Recent advances in heartworm disease

Veterinary Parasitology 125 (2004) 105–130 Recent advances in heartworm disease Edited by: J.W. McCall a,∗ , J. Guerrero b , C. Genchi c , L. Kramer d a Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA, USA b School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA c Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Milan, Italy d Dipartimento di Produzioni Animali, Università di Parma, Parma, Italy Contributing authors: American Heartworm Society, J. Guerrero, J.W. McCall, C. Genchi, C. Bazzocchi, L. Kramer, F. Simòn, M. Martarino Abstract This compilation of articles consists of four papers presented at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) (held in New Orleans, LA, USA, on 10–14 August 2003) in a symposium session titled “Recent Advances in Heartworm Disease,” organized and chaired by John W. McCall and Jorge Guerrero. The first paper (Guerrero) covered the American Heartworm Society’s most recent revision of their guidelines for the diagnosis, prevention, and management of heartworm infection in dogs, based on new research and clinical experience, particularly in the areas of heartworm chemoprophylaxis, adulticide therapy, and serologic testing and retesting. The entire updated 2003 “Guidelines” are presented herein. One paper (McCall) reviewed the “soft-kill” adulticidal and “safety-net” (reach-back, retroactive, clinical prophylactic) activity of prolonged dosing of prophylactic doses of macrocyclic lactones, concluding that ivermectin is the most effective in this way, milbemycin oxime is the least effective, and the activity of injectable moxidectin and selamectin lies between that of ivermectin and milbemycin oxime. The two remaining papers provided an overview of the discovery, rediscovery, phylogeny, and biological association between Wolbachia endosymbionts and filarial nematodes (Genchi and co-authors) and compelling evidence that Wolbachia may play a major role in the immunopathogenesis of filarial diseases of man and animals (Kramer and co-authors). Keywords: Dirofilaria immitis; Heartworms; Guidelines; Filariae; Wolbachia; Endosymbionts; Macrocyclic lactones; Immunopathology ∗ Corresponding editor. Tel.: +1 706 542 8449; fax: +1 706 542 8467. E-mail address: [email protected] (J.W. McCall). 0304-4017/$ – see front matter doi:10.1016/j.vetpar.2004.05.008 106 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 2003 Updated guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in dogs Prepared and approved by the Executive Board of the American Heartworm Society (Officers: Donald W. Doiron, President; Susan L. Longhofer, Vice President; Sheldon B. Rubin, Secretary-Treasurer; R. Lee Seward, Lynn F. Buzhardt, Charles Thomas Nelson, and John W. McCall, Board Members; Jorge Guerrero, Symposium Chair; and Allan Paul, Editor (paper presented by Jorge Guerrero) 1. Preamble These recommendations are based on the latest information presented at the 2001 Triennial Symposium of the American Heartworm Society. Revisions to the last recommendations published in 2002 are based on new research and additional clinical experience, particularly in the areas of heartworm epidemiology, chemoprophylaxis, adulticide therapy, and serologic testing and retesting. This document focuses primarily on procedures and largely omits discussion of the better known pathophysiologic mechanisms and clinical features of heartworm disease in dogs. Due to increasing recognition of feline heartworm disease as a distinct clinical entity, guidelines for the diagnosis, treatment and prevention of heartworm infection in this species are contained in a separate document. 2. Epidemiology Heartworm infection in dogs has been diagnosed around the globe, including all 50 of the United States. In the US, its territories and protectorates, heartworms are considered at least regionally endemic in each of the contiguous 48 states, Hawaii, Puerto Rico, US Virgin Islands and Guam. Heartworm transmission has not been documented in Alaska, and even with the importation of microfilaremic dogs it is doubtful the climate this far north will permit maturation of infective larvae. Relocation of infected, microfilaremic dogs appears to be the most important factor contributing to further dissemination of the parasite. The ubiquitous presence of one or more species of vector-competent mosquitoes makes transmission possible wherever a reservoir of infection and favorable climatic conditions co-exist. A climate that provides adequate temperature and humidity to support a viable mosquito population and also sustain sufficient heat to allow maturation of ingested microfilariae to infective, third-stage larvae (L3 ) within this intermediate host is a pivotal prerequisite for heartworm transmission to occur. Laboratory studies indicate that development and maturation require the equivalent of a steady 24 h daily temperature in excess of 64 ◦ F (18 ◦ C) for approximately 1 month. Intermittent diurnal declines in temperature below the developmental threshold of 57 ◦ F (14 ◦ C) for only a few hours retard maturation, even