/test), is technically simple to perform and results can be obtained within 5 minutes on the farm.
Pie Medical Scanners Vet Vision 100 S/L
REFERENCES 1. Baird AN, Pugh DG, Rupp GP, Shell JW: Detection of immunoglobulin G concentration in the neonate. J Equine Vet Sci 7:124129,1987. 2. Bertone JJ, Jones RL, Curtis CR: Evaluation of a test kit for determination of serum immunoglobulin G concentration in foals. J Vet /ntemal Med 2 :181-183,1988. 3. Brewer BD: Neonatal foal evaluation: sepsis and survival scoring in private practice. Proc 33rd Ann Cony of Am Assoc Equine Practitioners, New Orleans: p 917-922,1987. 4. Crawford TB, Perryman LE: Diagnosis and treatment of failure of passive transfer in the foal. Equine Pracdce 2:17-21,1980. 5. Cudd TA, Koterba AM: The ISVP survey of equine neonatal disease: 1987./SVP News/etter 1:3-6,1988 6. Jeffcott LB: Passive immunity and its transfer with special reference to the horse. Biol Review47:439-464,1972. 7. Jeffcott LB: Studies on passive immunity in the foal. J Comp Patho184:93-101,1974. 8. Koterba AM, Brewer BD, Tarplee FA: Clinical and clinicopathological characteristics of the septicemic neonatal foal: a review of 38 cases. Equine VetJ 16:376-383,1984. 9. Kruse-Elliott K, Wagner PC: Failure of passive antibody transferin the foal. CompendContinEduc Pract Vet6:S702-S707,1984. 10. LeBlanc MM, Pritchard EL: Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils. An Genetics 19:227-237,1988. 11. McGuire TC, Crawford TB: Passive immunity in the foal: measurement of immunoglobulin classes and specific antibody. Am J Vet Res 34:1299-1303,1973. 12. McGuire TC, Poppie MJ, Banks KL: Hypogammaglobulinemia predisposing to infection in foals. J Am Vet Med Assoc 166:7175,1975. 13. McGuire TC, Crawford TB, HaJlowell AL, Macomber LE: Failure ofcolostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. JAm VetMedAssoc 170:13021304,1977. 14. Morris DD, Meirs DA, Merryman GS: Passive transfer failure in horses: incidence and causative factors on a breeding farm. J Am Vet Med Assoc 46:2294-2299,1985. 15. Rumbaugh GE, Ardans hA, Ginno D, TrommershausenSmith A: Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with zinc sulfate turbidity test, serum eletrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test. J Am Vet Med Assoc 172:321-325,1978. 16. Rumbaugh GE, Ardans AA, Ginno D, TrommershausenSmith A: Identification and treatment of colostrum-deficient foals. JAm Vet Med Assoc 174:273-276,1979. 17. Steele TGD, Torrie JH: Principles and procedures of statistics: a biometrical approach. 2nd ed., McGraw-Hill Book Co New York, New York, 1980.
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39
A MODIFIEDZINC SULFATETURBIDITYTEST FOR THE DETECTION OF IMMUNE STATUSIN NEWLYBORNFOALS M.M. LeBlanc, DVM, ACT; 1 J.P. Hurtgen, DVM, PhD, ACT; 2 and S. Lyle, DVM 1
ter. Serum containing <400 mg of IgG/dl had no precipitate or slight precipitate and transmitted >80% light. SUMMARY
The accuracy of a modified zinc sulfate turbidity test, in estimating a serum IgG concentration of 400 mg/dl, was evaluated. Sera from 161 foals <18 hours of age were incubated for 15 and for 60 seconds with zinc sulfate. Turbidity was assessed both visually, in a veterinary practice and a laboratory, and with a spectrophotometer. Serum IgG concentration was determined by single radial immunodiffusion assay. Twenty five of the 161 foals (15.5%) had <400 mg of IgG/dl of serum. Failure of passive transfer (serum IgG concentration <400mg/dl) was correctly identified visually in 21 of the sera (84%) in the laboratory, and in 20 of the sera (80%) in the veterinary practice. Spectrophotometric values (light transmittance 550 nm) from zinc sulfate assays were inversely proportional to the serum IgG content (r= -0.88; p <0.0001). Serum containing <400 mg/dl transmitted >80% light. Evidence of adequate passive transfer was detected visually in 100% of the samples (136 serum samples) and96% of these samples transmitted <80% light (131 of 136). Seventy four percent of those foals sampled by 10 hours after foaling had >400 mg of IgG/dl. The modified zinc sulfate turbidity test detected accurately all foals with failure of passive transfer when sera was assessed visually and by spectrophotomeAuthors' address: 1 Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32610. 2120 Freedom Avenue, New Freedom, PA 17349. Acknowledgements: This study was completed with the financial support of the Equine Neonatal Study Group, University of Florida. The assistance of E. L. Pritchard is gratefully acknowledged.
