A Comparative Study of an Indirect Fluorescent Antibody Test and an ...

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Brief Communications

J Vet Diagn Invest 14:420–423 (2002)

A comparative study of an indirect fluorescent antibody test and an immunoperoxidase monolayer assay for the diagnosis of porcine proliferative enteropathy Roberto M. C. Guedes, Connie J. Gebhart, Nathan L. Winkelman, Rebecca A. Mackie-Nuss Abstract. The currently used indirect fluorescent antibody test (IFAT) for the detection of antibodies against porcine proliferative enteropathy (PPE) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used in this comparison were collected from 5-week-old pigs on day 0 (pre-experimental challenge) and on days 7, 14, 21, and 28 after oral inoculation with intestinal homogenate from pigs affected by PPE (28 challenged pigs) and sucrose phosphate glutamate solution (2 control pigs). All animals were euthanized 4 weeks after inoculation. Immunohistochemistry staining was applied to formalin-fixed, paraffin-embedded sections of ileum for the detection of Lawsonia intracellularis antigen. The serology results with each method agreed in all samples, except on days 0 and 7 in 1 control animal, which was positive by IPMA, but negative by IFAT. The percentage of agreement between IFAT and IPMA was 98.6%. Porcine proliferative enteropathy (PPE) is a widespread enteric disease affecting the aboral small intestine and, sporadically, the large intestine of pigs. The 2 available antemortem methods for diagnosis of PPE are polymerase chain reaction of fecal samples and serology. Serology presents a less expensive option for testing and is more amenable to high-volume testing. Enzyme-linked immunosorbent assay1 and indirect fluorescent antibody tests (IFATs)3 with Lawsonia intracellularis antigens prepared from intestines of infected pigs have shown variable and low antibody titers. In contrast, other investigators, who used cell culture–grown L. intracellularis in an IFAT, reported 90% seroconversion in pigs challenged with pure culture, 21–28 days after inoculation.2 The immunoperoxidase monolayer assay (IPMA) is a serologic enzyme immunoassay test commonly used for the diagnosis of viral infection, for instance, porcine respiratory and reproductive syndrome (PRRS).5,7 The obligate intracellular organism L. intracellularis infects cell line monolayers in vitro. Consequently, cell monolayers can be used in the same way as virus-infected cell line monolayers to perform serology with the IPMA. The goal of this study was to compare the accuracy of IFAT and IPMA serology methods for the diagnosis of PPE in experimentally infected pigs. Thirty 5-week-old pigs weighing between 20 and 30 pounds (between 9.1 and 13.6 kg) were obtained from a herd with no history of PPE. The source herd was also clinically negative for Salmonella cholerasuis, transmissible gastroenteritis, and pathogenic Brachyspira species, and serologically negative for PRRS and Actinobacillus pleuropneumoniae. Twenty-eight pigs were inoculated via stomach tube with a homogenate of scraped intestinal mucosa from PPEaffected pigs in sucrose–phosphate–glutamate (SPG) on days From the Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, 1971 Commonwealth Avenue #205, Saint Paul, MN 55108 (Guedes, Gebhart, MackieNuss), and Swine Services Unlimited Inc., 10 East 6th Street, Morris, MN 56167 (Winkelman). Received for publication October 5, 2001.

