WHAT EVERYONE SHOULD KNOW: BASICS OF HEARTWORM BIOLOGY CLARKE ATKINS, DVM

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1 WHAT EVERYONE SHOULD KNOW: BASICS OF HEARTWORM BIOLOGY CLARKE ATKINS, DVM Heartworm infection (HWI, dirofilariasis, dirofilarosis), caused by Dirofilaria immitis, primarily affects members of the family Canidae. Dirofilarosis is widely distributed, being recognized in northern and southern temperate zones, in the tropics, and in the subtropics. Infections are recognized in most of the United States, although the distribution favors the Southeast and Mississippi River Valley. In some endemic areas in the United States, infection rates approach 45%, and in some hyperendemic tropical regions, virtually all dogs are infected. Dirofilariosis is generally infrequent in Canada, but there are endemic areas of concern in southern Ontario. A 2001 survey of veterinarians indicated that there were approximately 240,000 cases diagnosed in the United States; but realistic estimates puts the prevalence at >1,000,000 cases. 1 Species known to have been infected with D. immitis include the domestic dog, wolves, foxes, coyotes, domestic cats, ferrets, muskrats, sea lions, nondomestic cats, coatimundi, and humans. The species of greatest interest to the practicing veterinarian include the dog and domestic cat. Because the consequences, treatment, and prognoses differ between the two species, clinical aspects of canine and feline heartworm disease (HWD) are discussed separately. When HWI is severe or prolonged, it may result in the pathologic process called heartworm disease (HWD). This may vary from asymptomatic (radiographic lesions only) to severe, lifethreatening, chronic pulmonary artery, lung, and cardiac disease. In chronic HWI, glomerulonephritis, anemia, and thrombocytopenia may also be recognized. Severe dirofilarosis may, in addition, produce acute and fulminant multi-systemic presentations, such as caval syndrome (CS) and disseminated intravascular coagulation (DIC). LIFE CYCLE D. immitis is transmitted by over 70 species of mosquitoes, although important mosquito vectors probably number fewer than 12. Understanding the complex life cycle of D. immitis is imperative for veterinary practitioners in heartworm endemic areas. The terminology for the larval stages can be confusing. For the final heartworm stage, L5 (last or 5 th larval stage) is no longer the preferred term. The nomenclature has been updated, because, although far smaller than a mature adult, this stage does not molt again so is not a larval stage. Preferred terminology for this pre-cardiac stage includes immature Stage 5 (S5), immature or juvenile adult).. For the purposes of this manuscript, L5, Stage 5, juvenile, juvenile adult, immature adult, and mature adult are terms used to describe the final stage in the heartworm development. Additionally, the term L1 (1 st larval stage) refers to the 1 st stage larvae after ingestion by the female mosquito, while prior to this, in the host, these earliest HW are termed microfilariae. Adult heartworms reside in the pulmonary arteries and, to a lesser extent (in heavy infections), the right ventricle. After mating, microfilariae (first stage larvae; Mf) are produced by mature adult female heartworms (mature S5) and are released into the circulation. These Mf are ingested by feeding female mosquitoes and undergo two molts (L1 to L2 to L3) over an 8- to 17-day period. It is important to note that this process is temperature dependent; in times of the year when insufficient numbers of days occur in which the ambient temperature is adequate, molting in the mosquito does not occur during the lifetime of the female mosquito and transmission cannot occur. 2,3 Larval molts and maturation are also dependent on the presence of an intracellular symbiotic bacterium, Wolbachia pipientis. 3a The resultant L3 is infective and is

2 254-2 transmitted by the feeding mosquito to the original or another host, most often a male dog. Another molt to L4 occurs in the subcutaneous, adipose, and skeletal muscular tissues shortly after infection (1 to 12 days), with a final molt to S5 (immature adult) occurring 2 to 3 months (50 to 68 days) after infection. This immature adult (1 to 2 cm in length) soon enters the vascular system, migrating to the heart and lungs, where final maturation (mature male adults range from 15 to 18 cm and females from 25 to 30 cm) and mating occurs. Under optimum conditions, completion of the life cycle takes 184 to 210 days. The canine host becomes microfilaremic as early as 6, but typically by 7 to 9 months after infection. Microfilariae, which are variably present in infected dogs, show both seasonal and diurnal periodicity, with greatest numbers appearing in the peripheral blood during the evening hours and during the summer. Adult heartworms in dogs are known to live 5 to 7 years and Mf up to 30 months. Dillon has emphasized that the disease process in HWD begins with the molt to S5 (as soon as 2 to 3 months post-infection), at which time these immature adults enter the vascular system, initiating vascular and possibly lung disease, with eosinophilia and eosinophilic infiltrates and signs of respiratory disease. 4 It is important to note that this antedates the profession s current ability to diagnose HWI. PATHOPHYSIOLOGY Heartworm is a misnomer because the adult actually resides in the pulmonary arterial system for the most part, and the primary insult to the health of the host is a manifestation of damage to the pulmonary arteries and lung. The severity of the lesions and hence clinical ramifications are related to the relative number of worms (ranging from one to 250), the duration of infection, and the host and parasite interaction. Immature and mature adult heartworms reside primarily in the caudal pulmonary vascular tree, occasionally migrating into the main pulmonary arteries, the right heart, and even the great veins in heavy infections. Obstruction of pulmonary vessels by living worms is of little clinical significance, unless worm burdens are high, or the patient small. The major effect on the pulmonary arteries is produced by worm-induced (toxic substances, immunological response, and physical trauma) villous myointimal proliferation, inflammation, pulmonary hypertension (PHT), disruption of vascular integrity, and fibrosis. This may be complicated by arterial obstruction and vasoconstriction caused by live worms, dead worm thromboemboli and HW products. Pulmonary vascular lesions begin to develop within days of worm arrival (as early as 3 months post-infection), with endothelial damage and sloughing, villous proliferation, and activation and attraction of leucocytes and platelets. The immigration of such cells and the release of trophic factors induce smooth muscle cell proliferation and migration, with collagen accumulation and fibrosis. Proliferative lesions eventually encroach upon and even occlude vascular lumina. Endothelial swelling with altered intracellular junctions increases the permeability of the pulmonary vasculature. Worms, which have died naturally or have been killed, elicit an even more severe reaction, inciting thrombosis, granulomatous inflammation, and rugous villous inflammation. Grossly, the pulmonary arteries are enlarged, thick-walled, and tortuous, with roughened endothelial surfaces. These changes are at least partially reversible. 