Heartworm-associated respiratory disease (HARD) induced by immature adult Dirofilaria immitis in cats

Abstract

Background: A controlled, blind research study was conducted to define the initial inflammatory response and lung damage associated with the death of immature adult Dirofilaria immitis in cats as compared with cats developing adult heartworm infections and cats on preventive medication.

Methods: Three groups of cats were utilized, 10 per group. All cats were infected with 100 third-stage (L3) larvae by subcutaneous injection. Group A cats were treated topically with selamectin (Revolution®; Zoetis) per label directions at 28 days post infection (PI) and once monthly for 8 months. Group B cats were treated orally with ivermectin (Ivomec®; Merial) at 150 μg/kg at 70 days PI, then every 2 weeks for 5 months. Group C cats were untreated PI. At baseline (Day 0) and on Days 70, 110, 168, and 240 PI, peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected on all cats. Upon completion of the study (Day 245), cats were euthanized and necropsies were conducted.

Results: Results were analyzed statistically between groups by ANOVA and by paired sample T testing for changes within the group over time. The selamectin-treated cats (Group A) did not develop radiographically evident changes throughout the study and were free of adult heartworms or worm fragments at necropsy. The heartworm life cycle was abbreviated with oral doses of ivermectin (Group B), shown by the absence of adult heartworms or worm fragments at necropsy. The early stage of immature adult worm in Group B cats, however, did induce severe pulmonary airway, interstitial, and arterial lung lesions, revealing that the abbreviated infection is a significant cause of respiratory pathology in cats. Cats in Groups B and C could not be differentiated based on radiographic changes, serologic antibody titers, complete blood count, or bronchoalveolar lavage cytology at any time point throughout the study. Eighty percent of cats in Group A and 100% of cats in Groups B and C became heartworm antibody positive at some time point post infection.

Conclusions: The clinical implications of this study are that cats that become infected with immature adult heartworms may not develop fully mature heartworms and are only transiently heartworm antibody positive, but do develop Heartworm-Associated Respiratory Disease (HARD).

Keywords: Dirofilaria immitis; Feline; HARD; Heartworm; Myofibrocyte; Respiratory disease.

Fig. 1

Lung of cat in Group B before pressure perfusion fixation. The lungs are turgid and especially the right and left caudal lung lobes will not deflate. Discoloration of surface can be visualized in the right caudal lung lobe. No adult HWs were present

Fig. 2

Lung lobes after fixed pressure perfusion inflation in a cat from Group B. The right caudal, right middle and portion of left caudal lung lobes after inflation with fixation solution at 14 cm H₂O pressure. Note the uneven inflation, especially of the right caudal lobe and discoloration of the lobes. No heartworms or worm segments were identified in this cat, which was HW antibody positive

Fig. 3

Heartworms in Group C cat after L3 D. immitis infection. The right ventricle is opened along the ventricular septum and reflected back. Two viable heartworms were noted in the right ventricle and extending into the pulmonary artery. The surface of the lungs was uneven and discolored. The valve leaflets were unaffected. The cat was heartworm antigen and antibody positive

Fig. 4

Percentage of cats heartworm antibody positive in each group at days after L3 D. immitis infection. Treatment groups: Group A, selamectin monthly initiated on Day 28; Group B, oral ivermectin every 2 weeks initiated on Day 70 PI; Group C, infected, untreated. Days after infection listed on bottom axis and RV 245 is sample collected from right ventricle immediately post mortem

Fig. 5

Serology results in cats after D. immitis infection. Treatment groups: Group A, selamectin monthly initiated on Day 28; Group B, oral ivermectin every 2 weeks initiated on Day 70 PI; Group C, infected, untreated. Percentage of cats in each group that were positive at any time point for heartworm antibody (AB Pos Days 70–240) or antigen (ANG Pos Days 70–240). Percentage of cats that had heartworms or worm fragments at necropsy (Adult HW)

Fig. 6

Percentage of cats with radiographic bronchial-interstitial scores of ≥1 (Score 0–3) after L3 D. immitis infection. Percentage of cats in each group with score ≥ 1 (Score 0–3). Treatment groups: Group A, selamectin monthly initiated on Day 28; Group B, oral ivermectin every 2 weeks initiated on Day 70 PI; Group C, infected, untreated. Days after infection listed on bottom axis

Fig. 7

Group A (infected, selamectin monthly initiated on Day 28) cat. Lateral Radiograph on Day 240. Normal lateral thoracic radiographs in a cat from Group A (selamectin monthly initiated Day 28 PI) on Day 240 after L3 D. immitis infection. There was no significant change in radiographs in any cat from Day 0, 110, 175, or 240. All Day 0 radiographs were normal for all cats

