This chapter provides comprehensive coverage of a wide range of diseases and other situations in which immunoassay analytes are applied in the veterinary field, and the technologies and science behind them.

This chapter explains the technology and chemistry behind veterinary immunoassays. There is extensive coverage of infectious disease diagnosis and assessment of immune status using immunoassays for feline, canine, porcine, equine, bovine and avian applications, including the assay technology commonly used. There is also a section on the assessment of the reproductive or metabolic status of cats and dogs.

Canine leptospirosis is a zoonotic bacterial infection with worldwide distribution that is caused by spirochetes of the genus Leptospira (Sykes et al., 2011). Currently, the microscopic agglutination test (MAT) is the most frequently used test in North America and is commonly used to detect antibodies to 6 or more serovars of Leptospira in a series of individual reactions. MAT testing is typically performed in a reference laboratory, requires the maintenance of individual Leptospira cultures and may often require at least 2 days before results are reported to the veterinary practice. 

The Lepto ELISA test is a rapid assay SNAP-format ELISA for the detection of Leptospira specific antibodies in canine serum. The assay utilizes the LipL-32 target which is an abundant, highly conserved protein found only in pathogenic Leptospira (Murray, 2013). The sensitivity and specificity of the Lepto ELISA test were evaluated using a total of 403 serum field samples that were originally submitted to IDEXX Reference Laboratories, Inc. for MAT testing (Curtis et al., 2014). Of these, 201 were MAT negative and 202 were MAT positive with a peak MAT titer greater than or equal to 1:800. The sensitivity and specificity of the Lepto ELISA test relative to MAT (1:800 titer) were 83.2% and 82.1%, respectively. The test detected samples with peak MAT titers to serovars Pomona, Icterohaemorrhagiae, Grippotyphosa, Canicola, Bratislava and Autumnalis. In a separate study, 150 MAT negative samples from healthy dogs in a non-endemic area (Alaska) were tested; the specificity of the Lepto ELISA test in this population was 96.0%. 

The Lepto ELISA test represents a significant advance in serologic assays for Leptospira antibody because it is a standardized assay that measures antibody to a specific target known to be found in pathogenic leptospira. The rapid assay format, qualitative (positive/negative) result and reduced cost make the test useful for practicing veterinarians.

Additional information is available on the IDEXX Web Site: www.idexx.com

Canine whipworm (Trichuris vulpis) infections occur primarily in dogs, foxes and coyotes. The life cycle is direct with infection occurring after ingestion of embryonated eggs. The most common method for diagnosing infection is by observing eggs in fecal samples following fecal flotation. In practice there are a number of issues that can arise from using fecal flotation alone, which limit test sensitivity and compromise the usefulness of this method. These include the relatively long pre-patent period observed following whipworm infection where no eggs are produced, the method  and conditions used to perform the flotation step, the uncertainty involved in correctly identifying  the egg, and lastly,  intermittent shedding of eggs during the course of the infection (Dryden et al., 2005).

IDEXX Laboratories has developed a monoclonal antibody based microtiter plate-format ELISA capable of identifying intestinal whipworms by detecting an antigen secreted by adult whipworms in the feces (Elsemore et al., 2014). The detection of the worm-derived antigen bypasses many of the issues involved in attempting to identify species-specific eggs in the feces. For instance, ELISA detects antigen produced and shed by adult worms before the parasite begins laying eggs, is not affected by intermittent egg shedding and does not react to antigens produced by other species of nematodes. The test is available through IDEXX Reference Laboratories and is performed as a reflex test when the fecal ova and parasite test is negative for whipworm eggs. The test is useful because it identifies whipworm positive samples that, for a variety of reasons, give false negative results in the standard ova and parasite test.

Additional information is available on the IDEXX Web Site: www.idexx.com

Brain natriuretic peptide (BNP) is a neuroendocrine hormone produced as a prohormone (proBNP) by the atrial myocytes in the heart muscle. With cardiac disease, proBNP is also produced by the ventricular myocytes and released in proportion to the degree of stress on the myocardium. Concentrations tend to increase with severity of cardiac disease. The importance of measuring brain natriuretic peptides in the diagnosis and prognosis of heart disease has been well documented (Oyama et al., 2013).

The SNAP Feline proBNP Test in a newly developed Point-of-Care ELISA designed to identify cats at increased risk of heart disease. It is particularly useful because it can be used to provide an objective measure of heart health in time-sensitive clinic situations (Machen et al., 2012). The test provides a semi-quantitative measurement of the concentration of a breakdown product of proBNP (N-terminal brain natriuretic peptide, NTproBNP) in feline serum and EDTA plasma.  The SNAP test uses the same biological reagents as the quantitative reference-laboratory based microtiter plate-format Cardiopet proBNP Test.

In a validation study using 100 feline samples which was designed to compare the performance of the SNAP proBNP Test to the Cardiopet proBNP Test, there was 100% agreement for 57 normal samples (< 100 pmol/L). All 23 samples with NTproBNP concentrations greater than 270 pmol/L tested as abnormal on the SNAP Feline proBNP Test. Seven of the remaining 20 samples, which fell between 100 and 270 pmol/L, were identified as abnormal. An abnormal result on the SNAP Feline proBNP Test indicates that an increased level of NTproBNP has been measured and that additional diagnostics for cardiac disease should be considered (Machen et al., 2013)

Additional information is available on the IDEXX Web Site: www.idexx.com

Curtis, K., Foster, P., Smith, P., Monn, M., Stillman, B., Chandrashekar, R., Tasse, S., Goldstein, R. Performance of an in-clinic ELISA for the detection of Leptospira specific antibody in dogs. ACVIM Meeting Abstract, Nashville, TN (2014).