when the average daily temperature supports continued development. At 80 ◦ F (27 ◦ C), 10–14 days are required for development of microfilariae to the infective stage. The length of the heartworm transmission season in the temperate latitudes is critically dependent on the accumulation of sufficient heat to incubate larvae to the infective stage J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 107 in the mosquito. The peak months for heartworm transmission in the Northern Hemisphere are July and August. Algorithmic predictions, based on analysis of historical temperature records, have consistently overestimated actual transmission periods confirmed independently by a variety of field studies and appear to represent conservative guidelines. Under the most favorable conditions, these estimates range from less than 4 months in southern Canada to potentially all year in the subtropical zones of southern Florida and the Gulf Coast. The model predicts that heartworm transmission in the continental US is limited to 6 months or less above the 37th parallel, i.e. Virginia–North Carolina state line. Where the prevalence is low, heartworm infection may be detected unexpectedly. Presumably, this represents both a focal spread of infection and heightened awareness through increased testing. Once a reservoir of microfilaremic domestic and wild canids is established beyond the reach of veterinary care, eradication becomes improbable. However, epidemiologic surveys and anecdotal experience suggest that concerted efforts to test and provide timely chemoprophylaxis can reduce significantly the prevalence of infection. 3. Primary diagnostic screening 3.1. Test timing for optimal results The earliest heartworm antigenemia and microfilaremia appear is about 5 and 6.5 months post-infection, respectively. Depending on the sensitivity of the particular heartworm antigen test, antigenemia may precede, but often lags, the appearance of microfilariae by a few weeks. This interval of time between infection and the expected first appearance of microfilariae is the prepatent period. To determine when testing might become useful, a predetection period should be added to the approximate date on which infection may have been possible. A reasonable interval is 7 months. Thus, there is generally no need or justification for testing a dog for antigen or microfilariae prior to about 7 months of age. To detect an infection occurring any time during the preceding transmission season, the predetection period should be added to the approximate end of that period. Indiscriminate testing at any time of the year in an effort to distribute the workload may put the date of testing within the predetection period and waste the test, as far as determining if infection occurred the preceding season. Puppies born during periods when no heartworm transmission is occurring do not need to be tested before starting chemoprophylaxis the following summer. In the cooler regions, transmission may cease in time to allow infections occurring late in the season to mature before transmission resumes. If so, testing late in the spring is likely to detect infections from the preceding year. However, where transmission continues late into the year, the predetection period may overlap the beginning of the next season. If so, monthly chemoprophylaxis should commence (or continue if never interrupted) within 30 days following the start of the new season. If the possibility of an infection occurring late in the preceding season is a concern, testing should be delayed until such time when a positive result is possible. When changing chemoprophylaxis products, dogs should be tested immediately prior to changing and approximately 4–7 months after initiation of the new chemoprophylaxis to evaluate the efficacy of the original and new products (see Section 5). 108 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 3.2. Microfilaria versus antigen testing Whether screening a population of asymptomatic dogs or seeking verification of a suspected heartworm infection, antigen testing is the most sensitive diagnostic method. Microfilaria testing is complementary and may be done in tandem with antigen testing to specifically determine whether this life-cycle stage is also present in dogs that are antigenemic. Even in areas where the prevalence of heartworm infection is high, many (∼20%) heartworm-infected dogs may not be microfilaremic. The current generation of heartworm antigen kits identify most “occult” (microfilaria negative) infections consisting of at least one mature female worm and are nearly 100% specific. Since less than 1% of infections are patent but not antigenemic, testing for antigen will detect more infections than testing only for microfilariae. 