5 Although the role of exercise in exacerbation of the signs of thromboembolic HWD is accepted, its role in the development of pulmonary vascular disease and PHT is less clear. While Rawlings 6 was unable to show an effect of 2.5 months controlled treadmill exercise on PHT in heavily infected dogs, Dillon 7 showed more severe PHT in lightly infected, mildly exercised dogs than in more heavily infected but unexercised dogs. Clinical observation suggests that 2

3 254-3 outdoor dogs, particularly hunting dogs, have more severe lesions and PHT, possibly related to exercise, but the role of increased exposure and worm burden must also be considered. Diseased pulmonary arteries are thrombosed, thickened, dilated, tortuous, noncompliant, and functionally incompetent, thereby resisting recruitment during increased demand; hence exercise capacity is diminished. Vessels to the caudal lung lobes are most severely affected. Pulmonary vasoconstriction results secondary to vasoactive substances likely released from heartworms, as well as endothelin1 produced in excess by vascular endothelial cells 7a, as well as vasoconstrictive substances, such as serotonin, ADP, and thromboxane A2, produced by activated platelets. Furthermore, hypoxia (induced by ventilation-perfusion mismatching secondary to pulmonary thromboembolism [PTE], eosinophilic pneumonitis, pulmonary consolidation, or all three), further contributes to vasoconstriction. The result is PHT, increased right ventricular afterload, and compromised cardiac output. 8 Pulmonary hypertension is exacerbated by exercise or other states of increased cardiac output. The right heart, which is an efficient volume pump, but does not withstand pressure overload, first compensates by eccentric hypertrophy (dilatation and wall thickening) and, in severe infections, decompensation (right heart failure). In addition, hemodynamic stresses, geometric changes, and cardiac remodeling may contribute to secondary tricuspid insufficiency, thereby complicating or precipitating cardiac decompensation. Further compromise occurs with the advent of cardiac arrhythmia. Pulmonary infarction is uncommon because of the extensive collateral circulation provided the lung and because of the gradual nature of vascular occlusion. Because of increased pulmonary vascular permeability, perivascular edema may develop. Although, along with an inflammatory infiltrate, this fluid accumulation may be evident radiographically as increased interstitial and even alveolar density, in and of itself, it is seemingly of minimal clinical significance and certainly does not indicate left heart failure (in other words, it is not cardiogenic pulmonary edema and furosemide is not indicated). Spontaneous or post-adulticidal PTE with dead worms may precipitate or worsen clinical signs, producing or aggravating PHT, right heart failure or, in rare instances, pulmonary infarction. Dying and disintegrating worms worsen vascular damage and enhance coagulation. Pulmonary blood flow is further compromised and consolidation of affected lung lobes may occur. With acute and massive worm death, this insult may be profound, particularly if associated with exercise. Exacerbation by exercise likely reflects increased pulmonary artery flow with escape of inflammatory mediators into the lung parenchyma through badly damaged and permeable pulmonary arteries. Dillon has suggested that the lung injury is similar to that seen in adult respiratory distress syndrome (ARDS). 4 Pulmonary parenchymal lesions also result by mechanisms other than post-thromboembolic consolidation. Eosinophilic pneumonitis is most often reported in true occult HWD, in which immune-mediated destruction of Mf in the pulmonary microcirculation produces amicrofilaremia. This syndrome results when antibody-coated Mf, entrapped in the pulmonary circulation, incite an inflammatory reaction (eosinophilic pneumonitis). 9 A more sinister, but rare form of parenchymal lung disease, termed pulmonary eosinophilic granulomatosis, has been associated with HWD. The exact cause and pathogenesis are unknown, but it is felt to be similar to HWD-related eosinophilic pneumonitis. 10 It is postulated that Mf trapped in the lungs are surrounded by neutrophils and eosinophils, eventually forming granulomas and associated bronchial lymphadenopathy. Antigen-antibody complexes, formed in response to heartworm antigens, can produce glomerulonephritis in heartworm-infected dogs. 11 The result is proteinuria (albuminuria), 3

4 254-4 uncommonly associated with renal failure. Heartworms may also produce disease by aberrant migration. This uncommon phenomenon has been associated with neuromuscular and ocular manifestations because worms have been described in tissues such as muscle, brain, spinal cord, and anterior chamber of the eye. In addition, systemic arterial thrombosis with S5 has been observed when worms migrate aberrantly to the aortic bifurcation or more distally in the digital arteries. 12 Adult heartworms may also passively migrate in a retrograde manner from the pulmonary arteries to the right heart and venae cavae, producing CS, a devastating complication, described later. 