36
INTRODUCTION
Foals rely on the adequate intake and absorption of colostral immunoglobulins (Ig) for protection against infectious diseases during the first few weeks of life.6,7,12,13'16IgG concentrations >400 mg/dl provide adequate humoral immunity to foals thus minimizing their susceptibility of developing an infectious disease, tz13,16Passive transfer status (serum IgG concentration) in the foal needs to be evaluated early to enable prophylactic management of the foal with inadequate IgG .1,s,10,13,16Foals are routinely tested at 18 to 36 hours of age to determine maximal absorption of colostral IgG. 1,6,7,9,12,13'16 Evaluating IgG status prior to 18 hours gives the veterinarian the option of supplementing hypogammaglobulinemic foals orally with either colostrum or an Ig product prior to gut closure. Timely identification of foals with failure of passive transfer requires an accurate test with rapidly available results. Single radial immunodiffusion assay is accurate but time consuming (48 hours).~,~5Also, it is expensive to perform and requires greater technical skill than other test kits. The zinc sulfate precipitation test is a semiquantitative assay of the globulin fraction in serum samples. 4,9,1sWhen combined with 0.025% zinc sulfate solution, serum Ig will precipitate at 400500 mg/dl. 4,9.15The test is rapid, technically simple, inexpensive, and will yield qualitative results in field situations. 9.~5 However, the one hour incubation required has made it less convenient to use than other tests. EQUINE VETERINARY SCIENCE
Objectives of the present study were to decrease the incubation time of the zinc sulfate turbidity test and determine if the modified test estimated accurately a serum IgG concentration of 400 mg/dl in foals <18 hours of age.
spectrophotometer~set at a wavelength of 550 nm. Test serum IgG concentrations were estimated by comparing the amount of light the serum transmitted to a standard curve calibrated for IgG content. The regression line for the standard curve was plotted by measuring the serum IgG concentrations of the first 21 foals studied in 1987 by SRID.
M A T E R I A L S AND METHODS
Serum Collection. One postsuckling serum sample was collected (age range, 5 to 18 hours) from 161 foals located on 10 farms. The blood tube was stored upright and 0.1 ml of serum was subjected to a zinc sulfate turbidity test within 30 minutes of collection. The remaining serum was decanted in 6 to 10 hours. Hemolysis, if present, was recorded. At that time, a second zinc sulfate turbidity test was performed by 1 of 3 individuals in the New Freedom Veterinary Hospital at 15 seconds and a spec.trophotometric reading was taken at one minute. The serum was then stored at -20C in labelled, one ml aliquots until shipped to the Equine Reproduction Laboratory, University of Florida. At the University of Florida, a third zinc sulfate turbidity test was performed at 60 seconds and serum IgG concentrations were quantified by radial immunodiffusion assays (SRID). Zinc Sulfate Turbidity Test. Zinc sulfate turbidity tests were prepared as described previously? Briefly, 250mg of zinc sulfate" were added to one L of boiled distilled water and stored in an air tight container in the dark. New zinc sulfate solution was made every four months at the veterinary hospital and was made in two batches, of one L each, at the University of Florida. For each assay one tenth ml of test serum was added to six ml of zinc sulfate solution. The tube was rotated three to five times. Turbidity was rated subjectively from 0 (no precipitate) to 3 (heavy precipitate). The turbidity rating given to each serum in the veterinary practice by one of three individuals at 15 seconds was compared to a rating determined by one individual at the Equine Reproduction Laboratory at 60 seconds. These time intervals were compared because turbidity was interpreted between 15 and 60 seconds on the farms. Results from zinc sulfate turbidity tests performed on farms were not included in this study due to incomplete farm records.