3.3. Antigen tests ELISA and immunochromatographic test systems are available for detecting circulating heartworm antigen. Each testing format has proved to be clinically useful. Differences in sensitivity exist, but these are statistically insignificant. False negative results also can occur erratically with any one test, which is why unexpected negative results can sometimes be reconciled by retesting with a different test. Specificity is consistently very high with all the antigen tests, and this is their most important attribute. Selection of a test kit should not be based solely on claims of comparative sensitivity, but also should consider practice preference for “batching” multiple, separate samples or individual, “in-room” sample testing, technician capabilities, technical support, critical timing for reading results, clarity of end result, and unit cost. Testing, usually with one of the commercial kits, also can be performed by all veterinary diagnostic reference laboratories. The amount of antigen in circulation bears a direct but imprecise relationship to the number of mature female heartworms. A graded test reaction can be recognized by ELISA test systems, but quantitative results are not displayed by immunochromatographic tests. The utility of the ELISA tests for assessing the degree of parasitism is limited by such confounding complications as the transient increase in antigenemia associated with recent worm death. Therefore, quantitative analysis of antigen results is highly speculative and requires correlation with other relevant information. For example, radiographic evidence of advanced pulmonary arterial disease typical of chronic heartworm disease and a low or absent antigenemia is consistent with the aftermath of a previous infection that has been cleared, either naturally or by treatment. Reference laboratories report results as either positive or negative. However, in-clinic test results may be subjectively interpreted in order to utilize the limited quantitative potential of the ELISA kits. To obtain reliable and reproducible results, antigen tests must be performed in strict compliance with the manufacturer’s instructions. This has been simplified for several tests that use devices that minimize the number of steps and partially automate the procedure. False positive results usually are due to technical error, such as inadequate washing steps or delay in reading the test. If the validity of a weakly positive result is in doubt, verification may be achieved by repeating the test, and if still ambiguous, independent confirmation by some other means, such as a second antigen test format, concentration tests for microfilariae, J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 109 thoracic radiography to detect signs of heartworm disease, or ultrasonographic visualization of worms. Also, upon request, test manufacturers will analyze ambiguous samples in their own laboratories. False-negative test results occur most commonly when infections are light, female worms are still immature, only male worms are present, and/or the test kit or sample has not been warmed to room temperature. Newer versions of some test kits can be stored at room temperature. Antigen test results should be interpreted carefully, taking other relevant clinical information into consideration. However, in general, it is better to trust rather than reject antigen test results, unless that interpretation is contradicted strongly by independent clinical evidence or circumstances influencing the probability of infection. 3.4. Microfilaria tests Most microfilaremic dogs can be detected by microscopically examining fresh blood for cell movement created by the motility of the microfilariae. A stationary rather than a migratory pattern of movement is indicative of a dirofilaria species, nearly always Dirofilaria immitis in the US. Movement beneath the buffy coat in a microhematocrit tube also may be visible microscopically. However, these are insensitive methods for examining blood in which low numbers (50–100/ml) of microfilariae are present. Therefore, it should not be assumed that no microfilariae are present until at least 1.0 ml of blood has been examined, using a concentration technique (modified Knott test or filtration). The modified Knott test is the preferred method for observing morphology and measuring body dimensions to differentiate D. immitis from nonpathogenic filarial species such as Acanthocheilonema (formerly Dipetalonema) reconditum. Although screening may be based entirely on antigen testing, antigen-positive dogs should also be microfilaria-tested, because a microfilaremia validates the serologic results and identifies the patient as a reservoir of infection. 