13 It has been recently been recognized as important that the bacterium W. pipientis inhabits filarid parasites, including D. immitis. Importantly, these bacteria live in a symbiotic relationship with the filarid parasites, being necessary for molts within the mosquito and the canine host (L3- L4, L4-S5). The exact role of Wolbachia in the pathogenesis of HWD is unclear, but Wolbachia proteins (Wolbachia surface proteins; WSP) have been identified in the glomerulus and lung of heartworm-infected dogs. 13a, 13b In addition, proteins produced by the bacteria are thought to contribute to the pulmonary lesions produced by living and dying heartworms. CANINE HEARTWORM DISEASE: CLINICAL SIGNS The clinical signs of chronic HWD depend on the severity and duration of infection and, in most chronic cases, reflect the effects of the parasite on the pulmonary arteries and lungs and, secondarily, the heart. It is important to point out that the vast majority of dogs with HWI are asymptomatic. Historical findings in affected dogs variably include weight loss, diminished exercise tolerance, lethargy, poor condition, cough, dyspnea, syncope, and abdominal distension (ascites). Physical examination may reveal evidence of weight loss, split-second heart sound (13%), right-sided heart murmur of tricuspid insufficiency (13%), and cardiac gallop. 14 If right heart failure is present, jugular venous distension and pulsation typically accompanies hepatosplenomegaly and ascites. Cardiac arrhythmias and conduction disturbances are uncommon in chronic HWD ( 10%). With pulmonary parenchymal manifestations of HWD, cough and pulmonary crackles may be noted and, with granulomatosis (a rare occurrence), muffled lung sounds, dyspnea, and cyanosis are also reported. When massive PTE occurs, the additional signs of dyspnea, fever, and hemoptysis may be present. 4

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6 HEARTWORM DIAGNOSTICS CLARKE ATKINS, DVM Microfilarial Detection Ideally, the diagnosis is made by routine evaluation prior to the onset of symptoms (i.e., HWD). Dogs in areas in which heartworms are endemic should undergo a heartworm test yearly, particularly if not receiving heartworm preventive. This was accomplished most commonly in the past by the microscopic identification of Mf on a direct blood smear, above the buffy coat in a microhematocrit tube, using the modified Knott test, or after millipore filtration. The accuracy of these tests, typically used for routine screening and for the diagnosis of suspected HWI, is improved by multiple testing. The modified Knott test and millipore filtration are more sensitive because they concentrate Mf, improving chances of diagnosis. The direct smear technique allows examination of larval motion, helping in the distinction of D. immitis from Dipetalonema reconditum (now termed Acanthocheilonema reconditum); other useful diagnostic criteria are included in Table This distinction is important because the presence of the latter parasite does not require expensive and potentially harmful arsenical therapy, as does D. immitis. None of these tests can rule out HWI conclusively because of the potential for amicrofilaremic infections (reported to be 5% to 67% of cases, with 10-20% being generally accepted 16 ) and the fact that false-negative results may occur, particularly if microfilarial numbers are small, a small amount of blood is collected, or direct smears are relied upon. The number of circulating Mf in the peripheral blood does not correlate well with the number of adult heartworms and therefore cannot be used to determine the severity of infection. In most practices, microfilarial testing has been largely supplanted by immunodiagnostic antigen testing (i.e., enzyme-linked immunosorbent assay [ELISA], lateral flow immunoassay, and rapid immunomigration techniques. The modified Knott test, millipore filter test, or wet mount direct smear should always be performed, however, in antigen-positive dogs to determine microfilarial status. The reasons for this include knowing if large numbers are present, which allows pre-treatment or a scheduled observation time after the first dose of a ML. Secondly, there is concern that HW resistance to ML may be favored when these drugs are administered to HW-positive, microfilaremic dogs. Some veterinarians choose to combine the antigen and microfilarial tests. This practice is most useful in dogs receiving no preventive (ML typically render the dog amicrofilaremic). The generally accepted 1% of infected dogs that are Mf positive and antigen negative 15 may be an underestimate, based on new information about antibody-antigen complexing in HWI. 15a,15b,15c Immunodiagnostic Antigen Tests In dogs not having received ML preventive therapies, the prevalence of amicrofilaremic infections is typically 10% to 20%. 16 This may be observed in prepatent (young) infections, in single-sex infections, with immune-mediated destruction of Mf, and with drug-induced amicrofilaremia. Dogs receiving ML preventives are typically amicrofilaremic. Hence immunodiagnostic tests are now regularly used for both screening and in suspected HWI. These tests are popular because of their high sensitivity and specificity and ease of performance (Table 255-2) The weakness of these tests is that they detect antigen from adult female heartworms and hence will produce negative results during the first 6 (5 to 8) months of any infection, in all-male infections, and in infections with low female worm burdens. In fact, in a study of the

7 254-2 performance of three commercial test kits in detecting low worm burden ( 4 females), naturally acquired infections demonstrated an overall (median for three test kits) sensitivity of 79% and a median specificity of 97%. 21 The sensitivity was relatively low (64%) for infections of only one female worm but improved with increasing female worm burden (median of 85%, 88%, and 89% for two, three, and four female worms, respectively). Of course with higher worm burdens, better sensitivity is experienced. Nevertheless, despite overall excellent results in detection of small worm burdens, false-negative results do occur. 21 Certain ELISA antigen tests are designed to quantitatively predict worm burdens, based on antigen concentrations. Semiquantitative ELISA (Snap Canine Heartworm PF ) has been used to successfully predict antigen load and hence approximate worm burden. Rawlings and colleagues 22 have shown this to be useful in predicting thromboembolic complications, with dogs bearing greater worm burdens being more likely to experience such complications after adulticide. This application is most useful, however, in instances of low antigen concentration (suggesting low worm burden) because high antigen concentrations might be recognized when all or most worms are dead, having released a large amount of antigen into the circulation. ELISA technology also allows determination of the efficacy of adulticide therapy. ELISA antigen concentration typically falls to undetectable levels 8 to 12 weeks after successful adulticide therapy, so a positive test persisting beyond 12 weeks post-therapy has been suggested to indicate persistent infection. 