IgG quantification. Foal serum IgGconcentrations were determined by SRID, as described previously,u Samples were evaluated in duplicate. Statistical analysis. Mean values + standard deviation are given throughout. Least squares linear regression and correlation analysis (p<0.05) were used to analyze the relationship between light transmittance and serum IgG concentrations. 17
RESULTS
Light transmitted through serum incubated with zinc sulfate was inversely proportional to the IgG content of the serum (r= -0.88; p < 0.0001; Fig 1). Twenty five of the 161 foals (15.5%) had <400 mg IgG/dl of serum. Sera from these foals transmitted >80% light. A turbidity rating of 0 was given to 21 of the 25 sera (84 %) when evaluated in the laboratory and to 20 (80%) of the sera when evaluated in the veterinary hospital (Table 1). Investigators in the veterinary hospital agreed on a turbidity rating of 0 in 19 of the 21 samples (86%) rated as 0 in the laboratory. Three of the 25 sera consistently had precipitate, however these sera were not hemolysed. Agreement on turbidity ratings of 1-3 was less consistent. Serum containing >400 mg of IgG/dl had visible precipitate and transmitted 10-80% light except for 5 sera. These 5 contained 400-460 mg of IgG and transmitted >80% light. Seventy four percent of those foals sampled by 10 hours had >400 mg of IgG/dl whereas 80% of those foals sampled by 14 hours had >400 mg IgG/dl (Table 2). Mean time from foaling to blood collection was 12 + 2 hours with a range of 5 to 18 hours. Mean serum IgG concentration of those foals evaluated by 12 hours was 1270 mg/dl. Mean time from foaling to nursing was 2 + 1 hours.
Spectrophotometric Determination. Optical density was measured by absorbance and light transmittance. Our results are expressed in light transmittance because the regression line generated for light transmittance had a higher correlation coefficient than that for absorbance (light transmittance r2 = 0.79; absorbance r 2 = 0.72). Correlation coefficients may have differed because the two parameters differ mathematically. Absorbance is a function of the log equation A=2 - log % transmittance whereas light transmittance is a linear function. Light transmittance was recorded at one minute on a
Accurate, rapid assessment of the immune status of a newlyborn foal is desirable for the detection of inadequate passive transfer of IgG. In this and previous studies, a serum IgG concentration of 400 mg/dl or less is considered to be indicative of a failure of passive transfer. 2'5's'1°This condition has been detected in 2.9-24% of foals evaluated and increases their susceptibility to infectious disease. 12'14'16There are
"Sigma Chemical Co, St. Louis, Missouri
bBaush and Lomb, Chicago, III.
Volume 10, Number 1, 1990
DISCUSSION
37
2400 2000.2200; ~
-
r+
.
÷
1600-
~'~" ~ ~ + + *
+~
~14o0~
't'
'4,
+ + .1~1. + " + + + +** +
÷
~3m1200.
+÷ +
~
'
"
+ + + +
800. ,
÷
+
:1:'~+÷
4e+ + ÷
600.
+
+
÷ 4 +
+ +
400,
200.
+
e++
+
++ 4 +
+
4+
++ +
++
4. . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . .
0,
.
.
.
.
.
.
.
.
.
|
20
.
.
.
.
.
.
.
.
.
i
.
.
.
.
.
.
.
.
.
,
40 60 LJghtTransmittance(%)
.
.
.
.
.
.
.
.
.
t
.
.
.
.
.
.
.
80
.
.