0 and 1.8 All pigs received 2 doses of 25 ml with a total dose given per pig of 7.55 ⫻ 109 L. intracellularis organisms. The 2 remaining pigs (controls) were allotted to a different pen separated by wood walls from the pens with challenged pigs. These 2 animals were inoculated on days 0 and 1 with 25 ml of SPG only, in the same manner used for challenged animals. Blood samples were collected from all animals on days 0, 7, 14, 22, and 28 after inoculation. Serum samples were tested by IFAT, as described elsewhere,2 and by IPMA. Acetone-fixed 96-well culture plates with McCoy cells highly infected with L. intracellularis were used for both serology methods. Wells were seeded with 5 ⫻ 103 McCoy cells and grown for 24 hours before infection. Pure cultures of L. intracellularis were added to Dulbecco’s modified Eagle’s mediuma with 5% fetal bovine serum (FBS), and 100 ␮l of this preparation containing about 105 L. intracellularis organisms was added to each well. The plates were incubated for 5 days in a gas concentration of 8.0% O2, 8.8% CO2, and 83.2% N2. Cold 50% acetone and 50% methanol solution was used to fix the cells. The plates were stored at ⫺20 C until use. For the IPMA, the plate was rehydrated with distilled water at 37 C for 10 minutes. The water was then discarded and 50 ␮l of diluted serum sample was added to the test well. The plate was incubated for 30 minutes at 37 C and was then washed 5 times with phosphate-buffered saline (PBS), pH 7.2. Anti-porcine immunoglobulin G (IgG)-peroxidase conjugate,b diluted 1:600 in buffer (PBS with 2.5% FBS, 1% inactivated porcine serum, and 0.08% Tween 80), was added at a concentration of 30 ␮l/well, and incubation proceeded for 45 minutes at 37 C. The plate was washed again and chromogena (3-amino-9-ethyl-carbazole) solution, prepared in accordance with the manufacturer’s instructions, was added to each well and incubated at room temperature for 20 minutes. The plate was washed with distilled water 3 times, allowed to dry, and examined with an inverted light microscope. Animals with a titer of 1:30 were considered positive in both tests. Specific antibody–positive and –negative pig sera were included on each plate as controls.


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Table 1. Serology results on days 0, 7, 14, 22, and 28 after challenge with immunoperoxidase monolayer assay (P) and direct fluorescent antibody test (F), and immunohistochemistry (IHC) of ileum sections from samples collected 28 days after challenge. Day 0 Sequence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Pig

36 132 23 47 137 158 64 13 152 35 1 149 100 81 28 55 106 98 135 24 84 115 57 172 71 34 138 113 90 16

Day 7

Day 14

Day 22

Group*

P

F

P

F

P

F

C C Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch Ch

⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹

P

Day 28 F

⫺ ⫺ ⫺ ⫺ Died day 17 Died day 21 ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

P

F

IHC

⫺ ⫺

⫺ ⫺

0 0 4⫹ 3⫹ 3⫹ 4⫹ 4⫹ 3⫹ 3⫹ 3⫹ 3⫹ 3⫹ 2⫹ 2⫹ 2⫹ 2⫹ 2⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 1⫹ 0 0

Died day 23 Died day 24 ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

* C ⫽ control group; Ch ⫽ challenged group.

All pigs were euthanized 28 days after inoculation. Samples of ileum from each pig were fixed in 10% neutral buffered formalin and processed routinely for histology. To confirm the infected status, 1 section was stained by immunohistochemistry (IHC) by using the immunoperoxidase method of labeled streptavidinc with mouse monoclonal antibodies to L. intracellularis.4 The concentration of positive antigen labeled was graded as follows: 0 ⫽ no positive antigen labeled; 1⫹ ⫽ 1 isolated focal area of antigen labeled; 2⫹ ⫽ multifocal areas of antigen labeled; 3⫹ ⫽ majority of the mucosa has positive antigen labeled; and 4⫹ ⫽ all of the mucosa has positive antigen labeled. The serology tests with both methods agreed in all samples, except on day 0 and 7 of control animal 132 (Table 1). When each sample tested was considered individually (144 samples), the percentage of agreement between IFAT and IPMA was 98.6%. Six of 28 challenged pigs were seropositive on day 14 in both serologic tests (21.4%); and 24 of 26 and 23 of 24 challenged pigs also were seropositive in both tests (92.3 and 95.8%) on days 22 and 28 after challenge, respectively. Negative control pig 132 was positive by IPMA in the first 2 bleedings and then became negative. Two explanations are possible for this finding. First, those 2 positive re-