23 However, antigen tests may remain positive for longer periods, and this author does not assume a failure in adulticidal therapy unless the antigen test is positive >6 months after adulticidal therapy and does not advocate routine retesting until 8-12 months post-treatment. The American Heartworm Society (AHS) now prefers the term below detectable limits or no antigen detected to the term negative, when referring to antigen test results that are not positive. This is to emphasize the fact that negative tests do not rule out immature, small, or allmale HWI. As previously suggested, ML therapy with either ivermectin, milbemycin oxime, moxidectin, or selamectin results in clearance of Mf within 6 to 8 months , 23b, 24a In addition, embryostasis may be permanent. Thus the sole use of direct smears, the modified Knott test, or the millipore filter test (ie, microfilarial tests) in dogs receiving ML heartworm preventives is inappropriate, though they certainly play a supplemental role. The only routinely effective testing modality in dogs receiving monthly preventive is the use of antigen assays. Radiography Although not an effective screening test for HWI, thoracic radiography offers an excellent method for detecting HWD, for determining its severity, for evaluating pulmonary parenchymal changes, and for discovery of differential diagnoses. Radiographic abnormalities, which develop relatively early in the disease course, are present in approximately 85% of cases. According to the study of 200 heartworm-infected dogs by Losonsky and colleagues, 26 radiographic features include right ventricular enlargement (60%), increased prominence of the main pulmonary artery segment (70%), increased size and density of the pulmonary arteries (50%), and pulmonary artery tortuosity and pruning (50%). If heart failure is present, enlargement of the caudal vena cava, liver, and spleen, as well as pleural effusion, ascites, or both, may be evident. Thrall and Calvert 27 suggested that pleural effusion is uncommon in heart failure due to HWD, demonstrating that marked enlargement of the cranial lobar pulmonary artery was a more sensitive indicator of HWD-associated heart failure than enlargement of the caudal vena cava. 2

8 254-3 Thoracic radiographs obtained in the ventrodorsal projection are preferable for cardiac silhouette evaluation and ease, and they often minimize patient stress. However, the dorsoventral projection is superior for the evaluation of the caudal lobar pulmonary vessels, which are considered abnormal if larger than the diameter of the ninth rib where the rib and artery intersect. The cranial pulmonary artery is best evaluated in the lateral projection and should normally not be larger than its accompanying vein or the proximal one third of the fourth rib. The pulmonary parenchyma can best be evaluated radiographically. With pneumonitis, the findings include a mixed interstitial to alveolar density, which is typically most severe in the caudal lung lobes. In eosinophilic nodular pulmonary granulomatosis, the inflammatory process is arranged into the interstitial nodules, associated with bronchial lymphadenopathy and, occasionally, pleural effusion. With pulmonary thromboembolism, the radiographic findings of coalescing interstitial and alveolar infiltrates, particularly in the caudal lung lobes, reflect the increased pulmonary vascular permeability and inflammation described previously. Consolidation may accompany massive embolization, pulmonary infarction, or both. Electrocardiography Electrocardiography is useful in detecting arrhythmias but is generally insensitive in detection of cardiac chamber enlargement in HWD, when compared with radiography and echocardiography. If radiography does not suggest HWD, it is unlikely that the electrocardiogram (ECG) will be useful in the absence of arrhythmias. With the exception of CS and heart failure, arrhythmias are rare (2% to 4%). 28 Nevertheless, the finding of a right ventricular enlargement pattern is supportive evidence for HWD. Lombard and Ackerman 28 demonstrated that ECG abnormalities were present in 38% to 62% of dogs with moderate and severe echocardiographic changes of HWD, while Calvert and Rawlings 29 found that only 6% of 276 dogs with dirofilarosis had ECG changes suggestive of right ventricular enlargement. These investigators 30 also showed that the most sensitive ECG parameters for detection of HWD are lead II S waves deeper than 0.8 mv, mean electrical axis greater than 103 degrees, and greater than three ECG parameters of right heart enlargement. The latter ECG finding ( three criteria) is considered to be the most accurate. P-pulmonale (tall P waves, indicative of right atrial enlargement) is unusual in HWD. Echocardiography Echocardiography is relatively sensitive in the detection of right heart enlargement because the right ventricular end-diastolic dimension and septal and right ventricular free wall thicknesses are all increased. Lombard reported abnormal (paradoxical) septal motion in 4 of 10 dogs with HWD. 31 The ratio of left-to-right ventricular internal dimensions is a useful calculation, being reduced from a normal value of 3 to 4 to a mean value of 0.7 in dogs with HWD. In some instances, two-dimensional echocardiography can be used to demonstrate worms in the pulmonary artery. Although heartworms can occasionally be demonstrated in the right ventricle, this method is insensitive except in dogs with CS or very heavy worm burdens because the worms infrequently inhabit this location. 31 Clinical Pathology Hematologic and serum chemical abnormalities, although of limited use in making a diagnosis of HWD, are frequently useful in providing supportive evidence and for evaluating concurrent disease processes that may or may not be related to HWD. Calvert and Rawlings 30 report that the dog with HWD in Georgia is typically found to have a low-grade, non-regenerative anemia 3