4. Heartworm chemoprophylaxis Heartworm infection is entirely preventable in dogs, despite their high susceptibility. Since most dogs living in heartworm endemic areas are at risk, chemoprophylaxis is a priority. Furthermore, some evidence strongly suggests that by reducing the reservoir population through increasing the number of dogs receiving chemoprophylaxis, a disproportionately large decrease in the prevalence of infection among unprotected dogs may occur, relative to the percentage of additional dogs receiving chemoprophylaxis. This collateral protection spreads the umbrella of chemoprophylaxis most effectively in communities where heartworm prevalence and dog population density are both relatively low. With the exception of the subtropical south, heartworm transmission has distinct seasonal parameters (see Section 2). Therefore, regional climate should be taken into consideration when evaluating the potential for heartworm transmission. Since there are times of the year when lapses in chemoprophylaxis entail considerable risk, the vulnerable period should be emphasized objectively to sensitize care givers to be particularly conscientious about treating their dogs when it is most important to do so. 110 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 In regions where heartworm transmission occurs more than half the year, seasonal chemoprophylaxis may be viewed as impractical to implement. However, continuous, year-round chemoprophylaxis may not be necessary throughout the northern half of the country, in which the prospects for transmission are limited to the months of May through October. Options for effective chemoprophylaxis include several drugs administered either in oral, topical, or parenteral formulations on a daily, monthly, or 6-month interval. Before starting a prophylactic regime, all mature dogs that may have been infected at least 7 months earlier should be antigen tested and, in appropriate instances, also tested for microfilariae (see Section 3). It is strategically important to determine heartworm status before starting chemoprophylaxis for the first time. This will avoid unnecessary delay in detecting subclinical infections and potential confusion concerning effectiveness of the prevention program, if a pre-existing infection becomes evident after beginning chemoprophylaxis (e.g. chemoprophylaxis initiated during the prepatent period). Heartworm chemoprophylaxis requires authorization by a licensed veterinarian having a valid relationship with the client and patient. To establish this relationship, heartworm prevention should be discussed with the client and, if a record of past treatment does not exist, it may be necessary to test the patient before dispensing or prescribing chemoprophylaxis. 4.1. Macrocyclic lactones The most commonly used heartworm chemoprophylactics are the macrocyclic lactones (ivermectin, milbemycin oxime, moxidectin, and selamectin). These drugs have exceptionally high therapeutic/toxic ratios and possess anthelmintic activity against microfilariae, 3rd- and 4th-stage larvae, and in some instances young adult heartworms. The filaricidal effect of oral and topical formulations on precardiac larvae is achieved by brief pulsing at very low doses, which makes these drugs virtually 100% effective at the prescribed doses and intervals of administration, and among the safest used in veterinary medicine. It is well known that some collie dogs are unusually sensitive to high doses of ivermectin (in excess of 16 times the minimum effective prophylactic dose), but toxicosis has been reported with overdosage of other macrocyclic lactones as well. Invariably, these instances have occurred when concentrated livestock preparations of these drugs have been ingested. Dose miscalculation with extra-label use makes livestock formulations hazardous for dogs. The single-dose retroactive efficacy of all these macrocyclic lactones is assured for 1 month and remains high for at least an additional month. However, efficacy against older larvae declines and requires progressively longer-term administration as the worms age to achieve a high level of protection. The extended post-infection efficacy of the macrocyclic lactones is a safeguard in the event of inadvertent delay or omission of regularly scheduled doses and does not justify lengthening the recommended 1-month interval of administration for the oral and topical formulations. The extent of retroactive efficacy has important implications for chemoprophylaxis in dogs that have either missed several doses during the transmission season or are already well into the transmission season before chemoprophylaxis is started and may already be infected. Short lapses in administration can be accommodated. However, when omissions exceed 10 weeks during the transmission period, continuing monthly prophylaxis through the off-season in cooler climates has merit, since substantial protection still may be provided. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 111 However, this precautionary practice is unnecessary if the integrity of a seasonal prophylaxis program remains intact. When chemoprophylaxis is extended to compensate for interruptions, antigen testing should be performed after the predetection period has passed (see Section 3.1). Testing prior to first starting chemoprophylaxis is advocated for the reasons stated previously. The macrocyclic lactones may be administered to heartworm-infected dogs with few or no microfilariae. However, dogs with moderate to high microfilarial levels should be carefully monitored following administration of these drugs, as they are the most effective microfilaricides available (see Section 11). Oral administration: Ivermectin, milbemycin oxime, and moxidectin are available for monthly oral administration. Some of these