Cranking the heat on feline heartworm testing

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Dirofilaria immitis infection is diagnosed rarely in cats. Lack of detection is attributed partly to scanty circulating antigen associated with low worm numbers typical of feline infections.^1,2

In dogs, current SNAP (Idexx Laboratories) tests for detection of heartworm antigen are both sensitive and specific. But when antigen-based canine assays were first developed for detecting D. immitis in the 1980s, serum was heated or treated with ethylenediaminetetraacetic acid (EDTA) prior to testing, thereby destroying the antigen-antibody complexes that hampered antigen recognition.^3-5

Breaking the bonds

To gauge the role antigen blocking plays in pinpointing occult D. immitis, investigators at Oklahoma State University obtained serum samples from cats experimentally infected with heartworms and tested the samples before and after heat treatment. The results, published several years ago but still quite relevant,⁶ establish the efficacy of heat treatment in revealing heartworm antigen in cats.

In the study, 6 domestic short-haired cats (three male, three female) underwent subcutaneous inguinal inoculation of third-stage D. immitis larvae at 10 months of age (study days 7, 14, 21, and 28). Missouri strain larvae (100 per cat) were harvested shortly before inoculation from infected Liverpool strain Aedes aegypti mosquitoes.

Whole blood samples were collected from the jugular or, alternatively, the cephalic vein of each cat on study days 84, 112, 140, 168, 196, and 224. Vacuum tubes, with or without EDTA, were used. Anti-coagulated blood was assayed for microfilaria by modified Knott tests.

Antibody and antigen testing were performed using commercial assays, according to manufacturer’s instructions. For antibody testing, a reference laboratory tested each sample in triplicate and provided optical density (O.D.) results for each micrometer well.

For antigen testing, heat treatment was implemented. Serum samples were placed in a heat block at 103 °C for 10 minutes and then centrifuged. The supernatant was then used in each commercial antigen assay. Test kits evaluated before and after heat treatment included lateral flow immunochromatographic assays and enzyme-linked immunosorbent assays (ELISA) in both membrane-bound and microtiter plate formats. For one of the micrometer plates, O.D. readings were performed by spectrophotometry before and after heat treatment. All tests used in this study were manufactured by either Idexx or Zoetis.

Antigen revealed

All 6 cats developed D. immitis infection, evidenced by the recovery of adult worms at necropsy or by histopathology of classic pulmonary lesions. A total of 1 to 6 worms were recovered from each of 5 cats; worms were not recovered from the sixth cat, but pathognomonic pulmonary lesions were present.

All Knott tests were negative. Antibodies to D. immitis were detected in 5 cats on day 84 and in all 6 cats on days 112, 140, 168, 196, and 224.

Antigen for D. immitis without prior heating was detected in 0, 1, and 1 cat, using any of the 3 assays types, on days 168, 196, and 224, respectively. After heat treatment, however, the story was very different: Antigen was identified on microtiter assay in 1, 5, and 5 cats on days 168, 196, and 224, respectively, and in 4 cats on day 224 for both the membrane-bound ELISA and lateral flow immunochromatographic testing.

Boiling down the data

Antigen detection is an excellent means for diagnosing heartworm in dogs.^1,2 Not so for cats, however.

The cat is not hospitable to the heartworm life cycle, and these parasites often die or become entrapped in abnormal places like the affected cat’s brain and skin. Furthermore, heartworm infections in cats are usually antigen-deficient because the parasites involved are typically small, few in number, and often do not include antigen-producing females.

To complicate matters further, cats infected with D. immitis typically develop severe lung pathology characterized by villous arteritis and pulmonary parenchymal damage. This chronic inflammatory process produces hypergammaglobulinemia, which may also hinder antigen detection.

For these reasons, heartworm-infected cats test antigen-negative in 25% to 50% of cases, even when they are symptomatic.⁷ To this day, diagnosis of feline heartworm relies largely on imaging sick cats.

This important study shows that heat treatment of feline serum prior to antigen testing improves detection of D. immitis, presumably due to destruction of antibody and release of antigen from antigen-antibody complexes. Likewise, antigen blocking^1,2 probably leads to false-negative results in antigen-positive serum samples that are not heat treated in advance.

The rate of D. immitis in feline serum samples in the United States is estimated at around 1%, but the data in the study herein suggest that the incidence of feline heartworm is higher.

References

1. McCall JW, Genchi C, Kramer LH, et al. Heartworm disease in animals and humans. Adv Parasitol. 2008;66:193-285. doi: 10.1016/S0065-308X(08)00204-2

2. Lee AC, Atkins CE. Understanding feline heartworm infection: disease, diagnosis, and treatment. Top Companion Anim Med. 2010;25(4):224-230. doi: 10.1053/j.tcam.2010.09.003

3. Weil GJ, Malane MS, Powers KG, et al. Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs. J Immunol. 1985;134:1185-1191.

4. Brunner CJ, Hendrix CM, Blagburn BL, et al. Comparison of serologic tests for detection of antigen in canine heartworm infections. J Am Vet Med Assoc. 1988;192(10):1423-1427.

5. Tonelli QJ, Quentin AB. Factors affecting the accuracy of enzyme immunoassays for Dirofilaria immitis adult antigen. Proc Am Heartworm Symp. 1989;161-165.

6. Little SE, Raymond MR, Thomas JE. et al. Heat treatment prior to testing allows detection of antigen of Dirofilaria immitis in feline serum. Parasit Vectors. 2014;7:1. doi: 10.1186/1756-3305-7-1

7. Heartworm in cats. American Heartworm Society. Accessed July 20, 2020. https:heartwormsociety.org/heartworms-in-cats