9 254-4 (present in 10% of mildly to moderately affected dogs and up to 60% of severely affected dogs), neutrophilia (20% to 80% of cases), eosinophilia ( 85% of cases), and basophilia ( 60% of cases). Thrombocytopenia, which may be noted in chronic HWD, CS, and DIC, is most common 1 to 2 weeks after adulticidal therapy. In severe HWD, especially if heart failure is present, liver enzyme activities may be increased (10% of cases) and occasionally hyperbilirubinemia is noted. Azotemia, seen in only 5% of cases, may be prerenal in origin if dehydration or heart failure is present or may be secondary to glomerulonephritis. In 10% to 30% of cases, albuminuria is noted. If glomerular disease is severe, hypoproteinemia (hypoalbuminemia) has the potential to complicate the clinical picture. Not surprisingly, the most severe clinicopathologic findings are associated with the most severe clinical findings. Evaluation of tracheobronchial cytology is at times useful, particularly in the coughing dog with pneumonitis, occult HWD, and minimal radiographic evidence of HWD. Microscopic examination reveals evidence of an eosinophilic infiltrate. In microfilaremic dogs, Mf may occasionally be detected in this manner. Abdominal fluid analysis in cases of congestive heart failure (CHF) typically reveals a modified transudate. Dogs with HWD and right heart failure have central venous pressure (CVP) that ranges from 12 to more than 20 cm H 2 O, but ascites may develop at lower CVPs, if hypoalbuminemia is present. Only recently has the use of biomarkers been evaluated expressly for HWI/HWD. Venco 31a reported on C-reactive protein (CRP), which binds to dead and dying cells in response to increased cytokine plasma concentrations. The CRP level in serum of dogs with and without HWI/HWD was shown to be higher in HWD than in controls and correlated to degree of pulmonary artery damage and PTE. The hope is that this biomarker, which rises and falls quickly, with disease and its resolution, respectively, may be useful in staging HWI, monitoring therapy, and evaluating therapeutic outcome. Carreton, et al. have shown that D- dimer, cardiac troponin I and myoglobin, are abnormally elevated in HWD, indicating PTE and myocardial damage, respectively. 31b,c, While the results of these preliminary studies are encouraging, the exact clinical use of biomarkers in diagnosis and evaluation of HWI/HWD requires further study. 4

10 Heartworm Resistance Clarke Atkins, DVM, Diplomate ACVIM (IM, Cardiology), North Carolina State University Prevention failure most often results from a lack of understanding on the part of owners as to the risk of HWI, a lack of owner compliance, or from inadequate instruction on preventive measures by the attending veterinarian. 1,46-48 Another potential source of prevention failure is the very real and frightening prospect of HW drug-resistance, with the potential loss of this important drug class from the armamentarium of the practicing veterinarian. Since the earliest part of this century, there has been growing concern that heartworms are becoming resistant to the macrocyclic lactone class. This concern is based on reports from the field of increasing instances of drug failure to prevent heartworm infection, termed LOEs (lack of efficacy reports). These reports have largely emanated from a region termed the Mississippi Delta, including parts of Tennessee, Arkansas, Louisiana, and Mississippi. Veterinarians in this area (Figure ) have reported increased drug failure in dogs considered to have received adequate ML preventives and an increase in the difficulty with which Mf are cleared, using this class of drugs. Based on the growing number of veterinary reports, the FDA-CVM, formally reported this concern in 2005, subsequently requiring that manufacturers remove product claims of 100% efficacy. 126 During the next decade, millions of dollars have been spent to explore the validity and suspected mechanisms behind LOEs In-vivo microfilarial resistance to ML was documented in a Hurricane Katrina canine survivor, which could not be cleared of Mf, regardless of dosage and combination of MLs (but not doxycycline) utilized. 127 Other early studies focused on a handful of dogs with medical records supporting claims of good preventive compliance and a history of difficulty in clearing Mf with ML. 128 Using an in-vitro assay, Mf isolated from 4 of these dogs were subsequently demonstrated to be less susceptible to all currently marketed ML molecules, as compared to Mf from control dogs. Subsequent genetic analysis showed a close relationship between these Mf isolates, with a particularly high degree of homozygosity in the P-gp gene. 129 Importantly, in 3 isolates studied, there was a significant negative correlation between the susceptibility of the Mf to ML and the percentage of homozygous (GG-GG) P-gp genes. 129 Independent of these findings, a HW isolate (MP3) from Athens, Georgia produced infection, despite treatment with either ivermectin or milbemycin oxime in the FDA-CVM (1-dose, 30 days post-infection) test of efficacy, the first ever recorded. 130 Expanding our understanding of the breadth of the problem and its therapeutic variability, Blagburn used a heavy MP3 challenge (100 L3) to demonstrate failure of onedose of ivermectin, milbemycin oxime, and selamectin, while topical imidacloprid-moxidectin experienced no failures in preventing HWI. 131 The infection rate was high (7/8 dogs in each group), but the worm burden of infected dogs was small. Subsequent tests showed that milbemycin oxime, given for 3 consecutive monthly doses, was 100% protective, temporarily alleviating some, but not all concern. 132 Furthermore, 2 of the aforementioned Blagburn isolates (Td2008 and Jd2009) and 2 separate LA isolates (LSU10 and LSU13) were used to challenge dogs receiving ivermectin. 133,134 All were successful in creating infection, as was Jd-2009 in dogs having received moxidectin in the six-month injectable formulation. 135 Finally, and most concerning, is the isolate JYD-34, whose L3 produced infection in 7 of 8 dogs (worm burdens 1/3 to ½ that of control group) per group, despite 3 monthly doses of ivermectin, milbemcycin oxime, or selamectin 136, while topical imidacloprid-moxidectin, with high and prolonged tissue levels, protected against JYD-34 in the FDA-CVM one-dose protocol. 136 It is noteworthy that, in a separate 1-dose study of JYD-34, 1 of 8 dogs was infected with 2 worms, despite having received imidaclopridmoxidectin. 136 Importantly, resistance to ML in HW-challenge was proven in all 6 isolates referred to above. Also, the MP3 strain is considered by some to be only partially resistant because it can be prevented with traditional preventive molecules, administered for 3 consecutive months after exposure. 122 The JYD strain is highly resistant to all ML molecules, but only partially resistant to topical imidacloprid-moxidectin. At the time of this writing, the concept of hereditary HW resistance to ML in certain isolates or biotypes is well accepted by clinicians and HW researchers. 122, It also is now clearly apparent that Mf exist which have reduced or absent susceptibility to this drug class. 127,128 Other areas remain controversial, unclear, or both. At least one expert believes that the off-label use of ML as a slow-kill HW adulticide has produced this resistance, with the MS Delta LOE explosion being the