formulations are flavored and chewable to increase patient acceptance and facilitate administration. Dose units are packaged for dogs within prescribed weight ranges. To be maximally effective, administration should begin within 1 month of the anticipated start of transmission and the last dose should be given within 1 month after transmission ceases. Topical administration: Selamectin is available as a topically applied liquid, which is a convenient alternative to orally administered products. The parameters for treatment with selamectin are the same as for monthly oral chemoprophylaxis. The FDA has approved a topical formulation of ivermectin combined with imidacloprid, but the product is not commercially available. Parenteral administration: A slow release (SR) formulation of subcutaneously injected moxidectin-impregnated, lipid microspheres provides single-dose, continuous protection in excess of 6 months. Moxidectin SR should be administered within 1 month of exposure to infective mosquitoes, but it is still more than 80% effective 4 months post-infection. Although information about the duration of back-end (>6 months post-treatment) efficacy is not presently in the public domain, full protection extends beyond 6 months. In areas where the risk of infection is limited to 5–6 months, a properly timed injection of moxidectin SR provides a comfortable margin of protection. 4.2. Diethylcarbamazine citrate (DEC) Though protective, the efficacy of DEC is critically dependent upon uninterrupted daily administration during the prescribed period of use. Discontinuation for only 2–3 days may void protection temporarily. Consequently, compliance with the administration regime is even more important with DEC than with the macrocyclic lactones. Due to the lack of any appreciable retroactive effect from DEC, administration must begin shortly before the anticipated start of the transmission season to ensure protection. Furthermore, since DEC is not immediately larvicidal, it is necessary to continue daily administration for two additional months after exposure to infective mosquitoes has ceased. Testing for microfilariae is mandatory before initiating or restarting prophylaxis with DEC. Non-dose-dependent gastrointestinal distress frequently develops shortly after administering DEC to previously untreated microfilaremic dogs. These reactions recur with each dose and, although usually self-limiting, may progress to hypovolemic shock and death. In dogs with occult infections, DEC may be started prior to adulticide therapy to prevent further infection. Dogs with an uncertain infection status, but having no microfilaremia, 112 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 may be started on DEC. If a microfilaremia develops after DEC administration has begun, daily treatment may continue. However, if later interrupted or discontinued, DEC administration should not be resumed, since these dogs then develop the reactions typical of those that have never been exposed to the drug. Chemoprophylaxis should be switched to one of the macrocyclic lactones, and adulticide and subsequent microfilaricide treatments must be completed before resuming DEC administration. After a lapse in DEC administration during the heartworm transmission season, a macrocyclic lactone should be started promptly for its retroactive chemoprophylactic effect. One dose should be sufficient to restore protection if the interruption was less than 2 months. If DEC administration is to be resumed, it should be restarted at the same time the bridging macrocyclic lactone dose is administered. When DEC is omitted for 3 months or longer, macrocyclic lactone administration (ivermectin or selamectin monthly or moxidectin SR every 6 months) should be extended for full-year coverage, as a precaution against latent infections. 5. Retesting Periodic but not necessarily annual retesting is an integral part of ensuring that prophylaxis is achieved and maintained. Where heartworm transmission has a local seasonal cycle, scheduling for retesting should take into consideration the 7-month predetection period used for primary screening. The frequency of testing should be based on several criteria: (1) the prevalence of heartworms in the area of residence or areas to which the patient may travel; (2) the chemoprophylaxis selected; (3) the purchase or administration (e.g. injection) history of the chemoprophylaxis; and (4) any change in the type of chemoprophylaxis prescribed. It is recommended to test at or about the time of any change in the type of chemoprophylaxis, and again 4–7 months after the change. Considering that an antigen test may be positive as early as 5 months after infection, a positive test 4 months after changing products indicates that infection was acquired during administration of the original product. Negative tests 4 and 7 months after changing products confirm efficacy of both products. Annual testing may be indicated in highly endemic areas such as the Gulf Coast. 