Featured News Monitoring Mycoplasmas in Poultry Farms Case Study 1 Case Study 2 Introducing New Label Format for IDEXX Europe Kits IDEXX Production Animal U.S. Reference Laboratory		MORE INFORMATION Newsletter Archive Subscribe now Print this page

Mycoplasma spp are primary bacterial pathogens that can cause respiratory problems in chickens and turkeys and/or be part of the chronic respiratory syndrome in chickens. Some of the most important causes of disease-associated loss in poultry production are related to infection with M. gallisepticum (MG) and/or M. synoviae (MS). Economic loss associated with Mycoplasma infection is often the result of a complex interaction between the Mycoplasma sp and co-infections with other bacteria, poor health management and/or poor environmental conditions. Studies have shown that over 80% of poultry operations worldwide are infected with MG and/or MS, making it one of the most prevalent and costly poultry diseases.1 Even at low levels of infection, this chronic respiratory disease presents significant added costs to poultry operations through reduced feed efficiency, repeated antibiotic treatments, poor chick quality and detrimental vaccine reactions.

Mycoplasma spp are maintained in many flocks by breeder-to-progeny transmission. Transmission within the flock occurs once infection is established in a few breeder chickens, especially when birds are under management conditions, such as individual vaccinations, live weight control and onset of egg production. In multi-age production systems, MG/MS and a number of other important respiratory pathogens may be transmitted in large numbers from older to younger birds. Overt signs of Mycoplasma infection are not usually seen until the flock experiences a stress condition that can facilitate the multiplication of MG/MS.

Although Mycoplasma infections are thought to be common, microbiologic evidence of the organism’s presence is difficult to confirm via isolation of the bacteria. Factors that may contribute to the prevalence of Mycoplasma spp are likely to include a long incubation period without clinical signs, a slow spread of the organism and delayed immune response detected by common serological methods (circulating antibodies can take at least two weeks to be detected at a high level). Detection of Mycoplasma infections can also be limited by the sample size used during flock surveillance. If the surveillance program includes fewer than 30 samples per population, the risk of missing the detection of the first infected animal (first index case) is increased. Early detection of a recent infection depends on the frequency of sampling, sample size and the sensitivity of the test. Also, there is not a serological test that has both 100% sensitivity and 100% specificity available for Mycoplasma spp.2

Based on the epidemiological distribution of these agents, the first target for a continuous surveillance program should be the great-grandparents, then the grandparents and the breeders to reduce the vertical transmission of the Mycoplasmas and introduction of the agent into MG/MS-free chicken houses.

To initiate a regular monitoring program in a unknown situation, the ideal first step should be to establish the prevalence of the Mycoplasma spp in each company or region. If this is not possible, some other guidelines, such as the National Poultry Improvement Program (NPIP), can be followed, starting with sampling 300 birds per flock of at least three different ages to be able to detect a 1% infection prevalence with 95% confidence.3 Where the rate of infection is higher and prevalence in early age flocks is 30% or more, the number of samples per population should also be between 30 and 60 samples per flock.

Dealing with presumed Mycoplasma-negative populations

The first step for controlling the respiratory complex in poultry operations is to define the prevalence of any of the potential agents that can be involved in the respiratory syndrome. In other words, one needs to look for the presence of those viruses and/or bacteria with low or high virulence affecting the upper respiratory tract and opening the door for other secondary pathogens to create a potentially devastating disease throughout the entire flock.

If a poultry operation or region is considered a Mycoplasma-free zone, a continuous program of screening both newly introduced and resident stock should be in place. These stocks should be screened with a very sensitive test to ensure with a high level of confidence that the results will not produce false-negative results. IDEXX ELISA tests are reliable tools for accomplishing the goals of first-stage screening. Two sensitive tests, such as the IDEXX FlockChek® ELISA kits for MS and MG, can detect recently acquired infections and indicate the need to proceed to more involved investigations with a more specific testing tool—such as polymerase chain reaction (PCR) assays to look for the presence of the organism, or hemagglutination inhibition (HI) and serum plate agglutination (SPA) to look for specific antibody reactivity against MS or MG.

A test with greater specificity should always be used to retest and confirm any single reactors from the screening test. But these single reactors should not be treated as false-positives until the more specific investigation is performed. A confirmatory test with a lower sensitivity will create the opportunity for a higher percentage of false-negatives at confirmation, and will increase the potential for the quick spread of the agent within the population and across the region/company.

The histogram analysis is also important for interpretation of results. This helps veterinarians analyze the population’s MG/MS status instead of looking at each bird’s individual results. Figure 1 shows ELISA results on SPA-negative flocks. The cutoff of MG and MS kits is 0.5. The mean S/P in the negative population was 0.058 for MS and 0.106 for MG. Both populations are positioned to the far left of the cutoff. Figure 2 shows ELISA results on a flock recently infected with MG (confirmed by PCR). Note that although only two samples were positive, the histogram has moved toward the right side of the population.

