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• The Latest News—Neospora caninum-Associated Abortion in Cattle—Diagnosis and Control



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• Technical Tip of the Month—Agar Preparation



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Neospora caninum-Associated Abortion in Cattle—Diagnosis and Control

Neospora caninum is a protozoal parasite, that can cause abortion and neonatal morbidity and mortality in cattle. It was first identified as an important cause of abortion in dairy cattle in New Mexico, and described as a major cause of abortion in California dairy cattle in 1991.^1,2 N. caninum is morphologically similar to Toxoplasma gondii, Sarcocystis spp. and Eimeria spp.

Neosporosis is considered a major cause of bovine abortion throughout the world.³ Cows seropositive for N. caninum have a decreased daily milk production compared to uninfected cows.⁴ Seropositive cows in N. caninum-infected herds also have a higher risk of abortion compared to seronegative cows.⁵

In cattle, N. caninum is transmitted transplacentally (vertical) with high efficiency to the progeny. N. caninum-associated abortions can be endemic or epidemic in a herd. There is also some epidemiological evidence of possible point exposure associated with herd abortion outbreak.⁶

N. caninum infection in cattle is associated with abortion around the fifth month of pregnancy. Congenital infection may cause mortality, ataxia and neurological disease in neonatal calves, as well as weak calves and stillbirth due to multifocal encephalitis. Natural infections are found in cattle, sheep, goats, horses, dogs and deer that can be intermediate hosts for N. caninum. To date, the dog has been identified as a definitive host for N. caninum.⁷

In aborted fetuses, there are no typical macroscopic lesions. Pathohistologically degenerative inflammation in heart, muscles, liver and CNS can be observed. Tissue cysts containing bradyzoites can be observed in neural tissues, and tachyzoites are found in many cell types.³

Direct detection of infection can be done by screening aborted fetal tissue for the presence of Neospora organisms using immunofluorescence assay (IFA), polymerase chain reaction (PCR) or by histological examination. In cows, indirect diagnosis for detection of antibodies against N. caninum can be done by serological testing using indirect IFA or enzyme-linked immunosorbent assay (ELISA).

The following diagnostic steps should be taken to investigate N. caninum as a potential cause of cases of bovine abortion within a herd:

• Test cows that aborted for N. caninum antibodies using ELISA or IFA. If any prove positive, screen all cows in the herd for N. caninum antibodies.
• Test aborted fetal tissue by PCR for the presence of N. caninum DNA, or perform histopathology analyses looking for changes compatible with N. caninum infection together with bradyzoite and tachyzoite detection.

It is important to remember that positive N. caninum antibody titers found in individual cows are indicative of infection, not of abortions due to neosporosis. The interpretation of serological results should always be done together with epidemiology.

The *HerdChek® Neospora caninum Antibody Test Kit is an IDEXX enzyme immunoassay for the detection of antibody to N. caninum in bovine serum. Using a cutoff of 0.50 S/P, the assay shows sensitivity of 98.6% and specificity of 98.9%, compared to IFA.⁸ Researchers in Germany have found an excellent agreement (kappa 0.95) between the IDEXX ELISA test and their immunoblot.⁹ The assay can be used for epidemiological studies and for management decisions to attempt to reduce the prevalence of neosporosis in cattle herds.

Recently developed vaccines derived from tissue culture-grown Neospora tachyzoites could be an aid in reducing abortions in healthy, pregnant cattle that are infected with Neospora caninum.¹⁰ The HerdChek® Neospora caninum Antibody Test Kit could also be an adjunct tool with vaccine compliance testing.

Seroepidemiological data can be an important tool for developing strategies to control neosporosis worldwide. IDEXX has the diagnostic and software tools to identify cattle and cattle herds infected with Neospora caninum.

REFERENCES:

1. Thilsted JP, Aubey JP. Neosporosis-like abortions in a herd of dairy cattle. J Vet Diagn Invest. July 1989;1(3):205–9.
2. Anderson ML, Blanchard PC, Barr BC, Dubey JP, Hoffman RL, Conrad PA. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J Am Vet Med Assoc. 1991;198(2):241–244.
3. Dubey JP, Lindsay DS. Neosporosis. Parasitol. Today. 1993:9,452–458.
4. Hernandez J, Risco C, Donovan A. Association between exposure to Neospora caninum and milk production in dairy cows. JAVMA. 2001;219(5):632–635.
5. Thurmond MC, Hietala SK. Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am. J. Vet. Res. 1997;58(12):1381–1385.
6. McAllister MM, Huffman EM, Hietala SK, Conrad PA, Anderson ML, Salman MD. Evidence suggesting a point source exposure in an outbreak of bovine abortion due to neosporosis. J. Vet. Diagn. Invest. 1996;8(3):355–357.
7. McAllister MM, Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 1998;28(9):1473–78.
8. IDEXX USDA data package. HerdChek® Neospora caninum Antibody Test Kit. 1996.
9. Schares G, Peters M, Wurm R, Bärwald A, Conraths FJ. The efficiency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet. Parasit. 1998;80:87–98.
10. Choromanski L, Block W. Humoral immune responses and safety of experimental formulations of inactivated Neospora vaccines. Parasitol Res. 2000;86(10):851–853.

