IDEXX Animal Health Updates: June 2008

BVDV can affect cattle of all ages, in every herd size, worldwide and in every U.S. state. The infection is one of the most costly diseases for the cattle industry worldwide, with losses reported at $10–$88 per head.

Bovine viral diarrhea virus (BVDV) causes a complex viral disease that suppresses a cow’s immune system, making it susceptible to a range of respiratory and reproductive illnesses. The virus can cross the placenta in infected pregnant cows, causing reproductive losses due to abortions, stillborn calves and calves that die early in life. Calves can be born with the infection and remain infected for life, continually spreading the disease to other animals. BVDV can affect cattle of all ages, in every herd size, worldwide and in every U.S. state. The infection is one of the most costly diseases for the cattle industry worldwide, with losses reported at $10–$88 per head.

Transmission
An exposed cow can become transiently infected by close contact with an infected cow. A transient infection lasts several days to a few weeks until the cow’s system fights off the virus. During this time, the animal is highly susceptible to other diseases.

A PI calf sheds 1,000 times more virus than an animal with a transient infection. PI animals are the main source of BVDV transmission.

Much more serious is a persistent infection. A calf is born with a persistent infection if its mother becomes infected between days 30 and 150 of gestation. Persistently infected (PI) calves remain infected for life and can continually infect other animals. Most calves with a persistent infection die within the first 18–24 months of life, but some live well into adulthood, appearing as healthy as the healthiest calf in the herd. A PI calf sheds 1,000 times more virus than an animal with a transient infection. PI animals are the main source of BVDV
transmission.

Disease Control
Vaccination alone cannot control BVDV because the large amount of virus shed by a PI animal can overwhelm even a vaccinated animal.

The IDEXX HerdChek* BVD Antigen Test Kit is the only USDA-licensed test for detecting BVDV infection in PI cattle.

BVDV control requires a combined program of vaccination, biosecurity and early testing specifically for PI animals. Testing is essential, so that PI animals can be identified and removed before they spread BVDV infection. The IDEXX HerdChek* BVD Antigen Test Kit is the only USDA-licensed test for detecting BVDV infection in PI cattle. Consult your veterinarian to determine the most appropriate measures to take.

*HerdChek is a trademark or registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries.

IDEXX HerdChek* BVD Antigen Test Kit: Summary of Results from Large-Scale BVDV Laboratory Testing

This article is adapted from an oral presentation by John Lawrence of IDEXX Laboratories (Westbrook, Maine, USA) and Chris McChure of Gold Standard Labs (Hereford, Texas, USA) at the 50th Annual Conference of the American Association of Veterinary Laboratory Diagnosticians in October 2007.¹

Background
From late 2005 through the end of 2007, ear-notch samples representing 866,602 North American animals were tested by a private, independent testing laboratory using the USDA-licensed IDEXX HerdChek* BVD Antigen Test Kit.

Ear-notch samples represented a variety of cattle types and sources of origin. There were 546,616 samples from 34 U.S. states, 36,345 samples from Mexico and 522 samples from Canada. An additional 283,119 samples were of undetermined origin.

Testing Routines
Testing was performed at three locations in Texas and one in Kansas. IDEXX trained all laboratory personnel and provided standard washers, readers, pipettes and data reduction software. Suspect and positive results were rerun to confirm initial status. Samples that were still suspect after retesting were resampled in 3 weeks to confirm BVDV persistently infected (PI) status.

Results
The overall prevalence of BVDV positives was 0.40% (3,489 out of 886,602) and ranged from 0.00% to 0.79% based on source of origin. (In addition, a single submission of 2 samples from one state yielded 2 positive results for 100% prevalence). Results for all tests by source of origin are summarized in Table 1.

Results by Cattle Group
A total of 11,857 distinct groups of cattle were represented, with a little over 20% of groups (2,470) containing at least 1 BVDV PI animal. Results for cattle groups are summarized in Figure 1.