5.1. Monthly ivermectin, milbemycin oxime, moxidectin and selamectin Macrocyclic lactone chemoprophylaxis will clear microfilariae from the blood of dogs with patent infections by exerting a direct or indirect microfilaricidal effect, depending on the specific product used, and retarding repopulation by gradually suppressing embryogenesis. With uninterrupted dosing, elimination of microfilariae is usually complete within 6–12 months of oral dosing with monthly macrocyclic lactones or 1 month following moxidectin SR injection, and once the adults are sterilized, clearance is permanent unless the dog is reinfected. In the event a pre-existing prepatent infection matures after starting macrocyclic lactone chemoprophylaxis, microfilariae are unlikely to be found or appear only transiently in small numbers. Since macrocyclic lactone chemoprophylaxis may negate microfilaria testing and microfilariae do not contribute to heartworm antigenemia, antigen testing is the most reliable method of retesting. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 113 To verify that a chemoprophylaxis program has been successfully started, retesting 7 months following the end of the first transmission season is advised. A negative antigen test at this time generally ensures that a prepatent infection did not precede initiation of chemoprophylaxis, and an adequate dose had been administered to dogs started on chemoprophylaxis before attaining their mature weight. The subsequent retesting interval depends on how faithfully the patient was administered the prescribed monthly medication. Client consultation and medical purchase and/or prescription records must be evaluated to objectively make the decision. If there is suspicion that one or more doses may have been missed, annual retesting is justified. When medical purchase and/or prescription records are satisfactory, testing intervals may be extended. Annual retesting will not fulfill the objective of early detection if performed indiscriminately within the calendar year without regard for the 7-month predetection period (see Section 3.1). For example, testing in early January will not detect an infection occurring in late July. If the next annual retest is performed the following January, the effective testing interval is 18 months. 5.2. Moxidectin SR injections Since administration of this form of chemoprophylaxis is completely under the control of a veterinarian, the medical record should leave no doubt about the timing and frequency of treatment. A retest should be performed after completion of the first cycle of protection to ensure that a prepatent infection was not present. As with all chemoprophylaxis products, periodic testing will ensure there have been no efficacy breaks, compliance failures, or treatment anomalies. 5.3. Daily DEC The chance is greater that brief interruptions in DEC administration will cause breaks in heartworm protection. In the event a microfilaremia should occur, these dogs are at serious risk of developing potentially fatal reactions following resumption of DEC chemoprophylaxis. Since antigen testing may miss an occasional microfilaremic dog, a microfilaria test must be run before resuming seasonal prophylaxis with this drug. Even if DEC is given daily throughout the year without interruption, it is still prudent to retest annually for microfilariae. Antigen testing is recommended highly for its greater sensitivity, but is not a substitute for microfilaria testing when DEC is used for chemoprophylaxis. 6. Other diagnostic aids Additional testing methods are useful for confirming the diagnosis and staging the severity of heartworm disease. 6.1. Radiography Radiography provides the most objective method of assessing the severity of heartworm cardiopulmonary disease. typical (nearly pathognomonic) signs of heartworm vascular 114 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 disease are enlarged, tortuous, and often truncated peripheral intralobar and interlobar branches of the pulmonary arteries, particularly in the diaphragmatic lobes. These findings are accompanied by variable degrees of pulmonary parenchymal disease. The earliest and most subtle pulmonary arterial changes are found in the dorsal caudal wedge of the diaphragmatic lung lobes. As the severity of infection and chronicity of disease progress, the pulmonary arterial signs are seen in successively larger branches, and in the worst cases, eventually the right heart also enlarges. 6.2. Echocardiography The body wall of adult heartworms is highly echogenic and produces distinctive, short parallel-sided images with the appearance of “equal signs” where the imaging plane cuts across loops of the parasite. Echocardiography can provide definitive evidence of heartworm infection as well as allow for assessment of cardiac anatomic and functional consequences of the disease. However, it is not an efficient method of making this diagnosis, particularly in lightly infected dogs, since the worms often are limited to the peripheral branches of the pulmonary arteries beyond the echographic field of view. When heartworms are numerous, they are more likely to be present in the main pulmonary artery, right and proximal left interlobar branches, or within the right side of the heart where they can be imaged easily. In dogs with hemoglobinuria, visualization of heartworms in the orifice of the tricuspid valve provides conclusive confirmation of the caval syndrome. 