100
Figure 1. Relationship between light transmittance and serum IgG concentrations of 161 foals. (y=2308 + (-23.7)x; r= -.0.88). numerous methods available for measuring serum IgG in the foal; each has its disadvantages. I.z4.9.11.14,15Although the zinc sulfate turbidity test is semiquantitative, it is an inexpensive, practical method for estimating serum Ig concentrations? .14.15 Its disadvantage is the one hour incubation recommended by previous workers. In the present study, serum IgG concentration was evaluated between 15-60 seconds with the zinc sulfate turbidity test. If less than 400 mg IgG/dl was present, there was no visible precipitate (turbidity rating of zero) in 80% of the cases. Sixteen percent of the samples evaluated visually in the laboratory and 20% of those evaluated in the veterinary practice overestimated IgG in sera containing <400mg IgG/dl. Howe~/er, if a test serum showing slight precipitation (turbidity ~ating of 1) was evaluated on the spectrophotometer and it transmitted >80% light, the serum contained <460mg of IgG/dl. Using this combined interpretation all sera containing <400rag IgG were correctly identified. Overestimating IgG in serum containing <400 mg/dl with the zinc sulfate turbidity test and one hour incubation has been reported previously. 8.14Possible reasons include differences in operators, field conditions, reagent quality, and the presence of hemolysis in the sample. Why the modified zinc sulfate turbidity test was as accurate as the original test at determining a IgG concentration of 400-500 mg is not clearly understood. The 15-60 second incubation time was chosen because we noticed when performing the original test that turbidity occurred within 15 seconds. Also, the number of false positives (test result indicating that a sample had >400 mg IgG/dl when in had <400 mg/dl) were lower when samples were incubated for 15 seconds versus 1 hour. This may be because precipitation continues during the 1 hour incubation and globulins, other than IgG, precipitate. Individuals did not consistently agree on turbidity ratings of 1 to 3. Unless individuals assess numerous precipitated samples together, differences between readings will occur. Therefore, the modified zinc sulfate turbidity test is probably most accurate if turbidity is recorded as present (+) or absent
(-).
A serum IgG concentration of 400 mg/dl may not ade38
Table 1. Comparison of turbidity ratings performed at a veterinary hospital and at a laboratory.
Field ratings
Laboratory ratings
0
1
0
19"
2
1
1
20
14
8
47
23
78
3
24
27
64
47
2 3 20
30
2
3 21 35
Total for each laboratory rating
Total for each field rating aNumberof observations
Table 2. Foals with failure of passive transfer at 5 to 18 hours of age. Time of blood collection (hrs) 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Serum IgG (mg/dl) < 400 2= 1 2 2 1 3 6 3 4 1 0 0 0 0
> 400 0 1 3 4 9 14 2O 25 14 18 10 7 7 5
Cumulative no. of samples 2 4 9 15 25 42 68 96 113 132 142 149 156 161
anumberof foals
EQUINE VETERINARY SCIENCE
quately protect those foals at high risk from the development of infection.3'4~'1°In foals admitted to an intensive care facility for treatment of neonatal disease, there was a relationship between survival rate and the level of IgG in the foal' s serum.5 Fifty-three percent of the foals having <200 mg/dl survived whereas 68% and 76% of the foals survived if serum IgG concentrations were 200-400 mg/dl or400-800 mg/dl, respectively. Furthermore, in foals with <400 mg/dl of IgG/dl, the opsonizing activity of serum is significantly lower than the opsonizing activity of that from adults or from foals with >600 mg/dl. 1° If such foals become ill they do not have adequate concentrations of circulating IgG for opsonizing invading bacteria effectivelyJ° Therefore, a serum IgG concentration of 400 mg/dl or less indicates a need for therapeutic intervention. In the present study, the modified zinc sulfate test allowed a way for rapid assessment of failure of passive transfer to be made. Since the critical level of 400 mg IgG/dl was never underestimated no foal would have been treated when therapy was unnecessary. Of those foals requiring treatment, 84% were identified by simple visual evaluation of serum turbidity. The remaining 16% of foals that required treatment could be identified if serum with a turbidity rating of one was also assessed with a spectrophotometer. Those sera that transmitted >80% light contained <460 mg IgG/dl. Therefore, there is the chance that a small percentage of foals with IgG levels slightly above 400 mg/dl may be treated unnecessarily. Passive transfer status of foals is routinely assessed when foals are 18 to 36 hours old. 2.4.9,12"14,16The minimum age for such tests has not been defined. Because absorption of immunoglobulins from the gastrointestinal tract begins to decrease rapidly beyond 12 hours after birth,6 the best time to evaluate passive transfer status based on a small amount of data collected in out laboratory may be, in our opinion, between 9 and 12 hours. This would give the foal time to absorb Ig but would also allow sufficient time for the veterinarian to treat the foal orally. In the present study, 9 of the 10 foals evaluated at 9 hours had > 400 mg IgG/dl. The modified zinc sulfate turbidity test should be a useful assay for rapidly determining failure of passive transfer in the foal. It is inexpensive (approximately $0.50/test), is technically simple to perform and results can be obtained within 5 minutes on the farm.