Results by Cattle Type
Samples from sale barn cattle accounted for the majority of samples tested (58.11%), with 0.40% yielding positive test results.

Other classes of cattle included Mexican (4.58% of total, 0.448% positive), dairy (1.14% of total, 0.375% positive), preconditioned (7.62% of total, 0.298% positive), ranch (1.62% of total, 0.314% positive), and natural and show (0.07% of total, no positives).

Samples from animals of undetermined class (“Unknown” and “Other”) represented approximately a quarter of the total (26.87%), with 0.442% yielding positive test results.

Figure 2 and Table 2 show the prevalence and 95% confidence interval for each cattle type.

Results by Weight Class
The weight classes for cattle ranged from <300 lb. (<136 kg), with 0.709% yielding positive test results, to ≥900 lb. (≥409 kg), with 0.083% yielding positive test results.

The largest number of cattle where weights were known was in the 400–499 lb. (182–226 kg) class (22.75% of total, 0.318% positive). There were 345,853 samples from cattle of unknown weight class (39.91% of total, 0.409% positive).

Figure 3 and Table 3 shows the average prevalence and the range of prevalence at 95% confidence interval by weight class. Figure 3 also displays a regression line showing a general trend of lower prevalence in heavier class animals (R² = 0.668).

Conclusions
The testing results of this large private laboratory are similar to previous reports of overall BVDV prevalence and differences in prevalence by weight class.^2,3 This report demonstrates that BVDV is prevalent across all types of cattle and sites of origin within North America.

BVDV ELISA testing using ear-notch samples and the IDEXX HerdChek BVD Antigen Test Kit continues to be a valuable method for identifying BVDV PI cattle and for understanding the prevalence of BVDV infection within various segments of the cattle industry.

Notes:

* HerdChek is a trademark or registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries.

Informational Tools on idexx.com

Have you visited the livestock and poultry technology page on idexx.com? If not, please take a look. You’ll find valuable information that can make a difference in the performance of your IDEXX test kits.

Do you want a new lab technician to learn how to run a FlockChek* ELISA like a pro and learn important tips along the way? View the ELISA video presentation.

Do you need to know how to prepare a milk sample or which layer to draw the sample from for an assay? The ELISA Technical Guide, which includes a troubleshooting section, has the answer for you.

Would you like a handy list of tips for ELISA testing that can be posted in the laboratory for quick reference? Print out our ELISA Tips.

We hope these informational tools help you get the most value from your IDEXX products.

*FlockChek is a trademark or registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries.

Events Around the World

Latest BVDV Publications and Ring Trial Review (2006/2007)

The IDEXX HerdChek* BVD Antigen Test Kit (available in the U.S.) and the IDEXX HerdChek BVDV Antigen Serum Plus Test Kit have received excellent marks from U.S. and European studies and ring trials. A list of publications is shown below; a summary of each study follows the list.

1. Edmondson MA, Givens MD, Walz PH, et al. Comparison of tests for detection of bovine viral diarrhea virus in diagnostic samples. J Vet Diagn Invest. 2007;19:376-381.

In her study, Dr. Edmondson examined how the lack of standard test methods might prevent the consistent identification of animals affected with bovine viral diarrhea virus (BVDV). Dr. Edmondson used intra- and interlaboratory comparisons to demonstrate distinct differences in diagnostic methods for the detection of BVDV infection.

The following diagnostic methods were used in the study: immunohistochemical staining (IHC), antigen-capture enzyme-linked immunosorbent assay (IDEXX HerdChek BVD Antigen Test Kit), virus isolation (VI), and reverse transcription polymerase chain reaction (RT-PCR).