Fig. 8

Group A cat (infected, selamectin monthly initiated on Day 28). Ventrodorsal radiograph on Day 240. Normal lateral thoracic radiographs in a Group A cat (selamectin monthly initiated on Day 28 PI) on Day 240 after L3 D. immitis infection. There was no significant change in radiographs in any cat from Day 0, 110, 175, or 240. All Day 0 radiographs were normal for all cats

Fig. 9

Radiographs of a cat from Group B: Day 110. Left lateral and ventrodorsal radiographs of a cat from Group B (oral ivermectin every 2 weeks initiated on Day 70 PI) with no heartworms at necropsy on Day 240. This cat was heartworm antibody and antigen negative. Day 0 radiographs were normal, and did not differ from Group A cats on Day 0–240 and Group C cats on Day 0. The lungs revealed an interstitial and bronchial pattern on Day 110 more marked in the caudal lung lobes which became more severe on Day 175, with anterior lobes becoming more involved. The pulmonary arteries could not be clearly defined because of the severity of the lung parenchymal disease. On Day 240, the lung disease continued to be evident and the left caudal pulmonary artery was slightly more pronounced beyond the cardiac silhouette. The heart and main pulmonary artery were not enlarged

Fig. 10

Radiographs of a cat from Group B: Day 110. (See Fig. 9 caption for details)

Fig. 11

Radiographs of a cat from Group B: Day 175. (See Fig. 9 caption for details)

Fig. 12

Radiographs of a cat from Group B: Day 175. (See Fig. 9 caption for details)

Fig. 13

Radiographs of a cat from Group B: Day 240. (See Fig. 9 caption for details)

Fig. 14

Radiographs of a cat from Group B: Day 240. (See Fig. 9 caption for details)

Fig. 15

Radiographs of a cat from Group C: Day 110. Left lateral and ventrodorsal radiographs of a cat from Group C (infected no treatment, with two heartworms at necropsy) that was heartworm antibody and antigen positive. Day 0 radiographs were normal, and did not differ from Group A cats on Days 0–240, and Group B on Day 0. The lungs revealed an interstitial and bronchial pattern on Day 110, more marked in the caudal lung lobes, which became more severe on Day 175 with anterior lobes becoming more involved. The pulmonary arteries could not be clearly defined because of the severity of the lung parenchymal disease. On Day 240, the lung disease continued to be evident and the left caudal pulmonary artery was slightly more pronounced beyond the cardiac silhouette. The heart and main pulmonary artery was not enlarged

Fig. 16

Radiographs of a cat from Group C: Day 110. (See Fig. 15 caption for details)

Fig. 17

Radiographs of a cat from Group C: Day 175. (See Fig. 15 caption for details)

Fig. 18

Radiographs of a cat from Group C: Day 175. (See Fig. 15 caption for details)

Fig. 19

Radiographs of a cat from Group C: Day 240. (See Fig. 15 caption for details)

Fig. 20

Radiographs of a cat from Group C: Day 240. (See Fig. 15 caption for details)

Fig. 21

a (left) and b (right). . Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. The cats in Group A were generally normal. a (left), b (right)] with occasional isolated area of infiltration of smooth muscle [Fig. 22 a (left), B (right)], especially in small arterioles. Cats in Group B had severe inflammation around airways [Fig. 23 a (left), b (right)] and interstitial and pulmonary arterial proliferation of smooth muscle (Figs. 22–24) not directly associated with bronchioles or arterioles. The pulmonary arterial smooth muscle proliferation was consistent with heartworm disease. Although all cats in Group A did not consistently have lung pathology (Fig. 25 a, left), in some lobes isolated areas of myofibrocyte proliferation were observed (Fig. 25 b, right). Cats in Group C had disease similar to Group B cats except that the endothelial proliferation in pulmonary arteries (Figs. 26, 27) was noted in isolated major vessels and the airway and interstitial disease was more consistent that observed in Group B cats

Fig. 22

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 23

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 24

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 25

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 26

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 27

a (left) and b (right). Histopathology of feline lung with H & E stain or alpha-smooth muscle actin stain after L3 D. immitis infection. (See Fig. 21 caption for details)

Fig. 28

Diagram of feline heartworm disease and HARD. Heartworm-associated respiratory disease (HARD) is induced at the first arrival of immature adult worms as early as 70 to 90 days after infection. The inflammation develops even if the cat “self-cures” and all immature adults die, and no adult heartworms develop. The lung lesions continue for up to 8 months after infection and perhaps longer. The antibody (Ab) response is present if cats are started on selamectin 28 days after the infection even if immature adults do not reach the heart. The antibody response continues after the arrival of immature adults; in cats which develop only the immature adults with HARD, the antibody response is present in 50% of the cats 8 months after the infection. Cats that develop the adult heartworms continue to live for up to 4 years, often with no symptoms, and the cats develop a decreased responsiveness of their pulmonary intravascular macrophages (PIM). When adult heartworms eventually die, acute symptoms may develop