11 result (Dwight Bowman, personal communication, 2014). Bowman, who has long warned against use of the soft-kill technique and long predicted the development of resistance, succinctly explains the hypothesized role treating microfilarmeic dogs with ML. The concern with the extended presence of microfilariae in dogs on a long-term preventive regimen (is that) these microfilariae are persisting in the presence of the macrocyclic lactones. The worry is that these microfilariae have been selected for resistance to these molecules, and the mosquitoes have the ability to transfer worms that have already been selected for their ability to survive in the presence of these products. Some parasitologists, veterinarians, and lay people also believe, or are concerned, that the problem is not just wide-spread, but also spreading north from the Delta While believing in the existence of HW resistance and advocating against the use of the slow-kill approach, this author takes a somewhat less alarmist perspective on the range and immediate danger of resistance. This is based on published and unpublished data and personal observation. Importantly, however, he does strongly advocate for careful surveillance and taking appropriate steps to do what we can to prevent further resistance development and spread, which at its worst, has the potential to render this class of drugs useless to us for HW control (See What Do We Do?). While it is accepted that there are HW biotypes that are resistant or less susceptible to ML, when compared to our historical perspective, there is an alternative to the man-made resistance explanation. This is the possibility that a bell-shaped curve of susceptibility, due to spontaneous mutation 122,137 naturally exists, with HW biotypes being variably susceptible to ML. This possibility is supported by experts on resistance and by the fact that early (1980 s) range-finding studies of ivermectin showed that some heartworms were able to break-through at a dosage of 3 ug/kg (one-half the recommended dosage), even though the majority of infections were prevented. 134 Indeed, it is at least conceivable, since no one had ever looked for resistant biotypes until circa 2005, that less susceptible HW have always been there. If true, this neither reduces the risk, nor diminishes the importance of these recently discovered resistant HW. While not an advocate of the soft- or slow-kill adulticidal approach, this author does not feel that the LOE rise reported from the Delta is likely due solely to resistance arising from off-label use of ML as adulticides. First, the validity of the vast majority of such claims has been called into question. In a study carried out by the author and colleagues, 301 cases of HWI in 271 proven HW-infected dogs from 19 Delta practices expressing concern regarding resistance were scrutinized. 143 Each of the dogs evaluated was the source of an owner satisfaction guarantee claim to the pharmaceutical company making the heartworm preventive in question. Computerized analysis (Window of Infection Tool, Merial, Ltd.) of the medical records from the dogs involved in the claims revealed that only 1.7% of the 301 infections had a perfect purchase history (no lapses in purchasing HW preventive >45 days in duration), evident in th medical record. Another 19% of claims, while they did not have purchase gaps >45 days, within the window of infection, were associated with multiple HWI per dog, sporadic purchase histories, and significant (often prolonged and multiple) purchase gaps outside the window of infection, casting aspersions on the reliability of the preventive care provided to these dogs. Nearly 80% of the claims were invalidated by careful investigation of the owners purchase histories and over 98% drawn into question. Several conclusions can be drawn from these data. First, compliance is a problem. Second, the majority of claims are invalid and the vast majority are, at the very least, questionable. And third, the LOE problem has been vastly overreported. Still another argument against slow-kill driven resistance being the cause of the Delta epidemic is that in 10 years exploration, only 6 dogs have been discovered from which Mf have been able to develop into proven resistant L3. 130,133,134,136 The origin of these dogs has been varied and somewhat scattered (Figure 19), only 3 of which were isolated from the Delta. It is difficult to look at this number and distribution and conclude resistance is wide-spread, with scattered and sporadic being more appropriate adjectives. Furthermore, 2 of the 6 (MP3 and JYD-34) were found in geographical sites without resistance concerns and far from the MS Delta (Illinois and Georgia). That the MS Delta s increase in LOEs is resistance-driven is also challenged by the fact that the three major tenets of resistance have not been met. 122, These tenets are: 1) Resistance does not develop where there is a large refugia (susceptible animals not receiving preventive) - The Delta has likely the largest refugia in the nation, with wildlife, a high percentage of unprotected pets, a low per-capita income, and a massive mosquito population with a high infection rate ,148

12 2) Resistance spreads rapidly 122,147 - The lack of spread of resistance beyond the Delta over the last 10 years is impressive, with cities 1-2 hours beyond its boundaries (e.g., Tupelo, MS, Mobile, AL, and Pensacola, FL) voicing little concern over escalating LOEs (Atkins, personal observation). Furthermore, while most of the resistant isolates are similar in that they share the GG-GG P-gp genotype, two isolates (MP3 and JYD34), which came from dogs far from the Delta and from areas with no current resistance concerns, do not exhibit this polymorphism, arguing that these isolates did not originate in the Delta. Three of 4 remaining resistant isolates had the P-gp genotype and were found in the Delta, supporting the viewpoint that resistance has been largely static and is not spreading. 3) Resistance is forever. 147 Since peaking in 2009, the number of Delta LOE claims made to manufacturers has dropped substantially, according to industry sources. Pulaski has confirmed this in Louisiana, where the peak was in This decline is not consistent with drug resistance being the primary driver of LOE claims. 122 The Delta LOE spike appears to meet none of these 3 necessary criteria needed to support ascribing this increase in LOEs solely to resistance. There are, however, potential alternative explanations for failure of these tenants to hold true for HW resistance in the MS Delta. 