and exercise intolerance. In the most advanced cases where many adult worms have built up in the heart without treatment, signs progress to severe weight loss, fainting, coughing up blood, and finally, congestive heart failure. There are 4 different classes of symptoms: Class 1 – no or mild symptoms with occasional cough. Class 2 – mild symptoms with occasional cough and tiredness after moderate activity. Class 3 – more severe symptoms, including a generally sick appearance, persistent cough, difficulty breathing, and tiredness after mild activity. Heart and lung changes may be seen with a chest x-ray. Class 4 – also called caval syndrome. The blood flowing back to the heart is blocked due to the large mass of worms. This is life-threatening and the only treatment option is surgery. Role of Wolbachia pipientis Wolbachia pipientis is an intracellular bacterium that is an endosymbiont of D. immitis. All heartworms are thought to be infected with Wolbachia to some degree. The inflammation occurring at the die-off of adult heartworms or larvae is in part due to the release of Wolbachia bacteria or protein into the tissues. This may be particularly significant in cats, in which the disease seems to be more related to larval death than living adult heartworms. Treating heartworm-positive animals with an antibiotic such as doxycycline to remove Wolbachia may prove to be beneficial, but further studies are necessary. Diagnosis in dogs Microfilarial detection is accomplished by the using one of the following methods: Direct blood smear A blood sample is collected and viewed under the microscope. The direct smear technique allows examination of larval motion, confirming the presence of microfilaria. It also helps in the distinction of D. immitis from Acanthocheilonema reconditum. This distinction is important because the presence of the latter parasite does not pose a health risk to the host. D. immitis usually has stationary body movement, while A. reconditum has progressive movement. However, this method often misses light infections because only a small amount of blood sample is used. Hematocrit tube method This method uses a microhematocrit (or capillary tube) filled with a blood sample that has been centrifuged, separating the plasma from the red blood cells. These layers are divided by the buffy coat. The buffy coat consists of the leukocytes and platelets that are in the sample. The tube is snapped at the buffy coat and added to a slide for microscopic examination. Adding