sults are falsely positive. Second, those results are due to maternal antibodies and the IPMA is slightly more sensitive than the IFAT. To investigate the possible cause of those 2 positive results in a control pig, a random bleeding of 10 sows from different parity orders in the source herd was performed. One of the 10 sows was seropositive by both IFAT and IPMA. Because control pig 132 was positive in 2 consecutive tests and then became negative, the positive results on days 0 and 7 probably were due to the presence of maternal antibodies. Only 2 of the 28 challenged animals did not have detectable antibodies (IgG) with both serologic methods (sensitivity of 92.9%). Knittel et al.2 showed a similar sensitivity level (90%) with the same tissue-culture IFAT 3 weeks after experimentally infecting pigs with pure culture of L. intracellularis. In this study, 1 challenged animal (pig 106, sequence 17 in Table 1) was seronegative throughout the study in both tests; however, IHC showed multifocal areas of antigen label in some mucosal glands (IHC grade 2⫹). The humoral immune response of this pig was either low and not detectable by IFAT and IPMA or this animal became infected by exposure to feces of infected pen mates and not as a result of direct experimental inoculation. The other pig that did not seroconvert (pig 47, sequence 4 in Table 1) died

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Figure 1. a, negative and b, positive indirect fluorescent antibody test and c, negative and d, positive immunoperoxidase monolayer assay for Lawsonia intracellularis with infected cell culture monolayer in 96-well plates. Notice in b the fluorescence in the cytoplasm of infected cells and in d the red-labeled bacteria in the cytoplasm and also extracellularly.

as a result of PPE on day 21 after challenge and also probably did not have time to develop a detectable IgG level when tested on day 14 after challenge. Two challenged animals were seropositive on days 14, 21, and 28 after challenge in both tests, but did not have positive antigen label in the ileum sections. Possible explanations for these results include that these animals seroconverted without any lesion, the lesions were resolved before the day of euthanasia, or the lesions were restricted to the jejunum and were not detected upon IHC on ileum sections. Four challenged animals (14.3%) died during the experiment between days 17 and 24 after challenge. All had severe necrosis of the superficial mucosa and extensive positive L. intracellularis–specific antigen label reaction (grade 3⫹ or 4⫹) in the ileum as detected by IHC. Unlike IFAT preparations, IPMA preparations can be examined only with an inverted light microscope. The color reactions in the IPMA are stable for several months, thereby providing a lasting record of the results.6 In addition, L. intracellularis IPMA-positive preparations of red-stained bacteria can be visualized intra- and extracellularly, in contrast to L. intracellularis IFAT-positive preparation where fluorescent clusters of bacteria can only be confidently detected in the cytoplasm of infected cells (Fig. 1). This is probably because only one fluorescein isothiocyanate molecule is pre-

sent per antibody binding site in the IFAT, whereas many chromogen molecules are present per each bound peroxidase-conjugated antibody in the IPMA. Therefore, the technical skills required for accurate interpretation of the IPMA results may be less than those for the IFAT. The IPMA requires 1 more step than the IFAT, but this additional step takes only 20 more minutes. A small additional cost is involved in the IPMA procedure because of the price of the chromogen, H2O2, and the inactivated porcine serum and FBS used in the buffer of the secondary antibody. However, this additional cost is negated, because the dilution of the anti-porcine IgG-peroxidase conjugate for IPMA is 1:600 in comparison to the anti-porcine IgG-fluorescein isothiocyanate conjugate for IFA diluted 1:10. In addition, the porcine serum and FBS concentrations used for the buffer are low and the buffer can be stored at ⫺20 C for a long period of time. As a result, the price of the IPMA was kept the same as for the IFAT. In conclusion, IPMA and IFAT showed similar results in experimentally infected pigs; however, IPMA preparations do not require the use of a fluorescent microscope, can be stored for a long time, and, based on this study, are considered to be easier to interpret. The obvious next steps are to validate the IPMA by testing specificity and sensitivity in experimental and field conditions, and to examine the pos-


sible effect of maternal immunity in piglets on test performance. Acknowledgements. We thank Evelyn Townsend for technical assistance and Dr. John Deen for critical review of the manuscript. R. M. C. Guedes was supported by the Brazilian government sponsoring agency Conselho Nacional de Desenvolvimento Cientifico e Tecnologico and Universidade Federal de Minas Gerais.

3.

4.

5.

Sources and manufacturers a. JRH Biosciences, Lenexa, KS. b. Sigma-Aldrich, St. Louis, MO. c. Dako Corporation, Carpinteria, CA.