Dryden, M.E., Payne, P.A., Ridley, R., Smith, V. Comparison of common fecal floatation techniques for the recovery of parasite eggs and oocysts. Vet. Ther. 6, 15–28 (2005).

Elsemore, D.A., Geng, J., Flynn, L., Cruthers, L., Lucio-Forster, A., Bowman, D.D. Enzyme-linked immunosorbent assay for coproantigen detection of Trichuris vulpis in dogs. J. Vet. Diagn. Invest. 26, 404­­­­­­­­­­–411(2014).

Machen, M.C., Gordon, S.G., Buch, J.S., Beall, M.J., Oyama, M.A. Detection of occult feline cardiomyopathy using a pet-side point-of-care NT-proBNP ELISA assay. J. Vet. Intern. Med. 26, 724 (2012).

Machen, M.C., Gordon, S.G., Rush, J.E., Achen, S.E., Stepien, R.L., Fox, P.R., Lee, P.M., Oyama, M.A. Multicentered investigation of NT-proBNP point-of-care ELISA assay to detect moderate to severe occult (asymptomatic) feline heart disease. J. Vet. Intern. Med. 27, 630 (2013)

Murray, G.L. The lipoprotein LipL32, an enigma of leptospiral biology. Vet. Microbiol. 162, 305–314 (2013). 

Oyama, M.A., Boswood, A., Connolly, D.J., et al. Clinical usefulness of an assay for measurement of circulating N-terminal pro-B–type natriuetic peptide concentration in dogs and cats with heart disease.   J. Am. Vet. Med. Assoc. 243, 71–82 (2013).

Sykes, J.E., Hartman, K., Lunn, K.F., Moore, G.E., Stoddard, R.A., Goldstein, R.E. 2010 ACVIM small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment and prevention. J. Vet. Intern. Med. 25, 1–13 (2011).

Dr. Tom O’Connor is currently a Research Fellow in the Immunoassay R&D Department at IDEXX Laboratories, Inc. in Westbrook, Maine. Dr. O’Connor has been employed at IDEXX Laboratories for over 25 years and has worked on many of the in-office rapid assays and microtiter plate-format assays developed at IDEXX Laboratories in the companion animal group. He has received 23 patents and is the author or co-author of over 25 publications dealing primarily with the development and use of immunoassays in the companion animal market. He received a B.S. in Biochemistry from Michigan State University, a Ph.D. in Biochemistry from Kansas State University and was a postdoctoral fellow in the Molecular Biophysics and Biochemistry Department at Yale University.

John Lawrence is currently a Senior Research Scientist in the Livestock, Poultry and Dairy RD group at IDEXX Laboratories, Inc. in Westbrook, Maine. Mr. Lawrence has been employed at IDEXX Laboratories for 29 years and has held positions in Quality, Manufacturing, Technical Support, Marketing and Product Development/Technology. During this time he has developed over 15 immunoassays for veterinary applications and has participated in 5 company or technology acquisitions. Previously, he held manufacturing and RD positions at Becton-Dickinson Immunodiagnostics in Orangeburg, New York. He received his BS in Biology and Natural Science from Muhlenberg College in Allentown, PA.

Erwin Workman is currently retired and resides on Hilton Head Island in South Carolina. He retired in 2003 from IDEXX Laboratories, Inc., a company he helped create in 1984. From 1977 to 1984, he was employed by Abbott Laboratories where he developed cardiovascular and hepatitis products in the Diagnostics Division. He received a BA in Zoology from Duke University and a PhD in Biochemistry from Texas A&M University. Erwin wrote the versions of this chapter in previous editions.

Philip Andersen is a Senior Scientist at IDEXX Laboratories.

Valerie Leathers is an R&D Manager at IDEXX Laboratories.

Virus, bacteria, protozoa, parasite, enzyme immunoassay, enzyme-linked immunosorbent assay (ELISA), fluorescence, microparticles, colloidal particle-based immunoassay, microwell, cat, feline, dog, canine, pig, porcine, horse, equine, cattle, bovine, bird, chicken, avian, CITE, SNAP, feline leukemia virus, feline immunodeficiency virus, Dirofilaria immitis, heartworm, canine parvovirus, giardia, Borrelia burgdorferi, Lyme disease, Leishmania infantum, Leishmania donovani, Leishmaniasis, recombinant, fluorescence, Ehrlichia canis, ehrlichiosis, Ehrlichia ewingii, Anaplasma phagocytophilum, anaplasmosis, pseudorabies, Aujeszky’s disease, porcine reproductive and respiratory syndrome, swine fever, hog cholera, Mycoplasma hyopneumoniae, pneumonia, equine infectious anemia, foal immunoglobulin G, Brucella abortus, brucellosis, abortion, bovine herpes, bovine leukemia, enzootic bovine leukosis, Mycobacterium avium, paratuberculosis, Johne’s disease, bovine viral diarrhea, Neospora caninum, neosporosis, foot-and-mouth disease, transmissible spongiform encephalopathy, prion, Mycobacterium bovis, tuberculosis, avian encephalomyelitis, avian leukosis, avian reovirus, infectious bronchitis, infectious bursal disease, avian Mycoplasma, Newcastle disease, Pasteurella multocida, reticuloendotheliosis, chicken anemia, Salmonella, avian influenza, reproduction, pancreatitis, thyroxine, hypothyroidism, hyperthyroidism, cortisol, Cushing’s syndrome, adrenocorticotropic hormone, bile acids, heart disease, cardiac, progesterone, pregnancy-associated glycoprotein, immunometric, antibody, antigen.