JOB OPENINGS

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VISIT US AT THE FOLLOWING EVENTS

Des Moines, Iowa, United States—June 5–7, 2003
World Pork Expo, at the Iowa State Fair Grounds, Booth #2463.

Rome, Italy—June 29–July 2, 2003
4th International Symposium on Emerging & Re-emerging Pig Diseases, Palazzo dei Congressi. Booth #9.

Denver, Colorado—July 19–23, 2003
AAAP/AVMA Annual Meeting/XIII Congress of the WVPA, Colorado Convention Center.

TECHNICAL TIP OF THE MONTH

Agar Preparation—the quality and consistency of the Noble agar media utilized has a direct and significant impact on the clarity and readability of the precipitin lines formed in the immunodiffusion reaction. Attention to the following points will ensure optimal agar quality for consistent assay performance.

Borate Buffer Preparation. Careful attention to weighing and adding the correct quantities of sodium hydroxide and boric acid will yield a buffer near the target pH of 8.6. The pH should always be verified with a calibrated pH meter. Filtration of the buffer with a sterile 0.22-micron filter unit will ensure a solution free of particulate matter that can be stored for multiple agar preparations.

Media Choice. Noble agar is the standard dry medium required for this protocol. Use of the correct, high purity agar is essential for optimal assay results.

Heating the Agar. A 1% solution of Noble agar is prepared in the borate buffer by gradually heating the mixture to approximately 100°C. The use of a Pyrex® flask, thermometer, stir bar and a heated magnetic stir plate offers the best technique for reaching the required melting point of the agar without overheating the solution or losing an excessive quantity of moisture. The use of an autoclave has been a common technique for agar preparation, but this method is subject to variations in equipment, and there is the potential risk of overheating the agar.

"Overheating or prolonged heating will change the composition of the medium...Agar media on prolonged sterilization or heating are likely to show a precipitate." "Repeated melting of solidified agar, or long holding of melted agar at high temperature may likewise cause a precipitate to form in the media...Excessive heating of the media also results in an increase in acidity." (Difco manual, 10th edition).

Discoloration of the agar (a light-brown caste) may indicate that overheating has occurred. The use of a microwave oven for melting the agar is also subject to equipment variability, and the possibility of either overheating or underheating the agar solution. Due to the rapid effect of microwave heating, there is little opportunity to adjust the heating cycle in response to the measured temperature of solution. Remelting previously prepared media will sometimes produce suboptimal agar quality in comparison to fresh prepared media. The preparation of smaller, more frequent batches of agar will help to ensure a reliable medium.

Pouring Plates. Cooling the melted agar (with stirring) to a temperature of 55°–60°C prior to dispensing into petri dishes will result in a uniform agar consistency and a smooth top surface to the set gel. Cooling below this range may cause reformation of solids as the agar sublimates. Dispensing the agar into petri dishes should be performed on a flat, level surface. Careful measurement of the exact volume for each plate will ensure uniform gel depth from plate to plate. The standard protocol for the EIA AGID assay requires 15 mL of heated agar solution per each 100 X 15 mm petri dish. Poured plates should be allowed to gel (uncovered) at room temperature for approximately one hour before either storing or cutting for the assay.

Storage. Preventing the loss of moisture from the agar will produce the brightest, sharpest precipitin lines in the immunodiffusion reaction. Maintaining a saturated environment for the storage and incubation of the agar will help to minimize any environmental variability related to humidity. Storage of the poured agar for more than five to seven days is not advised. Poured plates should be stored in two layers of polyethylene bags with a moistened gauze pad or paper towel in the inner bag. Short-term storage can be at either room temperature or 2°–8°C. Storage for a period of days is generally best at 2°–8°C.

Note: Refrigerated storage will increase moisture loss slightly due to condensation and low humidity conditions.

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