Dealing with Mycoplasma-positive populations

The most sensitive test is the best starting point for monitoring and establishing baselines for each farm/complex in a poultry operation. Using the ELISA as a tool for monitoring populations requires the testing of flocks at various ages using a statistically valid number of samples. We recommend that producers establish monitoring programs and choose a sampling plan that allows the veterinarian to rely on the results obtained. The veterinarian can analyze the data and arrive at accurate conclusions relating to the health management of the flock, and define strategies related to vaccine applications (e.g., age of application, routes of application, type of vaccine used, etc.).

IDEXX ELISA response to live MG vaccines

In a controlled study4, using specific-pathogen-free (SPF) birds with individual administration of live vaccines. The IDEXX ELISA has detected seroconvertion between 21 to 42 days after live vaccination. Most of the results were in group one, and up to 80% of the birds seroconverted. When live vaccines have been used in the field, the ELISA does not show a seroconversion in more than 30–40% of the birds tested. A revaccination of the same flock will probably increase the percent of positive birds.

The presence of birds in groups 2 and 3 would indicate that the vaccinated flock is facing a field challenge, and a correlation with the presence of clinical signs, including eggs per henhouse, should be measured.

IDEXX ELISA response to inactivated MG vaccines

In flocks that have been vaccinated with two inactivated vaccines, an immune response can be detected by the ELISA 3–4 weeks after the second vaccination. This response should not be higher than titer groups 2–3 (i.e., from 2,000–4,000 titer). If the titers reach a different or higher level, a correlation with field infection needs to be ruled out.

Figures 3 and 4 show an expected histogram for an MG-vaccinated flock (2x live plus inactivated) and field infection, respectively. Notice that the histogram moved toward the right side of the population when infection took place indicating the presence of a field challenge.

Nonspecific reactions: In some serological tests,5 some inactivated vaccines can cause a cross-reaction for two to three weeks after vaccine application. These are typically associated with MG reactivity, and, therefore, we recommend waiting for at least six weeks after vaccination to conduct Mycoplasma serology screening on those flocks.

Fourteen broiler breeder flocks from different ages (6, 16, 24, 32, 36 40, 48, 50, 51 and 61 weeks of age) were bled to evaluate different screening methods for Mycoplasma gallisepticum and Mycoplasma synoviae, using the hemagglutination inhibition test (HI) for MG and MS, and the FlockChek® MG/MS Antibody Test Kit to perform a flock correlation of the two tests and chose a reliable Mycoplasma screening tool. Eighteen samples per flock were collected and analyzed with the IDEXX ELISA and fifteen of those were analyzed with the HI tests (one for MG and one for MS). A total of 252 samples were analyzed by the IDEXX-ELISA and 210 samples by the HI tests.

Results:

One hundred and fifty-four samples were positive by either MG or MS by HI tests. From those samples, 142 were positive by the FlockChek® MG/MS Antibody Test Kit. The number of samples analyzed by the ELISA was higher—252 vs. 210 analyzed with the HI tests (see Table 1). Based on the positive results by the HI tests, there were 13 discrepant samples (154 by HI vs. 141 by the IDEXX ELISA; see Table 2).

If we take out the number of samples from the six-week-old flocks that are considered negative for MG or MS, the specificity for the HI test was 100%, and 96.66% for the IDEXX ELISA. The sensitivity for the potential positive population (flocks after 16 weeks of age) was 85.55% for the HI tests and 78.9% for the IDEXX ELISA for the fourteen flocks in this study. Comparison of tests methods on samples tested yields a kappa value of 0.84, which is considered an excellent agreement (see Table 2).

Summary of the results of case study 1:

Considering the results of the study, and the issues with antigen preparation variability and inconsistent interpretation for HI, the company switched to the IDEXX ELISA combo kit (MS/MG) for Mycoplasma screening. HI or PCR is used as confirmatory tool.

The purpose of this study was to investigate the use of the IDEXX MS/MG ELISA as a screening method for the detection of antibodies in chicken and turkey serum. ELISA results were compared to serum plate agglutination (SPA) and hemagglutination inhibition (HI) tests (HI- 4 HI Units) to determinate relative sensitivity and specificity.6

Samples

270 samples from chickens and turkeys previously evaluated by SPA and HI tests, including 15 specific-pathogen-free (SPF) serum samples.

Results:

Summary of the results of case study 2:

Considering the 32 samples that were consensus-positive (SPA and ELISA) and HI-negative (dilution interpretation), the Kappa value of 0.90 represents excellent agreement between the IDEXX ELISA and traditional tests (see Table 8).