122 Certainly, while resistance does indeed exist, the possibility that it might not be the sole, or even major, reason for the rise in Delta LOEs, must at least be entertained. If the Delta LOE dilemma is not entirely due to resistance, as has been suggested herein, then what other factors have contributed? 1) As stated above, compliance failure is the biggest association/causative factor, explaining the vast majority of Delta LOEs ) Also, for maintaining owner satisfaction guarantees, yearly heartworm tests are required. It only stands to reason that if we test more frequently, we will find more heartworms. 3) Another important factor, in the author s opinion, is that the climatic disasters that hit the Gulf Coast, and particularly ravaged the MS Delta, contributed to the HWI explosion. The tremendous rise in the number of LOEs from 2004 to 2009 occurred during the heaviest Gulf Coast hurricane incidence in recorded history. During , the time of ramping up of LOE reports, there were 8 hurricanes, 2.2/year (nearly 3 times the average of 0.83/year). 150 Between , there were no hurricanes in the Gulf. The peak in LOE reports was in 2009, with a steady decline since then, through 2013 (Fig ). Though, clearly circumstantial, the relationship in timing between the major storms and a marked increase in LOEs suggests a cause and effect. There are several explanations as to how this might occur. First major storms leave broken trees, debris, and water in their wake to act as breeding grounds for mosquitoes. Mosquito populations may change after storms, with repopulation by different species, potentially increasing (or lowering) possible heartworm vectors. Finally (and possibly most importantly), the resultant devastation of storms such as Katrina leaves lost and abandoned animals (250,000 in New Orleans, alone) in a heavy heartworm endemic area without veterinary care, including heartworm preventive. This scenario is quite plausible as a (major) contributor to the upswing in LOE reports between 2000 and Lastly, if there is a reward for filing an owner satisfaction claim for failure of a ML, there will be more claims submitted. LOEs have been grossly over-reported. 82p As was shown in a study of over 300 dogs whose owners and veterinarians filed for reimbursement for product failure, between 80 and 98% could not be justified with careful examination of the purchase history. 143 The previous points are not intended to deny the existence of heartworm resistance, nor do they infer that resistance is an unimportant threat to our patients, clients and profession. But they offer evidence that heartworm resistance alone cannot explain the rise in LOEs in the MS Delta and that resistance likely cannot be ascribed to a single factor such as the overuse or misuse of slow-kill methodology. What Do We Do? There are numerous ways in which the practicing veterinarian can help slow the spread of resistance. The most important step that veterinarians can take is to ensure that ML are used appropriately, year-around, at adequate dosage, and without lapses in administration. This effort should be buttressed by efforts to ensure compliance for those clients who purchase preventive, both in refilling and administering preventive. This can be done in a number of ways, including reminders using smart phone apps, the telephone, and post card mailers. If compliance is an impossibility, consideration should be given to the use of 6-month injectable moxidectin. In addition, yearly testing is imperative because if a drug or an owner fails, the resultant HW continues to be exposed to ML, which has been shown to change the genetic make-up of the Mf,

13 with greater expression of genetic markers associated with resistance. 149 In addition, this testing practice will reveal infected dogs so that adulticide can be administered, rather than allowing unrecognized HWI to progress into HWD. The unwanted results of this being the ultimate development of clinical signs, with the conditions for potential spread of HW, rendered less MLsusceptible, after having been exposed to ML. Adulticidal therapy with melarsomine (after 30 days doxycycline and monthly ivermectin) should be administered as soon after diagnosis as is medically and logistically possible. Because administration of ML to microfilaremic dogs risks pushing HW toward ML resistance, all HW antigen-positive dogs should be tested for Mf and, when present, Mf should be eliminated. This can be accomplished with either doxycycline and ivermectin or with imidaclopridmoxidectin. While the AHS recommends either a modified Knott or Millipore filter test, the less arduous direct smear also gives valuable information as to the presence and numbers of Mf. Knowledge of the presence of Mf is important in the fight against resistance and the numbers are important in assessing if precautions need be employed to avoid an adverse reaction. Slow-kill approach to HWI eradication should be avoided! If mitigating circumstances dictate otherwise, doxycycline should be administered and eradication of Mf confirmed (See Macrocyclic Lactones as Adulticides in Controversies). The use of doxycycline in antigen-positive dogs plays another role in reducing infectivity of L3 by eliminating Wolbachia from all HW stages. This, of course, reduces not only the potential of resistant HW being propagated, but reduces the spread of HWI in general. Puppies should be started on preventive as early as drug label claims allow, and kept indoors or in screened enclosures prior to institution of preventive therapy. Doing this alone, has the potential to eliminate nearly 15% of the MS Delta LOE reports, as was shown in the study by Atkins, et al., described above. 143 Ancillary measures include topical or oral repellents/insecticides, mosquito abatement programs, avoiding walking dogs at dawn and dusk, and keeping dogs indoors or in screened enclosures at night. The profession could also lobby to require negative microfilarial status for health certificates, needed for interstate travel or relocation. Research into the incidence and spread of resistance should continue to be studied and the development of genetic markers of resistance in Mf, as is being done at McGill University should be encouraged. 138 Finally, in practices experiencing true LOEs, changing to a preventive with the best spectrum for known resistant biotypes is logical, as is emphasizing reduced mosquito exposure through changes in housing or via permithron-containing, knock-down repellants References available upon request.