Samples were collected from 4 animals older than 7 months, including 2 BVDV negative animals, a persistently infected (PI) animal, and a PI animal that previously lacked detectable virus in serum, as determined by VI. A total of 890 samples were submitted to 23 participating diagnostic laboratories. Samples included serum (for the IDEXX HerdChek BVD Antigen Test, RT-PCR and VI), whole blood (for RT-PCR and VI) and skin biopsies (for IDEXX HerdChek BVD Antigen Test and IHC).

The IDEXX HerdChek BVD Antigen Test Kit, performed on skin, correctly identified 100% of both positive and negative samples and demonstrated the greatest agreement among laboratories.

The IDEXX HerdChek BVD Antigen Test Kit, performed on serum, correctly identified 91% of positive samples as positive.

RT-PCR (serum) and IHC (skin) performed well by correctly identifying ≥ 85% of positive samples.

VI, performed on serum, yielded the lowest percentage of correct positive identifications and the lowest level of agreement among laboratories.

Dr. Edmondson concluded that the IDEXX HerdChek BVD Antigen Test (performed on skin) was the most capable of accurately identifying animals both infected with BVDV (positive predictive value = 100%) and truly negative for BVDV (negative predictive value = 100%).

She also indicated that the level of agreement among laboratories for detecting BVDV in PI cattle ranged from perfect to less than expected by chance. In her opinion, the variation among laboratories suggests a need for training in standardized laboratory protocols and proficiency testing.

2. Fux, RG. Development and evaluation of diagnostic methods for detecting bovine viral diarrhea virus in dried ear biopsy samples using antigen-ELISAs and real-time RT-PCR [dissertation]. Munich, Germany: University of Munich Veterinary Department; 2007.

Early detection and elimination of cattle persistently infected with BVDV are key elements for BVDV eradication programs. Testing dried skin biopsies derived from ear tagging could be useful for detecting BVDV in newborn calves. The aims of this study were to develop methods for antigen solubilization and RNA preparation, to investigate the stability of these viral components, and to compare the analytical sensitivity of different tests. Tests included an enzyme-linked immunosorbent assay (ELISA) based on the E^rns antigen (the IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit), ELISAs based on the NS2/3 (p80) antigen and real-time RT-PCR.

These results agreed with the results of a study by Hilbe et al¹ that found that maternal antibodies usually do not interfere with the IDEXX ELISA.

The BVDV NS2/3 (p80) antigen was not suitable for BVDV detection by ELISA because a) solubilization of the NS2/3 (p80) protein was very difficult, b) detectable amounts of NS2/3 (p80) antigen strongly varied, c) storage at 50° C and 37° C led to loss of sensitivity resulting in either false-negative or doubtful results respectively and d) in ear notch tissues from calves with maternal antibodies NS2/3 (p80) antigen detection could be completely inhibited.

The study concluded that the sensitivity of available NS2/3 (p80) ELISAs was too low to safely detect BVDV antigen in dried ear notch samples of young calves and therefore BVDV NS2/3 (p80) antigen tests should not be recommended for ear notch testing of young calves.

3. Ridpath JF, Hessman BE, Neill JD, et al. Parameters of ear notch samples for BVDV testing: stability, size requirements and viral load. Proceedings from: 39^th Annual Convention of the American Association of Bovine Practitioners; Semptember 2006; St Paul, Minn, USA

In her presentation, Dr. Ridpath discussed her efforts to establish working parameters for sample size, viral detection limit and sample storage conditions for real-time PCR and antigen-capture ELISA tests for bovine viral diarrhea virus (BVDV). Her study also investigated the reproducibility of tests across three laboratories, based on a blinded panel of pooled and unpooled samples.

Dr. Ridpath noted that the most consistent results were observed among laboratories using the IDEXX HerdChek BVD Antigen Test Kit on unpooled tissues. The IDEXX HerdChek BVD Antigen Test correctly identified all samples (100%) as positive in all three laboratories.

The next most consistent results were those from the HerdChek BVD Antigen Test Kit performed on samples pooled 1:5 and on samples pooled 1:10.