Based on the results of this study, this company chose the IDEXX MG/MS ELISA as its screening method for the detection of antibodies against MG and/or MS. Specifically, the diagnostic performance and standardized nature of the ELISA were considered superior to the high antigen variability and subjective interpretations inherent in SPA or HI tests methods.

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Introducing New Label Format for IDEXX Europe Kits

As a part of our commitment to providing innovative products and services to our customers, IDEXX is pleased to announce a new format for kit labels and inserts for all HerdChek®, FlockChek® and CHEKIT™ kits produced at our European production facility in Bern, Switzerland. The new labels offer more complete and useful product information.

With the new labels, IDEXX customers will benefit from:

• Standardized ISO symbols for important kit handling and batch information
• Distinctive, easy-to-read formats that enable users to more quickly find the information they need.
• Up to five languages (determined by primary markets for each kit)
  • English, German, French, Spanish and Portuguese
    
    

The new label updates will be implemented over the next twelve months, starting with HerdChek® and FlockChek® products, followed by CHEKIT™ kits.

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IDEXX Production Animal U.S. Reference Laboratory

Over the past year, IDEXX has phased out the production animal reference laboratory services that had been offered at our Westbrook, Maine, location. This process is complete, and the service is no longer available as of April 1, 2006.

We regret any inconvenience that this may cause our customers. For questions about alternative testing services for livestock and poultry serum samples, you can contact our technical service department at: 1-800-548 9997, ext: 4895 for assistance in locating a facility to process your samples.

For customers who have used our IBD-PCR typing service, we have arranged for the lab at Ohio State University is offering this testing. Please contact:

IBDV Typing Laboratory
Food Animal Health Research Program
The Ohio State University/OARDC
1680 Madison Ave
Wooster, OH 44691 USA

Contacts:
Daral J. Jackwood, jackwood.2@osu.edu
Susan Sommer-Wagner, sommer-wagner.1@osu.edu

Telephone: 1-330-263-3749
Fax: 1-330-263-3760

Web site: www.oardc.ohio-state.edu/ibdv/IBDVDiagnosticServices.asp

Again, we regret any inconvenience that this may cause you and your laboratory. Feel free to contact us with any questions.

 

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Technical Tip

Pipetting to Avoid Bubbles

Careful pipetting is crucial in obtaining accurate test results when performing any ELISA test. Sometimes air, resulting in bubbles, can be drawn up into the pipette or dispensed into the wells. If this happens, bubbles can influence optical density values and results. To minimize or eliminate this problem, reverse pipetting is recommended for the addition of reagents to the ELISA plate.

Reverse Pipetting with a Multichannel Pipette

1. Put new tips on the pipette, ensuring they are on tight and straight.
2. Press the plunger past the first stop and halfway to the second stop.
3. Draw the liquid in a slow motion, being careful that no air bubbles are drawn into the tip. Check for consistency of volume in the tips.
4. Touch the tips to the edge of the reagent reservoir to remove excess liquid on the outside of the tips.
   • If the wells on your plate are empty, position the tips into the lower corner of the wells, making contact with the plastic.
   • If the wells on your plate contain liquid, position the tips above the liquid, making contact with the plastic.
5. Slowly dispense the liquid into the wells by depressing the plunger to the first stop. Be careful not to splash liquid out of the wells, and make sure there are no drops left on the tips.
6. To repeat, hold the plunger at the first stop and continue with step 3.
7. Eject the tips into an appropriate waste container.
   NOTE: Reverse pipetting uses more reagent/volume (="dead volume").

 

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Learn More

To learn more about our products and services, innovative technologies, trusted service and support, and our global offices, visit our Web site.

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Visit Us at the Following Events

San Antonio, Texas USA—April 18–20, 2006
Southern Animal Heath Association (SAHA)

Bad Staffelstein, Germany—April 23–25, 2006
BbT-Kongress

Ixtapa, Mexico—April 26–29, 2006
XXXI Convencion Anual ANECA

Santos, Brazil—May 3–5, 2006
Conferência Apinco de Ciência e Tecnologia

St. Malo, France—May 10–12, 2006
International Symposium Salmonella and Salmonellosis I3S 2006

The Netherlands—May 16–18, 2006
VIV Europe Utrecht

Athens, Georgia USA—May 22–26, 2006
AMEVEA

London, England—May 28–30, 2006
International Conference: Prion Diseases of Domestic Livestock

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...and we want you to hear from us! Pass the following link along to your friends and colleagues. They can register for Animal Health Updates to stay informed of the latest IDEXX products and services and respond to various customer and market surveys. Registration is easy at www.idexx.com/production/livestockpoultrynews.

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*HerdChek, FlockChek and Chekit are trademarks or registered trademarks of IDEXX Laboratories, Inc. in the United States and/or other countries. All other product and company names are trademarks or registered trademarks of their respective holders.