References 1. Holyoake PK, Cutler RS, Caple IW, Monckton RP: 1994, Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis–specific immunoglobulin G response in sera of pigs. J Clin Microbiol 32:1980–1985. 2. Knittel JP, Jordan DM, Schwarz KJ, et al.: 1998, Evaluation of ante-mortem polymerase chain reaction and serologic methods

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for detection of Lawsonia intracellularis–exposed pigs. Am J Vet Res 59:722–726. Lawson GHK, McOrist S, Rowland AC, et al.: 1988, Serological diagnosis of the porcine proliferative enteropathies: implications for aetiology and epidemiology. Vet Rec 122:554–557. McOrist S, Boid R, Lawson GHK, McConell I: 1987, Monoclonal antibodies to intracellular campylobacter-like organisms of the porcine proliferative enteropathies. Vet Rec 121:421–422. Nodelijk G, Wensvoort G, Kroese B, et al.: 1996, Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus. Vet Microbiol 49:285–295. Soliman AK, Watts DM, Salib AW, et al.: 1997, Application of an immunoperoxidase monolayer assay for the detection of arboviral antibodies. J Virol Methods 65:147–151. Sorensen KJ, Strandbygaard B, Botner A, et al.: 1998, Blocking ELISA’s for the distinction between antibodies against European and American strains of porcine reproductive and respiratory syndrome virus. Vet Microbiol 60:169–177. Winkelman NL, Pauling GE, Bagg RN, et al.: 1998, Use of a challenge model to measure the impact of subclinical porcine proliferative enteritis on growth performance in pigs. Proc Am Assoc Swine Pract Meet, Des Moines, IA, pp. 209–211.

J Vet Diagn Invest 14:423–426 (2002)

Evaluation of result variability with a commercial Johne’s disease enzyme-linked immunosorbent assay kit and repeat testing of samples John M. Adaska, Claudia A. Mun˜oz-Zanzi, Sharon K. Hietala Abstract. Two hundred one serum samples from individual dairy cows with a range of results on initial testing with a commercial Johne’s disease enzyme-linked immunosorbent assay (ELISA) kit were repeat tested 5 times in each of 2 laboratories with kits produced by the same manufacturer. The results for the samples with all 10 replicates showed that the values for individual samples often had a coefficient of variation greater than 20%. As expected, the standard deviation for the results increased as the average value increased and the coefficient of variation was greater in samples with low mean values. The different lots of the commercial ELISA kit used in this study had a significant effect on both the optical density and the calculated sample to positive (S/P) ratio for test replicates. Based on the variability detected in S/P ratios of replicate samples, application of a single cutoff point to interpret individual test results as positive or negative for antibodies to Mycobacterium avium subspecies paratuberculosis could result in inconsistent classification of animals as positive or negative for Johne’s disease. Such inconsistency in test interpretation leads to frustration in large animal veterinarians or producers trying to make management decisions based on individual test results. Instead of dichotomizing the test results as positive or negative based on a single cutoff value, reporting numerical values and supplying a classification scheme that includes a suspect category reflecting the uncertainty inherent in the test is recommended to provide more reliable result interpretation. As disease and health management in both the dairy and From the California Animal Health and Food Safety Laboratory, Tulare Facility, 18830 Road 112, Tulare, CA 93274 (Adaska), the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, One Shields Ave, Davis, CA 95616 (Mun˜oz-Zanzi), and the California Animal Health and Food Safety Laboratory, Davis Facility, West Health Sciences Drive, Davis, CA 95616 (Hietala). Received for publication June 22, 2001.

beef industries continues to improve, Johne’s disease is becoming of increasing concern to veterinarians and producers. This disease has a slow insidious onset, which may result in significant production losses before the infected animal has overt clinical signs.1,2,6,10 In addition, animals can often shed the causal organism, Mycobacterium avium subspecies paratuberculosis (MAP), in their feces before showing clinical signs, resulting in contamination of the production environment and increased likelihood for the infection of other animals in the herd.8,9 As a result of these factors, and the