The least consistency was observed among laboratories using PCR tests on sample pools of 1:100 or greater.

The IDEXX HerdChek BVD Antigen Test Kit, used with single samples, correctly identified 100% of positive samples.

PCR tests, used with single samples, correctly identified 78% of positive samples. The percentage of correct identifications by PCR declined with sample pooling. PCR tests on sample pools of 1:100 or greater correctly identified only 17.5% of positives.

Combined test results from three laboratories using antigen-capture ELISA vs real-time PCR
IDEXX HerdChek BVD Antigen Test Kit	Total Samples	Number Correct	% Correct
1:1 (no pooling)	60	60	100.00%
Note: The IDEXX HerdChek BVD Antigen Test Kit is not approved for pooled samples, therefore results from pooled samples are not shown.
Real-time PCR	Total Samples	Number Correct	% Correct
1:1 (no pooling)	80	62	77.50%
1:10 pool	80	31	38.75%
1:100 pool	80	14	17.50%

Dr. Ridpath advised that “The concentration range of virus in ear notch extractions and the detection limits of real-time PCR suggest that pooling of samples in surveillance programs must be approached cautiously. Pooling of 10 samples, where a sample pool includes one positive and nine negative samples, could result in the failure to detect 10% of the samples used in this study. Pooling of 100 samples, where sample pool includes one positive and 99 negative samples, could result in failure to detect over 50% of the samples used in this study.” Dr. Ridpath concluded that pooling of 1:10 and 1:100 will result in missed positives.

The HerdChek BVD Antigen Test Kit was the more robust of the two tests when used on ear notch samples.

4. Schirrmeier H, Teifke JP. BVD interlaboratory comparison trial 2006—a contribution to quality assurance in BVD diagnosis. Tierarztliche Umschau. 2007; 62:467–479.

The German National Reference Laboratory for BVD/MD organized an interlaboratory comparison trial involving 38 laboratories (including international laboratories). Identical sets of 29 coded samples (23 sera, 4 ear notches and 2 cryosections) were provided to the laboratories with a request that the samples be tested using antigen, antibody or genome detection methods.

The results indicated the high standard of bovine viral diarrhea virus (BVDV) laboratory diagnosis. The IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit, based on the E^rns antigen, was the most-used test. Some laboratories used an ELISA based on the NS2/3 (p80) antigen, and some used reverse-transcription PCR (RT-PCR).

Results for the panel of 23 serum and plasma samples (antigen-detection ELISAs)

The HerdChek BVDV Antigen/Serum Plus Test Kit was used in 32 out of 34 laboratories performing antigen-detection ELISA tests. The test delivered 100% correct results.

One NS2/3 (p80) Ag ELISA produced 3 false-negative results. The authors commented that “In one laboratory three positive samples scored negative in a test which is based on the detection of NS2/3 and which is not suitable for analysis of serum samples.” ²

The IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit was tested in 24 out of the 31 laboratories that tested ear notches. All but one of the HerdChek test results were correct; the incorrect result was a false positive very close to the test cutoff (S/N = 0.32, cutoff = 0.30).

Ten RT-PCR results for ear notches included 3 false negatives and 1 false positive.

The results of the German international interlaboratory BVDV trial indicate that the IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit is a highly standardized, reliable, accurate and efficient tool for detection of BVDV infection.

*HerdChek is a trademark or registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries.

The IDEXX HerdChek BVD Antigen Test Kit is available only in the United States.
The IDEXX HerdChek BVDV Antigen/Serum Plus Test Kit is available outside the United States.

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June 2008 Issue

BVDV—Signs, Transmission and Control

IDEXX HerdChek* BVD Antigen Test Kit: Summary of Results from Large-Scale BVDV Laboratory Testing

Informational Tools on idexx.com

Events Around the World

Latest BVDV Publications and Ring Trial Review (2006/2007)

We want to hear from you... and we want you to hear from us!