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15 # Hurricanes 8 Hurricanes vs Estimated LOE Trend

16 HEARTWORM INFECTION IN DOGS Clarke Atkins, DVM, Diplomate ACVIM (IM, Cardiology) Raleigh, NC PROPHYLAXIS Prevention of HWI is an obvious and attainable goal for the veterinary profession. Prevention failure results from ignorance on the part of owners as to the presence or potential severity of HWI, lack of owner compliance, or from inadequate instruction on preventative measures by the attending veterinarian. 1,32-34 Studies of owner compliance have revealed that approximately 55% of dog owners that use veterinary care purchase heartworm preventative, and enough medication is dispensed only to meet the needs of approximately 56% of those dogs. Hence the proportion of cared for dogs in the population that receive adequate heartworm prophylaxis is less than one third. 33 If one takes into consideration doses purchased but not administered and dogs that are never taken to a veterinarian, the percentage of protected dogs falls drastically. This was emphasized in North Carolina in 1999, when Hurricane Floyd caused extensive flooding and disruption in the poorest part of the state. Of dogs rescued from the floodwaters, 67% were infected with heartworms (personal communication, Dr. Kelli Ferris, North Carolina State University, 2003). In addition, evidence suggests that the veterinary profession is failing in its education of clients. New and colleagues, 35 upon questioning veterinary clients purchasing macrolide preventatives, found that 38% did not realize that their prescribed drug s spectrum was broader than solely preventing HWI. Macrocyclic Lactone (Macrolide) Antibiotics. The introduction of the macrocyclic lactone endectocides (macrolides), ivermectin (Heartgard, Iverhart, TriHeart ), ivermectin with pyrantel pamoate (Heartgard Plus, Iverhart Plus, TriHeart Plus), ivermectin with pyranel pamoate and praziquantel (Iverhart Max ), milbemycin oxime (Interceptor ), milbemycin with lufenuron (Sentinal ) and with spinosad (Trifexis ), selamectin (Revolution ), and moxidectin (ProHeart, ProHeart 6), and moxidectin with imidicoprid (Advantage/Multi TM ) has provided the veterinary profession with highly effective, incredibly safe heartworm preventatives in a variety of formulations and with a variety of spectra. These agents, because they interrupt larval development (L3 and L4) during the first 2 months after infection, have a large temporal window of efficacy and are administered monthly. These products have enjoyed great efficacy, virtually 100%, when used as directed. Recently, a single isolate (MP3) from north-eastern Georgia has shown restistance/tolerance to some macrocyclic lactones, when administered once 30 days after heavy experimental challenge. All are safe in collies when used as directed at preventive dosages. They each have microfilaricidal efficacy and render female heartworms sterile. Hence microfilarial tests for HWI cannot be reliably used in dogs receiving these products. Prophylaxis should be commenced no later than 6 to 8 weeks of age in endemic areas or as soon thereafter as climatic conditions dictate. 19,49 Macrolides should be administered precisely as indicated by the manufacturer. If accidental lapses of more than 10 weeks occur, the preventative should be reinstituted at recommended doses and maintained for at least12 consecutive months. 25 In the event of a lapse in preventative administration during a time of known exposure risk, an antigen test should be performed 7-8 months after the last possible exposure to determine if infection has occurred. It is recommended by the AHS and by CAPC that these agents be used year-around in all areas of the U.S. Off-Label Use. The macrolides are effective microfilaricides, with varying microfilarial kill rates, but microfilariae, in reduced numbers, are often found in the circulation for months after treatment has begun. Some but not all macrolides have adulticidal activity if used continuously for prolonged periods Macrocyclic Lactone Resistance/Tolerance. In 2005, the FDA-CVM reported an increase in the reports of LOEs (Lack of Effectiveness) for macrocyclic lactones and required that such agents no longer be labelled as perfect in terms of efficacy. This failure of complete rapid microfilarial clearing, coupled with concern in the Mississippi River delta region (areas of LA, AR, MS, TN), has caused concern that resistance to this class of drugs may be developing. 41a The proof of this is small, but taken together, the data argue that a small percentage of microfilariae, isolated from dogs in this region have characteristics suggesting tolerance to the drug group. A joint consensus of the AHS and CAPC stated the following (excerpts). There is evidence in some HW populations for genetic variations that are associated with decreased in vitro susceptibility to the macrocyclic lactones. Whether the observed genetic variations constitute heritable resistance is being investigated. Most credible reports of LOE that are not attributable to compliance failure are geographically limited at this time. The extent of the problem is obscured by demonstrated lack of owner and DVM compliance, possible changes in environmental/vector factors, and more effective antigen testing. The potential for resistance is not a reason to abandon use of approved preventive products. The concern relative to the presence of circulating microfilariae in dogs that are started and maintained on monthly preventives is that they could be a source of propagation of microfilariae that are preselected for resistance to macrocyclic lactones. In 2005, Prichard wrote Consideration of the proportion of the D. immitis population in refugia, the life cycle stage targeted, and the anthelmintic dosages used suggest that it is unlikely that significant avermectin/milbemycin [macrolides] resistance will be selected in D. immitis with current treatment strategies. 118 However, this belief was based upon the assumption that people were using the preventives as per label instructions, not using them as adulticides and microfilarial suppressants. The prudent approach is simply to administer the products as approved by the FDA: as preventives that should be given to microfilaria-negative dogs. This means that the soft or slow kill approach to adulticidal therapy should be avoided. Likewise, one could argue against the use of macrocyclic lactones in microfilaria-positive dogs prior to beginning them on adulticidal therapy (ie, a method advocated by this author; see below) as 10% to 20% of these dogs will have circulating microfilariae for months after they start this regimen microfilariae that have seen a macrocyclic lactone. If this approach is utilized, the clinician must ensure that microfilaria are eradicated in the first months of macrolide therapy. All current heartworm preventives belong to the same class of molecule, the macrocyclic lactones, and thus, we need to be very prudent in our long-term