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REFERENCES 1. Baird AN, Pugh DG, Rupp GP, Shell JW: Detection of immunoglobulin G concentration in the neonate. J Equine Vet Sci 7:124129,1987. 2. Bertone JJ, Jones RL, Curtis CR: Evaluation of a test kit for determination of serum immunoglobulin G concentration in foals. J Vet /ntemal Med 2 :181-183,1988. 3. Brewer BD: Neonatal foal evaluation: sepsis and survival scoring in private practice. Proc 33rd Ann Cony of Am Assoc Equine Practitioners, New Orleans: p 917-922,1987. 4. Crawford TB, Perryman LE: Diagnosis and treatment of failure of passive transfer in the foal. Equine Pracdce 2:17-21,1980. 5. Cudd TA, Koterba AM: The ISVP survey of equine neonatal disease: 1987./SVP News/etter 1:3-6,1988 6. Jeffcott LB: Passive immunity and its transfer with special reference to the horse. Biol Review47:439-464,1972. 7. Jeffcott LB: Studies on passive immunity in the foal. J Comp Patho184:93-101,1974. 8. Koterba AM, Brewer BD, Tarplee FA: Clinical and clinicopathological characteristics of the septicemic neonatal foal: a review of 38 cases. Equine VetJ 16:376-383,1984. 9. Kruse-Elliott K, Wagner PC: Failure of passive antibody transferin the foal. CompendContinEduc Pract Vet6:S702-S707,1984. 10. LeBlanc MM, Pritchard EL: Effects of bovine colostrum, foal serum immunoglobulin concentration and intravenous plasma transfusion on chemiluminescence response of foal neutrophils. An Genetics 19:227-237,1988. 11. McGuire TC, Crawford TB: Passive immunity in the foal: measurement of immunoglobulin classes and specific antibody. Am J Vet Res 34:1299-1303,1973. 12. McGuire TC, Poppie MJ, Banks KL: Hypogammaglobulinemia predisposing to infection in foals. J Am Vet Med Assoc 166:7175,1975. 13. McGuire TC, Crawford TB, HaJlowell AL, Macomber LE: Failure ofcolostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. JAm VetMedAssoc 170:13021304,1977. 14. Morris DD, Meirs DA, Merryman GS: Passive transfer failure in horses: incidence and causative factors on a breeding farm. J Am Vet Med Assoc 46:2294-2299,1985. 15. Rumbaugh GE, Ardans hA, Ginno D, TrommershausenSmith A: Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with zinc sulfate turbidity test, serum eletrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test. J Am Vet Med Assoc 172:321-325,1978. 16. Rumbaugh GE, Ardans AA, Ginno D, TrommershausenSmith A: Identification and treatment of colostrum-deficient foals. JAm Vet Med Assoc 174:273-276,1979. 17. Steele TGD, Torrie JH: Principles and procedures of statistics: a biometrical approach. 2nd ed., McGraw-Hill Book Co New York, New York, 1980.
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