7. Preadulticide evaluation The extent of the preadulticide evaluation will vary, depending on the clinical status of the patient and the likelihood of co-existing diseases that may affect the outcome of treatment. Clinical laboratory data should be collected selectively to complement information obtained from a thorough history, physical examination, antigen test, and usually thoracic radiography. The two most important variables influencing the probability of post-adulticide thromboembolic complications and the outcome of treatment are the extent of concurrent pulmonary vascular disease and the severity of infection. Assessment of cardiopulmonary status is indispensable for evaluating a patient’s prognosis. Post-adulticide pulmonary thromboembolic complications are most likely to occur in heavily infected dogs already exhibiting clinical and radiographic signs of severe pulmonary arterial vascular obstruction, especially if congestive heart failure is present. Although a very crude method of assessing the severity of infection, the strength of ELISA-based antigen test reactions may provide an indication of whether an infection is light or heavy (see Section 3.3). Since radiographic signs of advanced pulmonary vascular disease may persist long after an infection has run its course, some of the most severely diseased dogs may have disproportionately low levels of circulating antigen by the time they are tested. Also, some inactive dogs can have large worm burdens and be clinically asymptomatic with minimal radiographic changes. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 115 8. Adulticide therapy 8.1. Melarsomine dihydrochloride The organoarsenical adulticide, melarsomine, is less toxic and more effective than its predecessor, thiacetarsamide, which is no longer commercially available. Melarsomine is administered via deep intramuscular injection into the epaxial lumbar muscles. Mild swelling and some soreness at the injection site may be present for a few days, but this can be minimized by ensuring that the injection is deposited deeply with a needle of appropriate length and gauge for the size of dog and body condition. Strictly adhering to the manufacturer’s instructions and classifying the stage of disease will minimize local reactions and reduce the risk of pulmonary thromboembolism. Also, if adulticide treatment is elected for clinically ill dogs, an attempt should be made to stabilize the patient’s clinical signs with medical support before proceeding with treatment. Exercise restriction during the recovery period is essential for minimizing cardiopulmonary complications (see Section 8.3). The administration protocol of two injections separated by a 24 h interval (= standard protocol) is recommended by the manufacturer for dogs at low risk of thromboembolic complications. For dogs at greater risk, a gradual, two-stage elimination of worms is possible, using a three-injection treatment protocol of one dose initially, followed in 4–6 weeks with a two-dose treatment (= alternative protocol). By initially killing fewer worms and completing the treatment in two stages, the cumulative impact of worm emboli on severely diseased pulmonary arteries and lungs can be reduced. This three-injection protocol is the treatment of choice of the American Heartworm Society and several university teaching hospitals, regardless of stage of disease, due to the increased safety and efficacy benefits. It is also beneficial to administer a prophylactic dose of ivermectin for 1–6 months prior to administration of melarsomine, when the clinical presentation does not demand immediate intervention. The reasoning for this approach is to greatly reduce or eliminate circulating microfilariae and migrating D. immitis larvae, stunt immature D. immitis, and reduce female worm mass by destroying the reproductive system. This results in reduced antigenic mass, which in turns reduces the risk of pulmonary thromboembolism. Antigen testing should be repeated 6 months post-treatment as part of the adulticide protocol (see Section 10). 8.2. Ivermectin Continuous monthly administration of prophylactic doses of ivermectin, alone or in combination with pyrantel pamoate, is highly effective against late precardiac larvae and young (<7 month post-infection) adult heartworms. Comparable adulticide capability of the other macrocyclic lactones has not been reported. The adulticide effect of ivermectin generally requires more than a year of continuous monthly administrations and may take more than 2 years before heartworms are eliminated completely. The older the worms when first exposed to ivermectin, the slower they are to die. In the meantime, the infection persists and continues to cause disease. Therefore, long-term continuous administration of ivermectin generally is not a substitute for conventional arsenical adulticide treatment. If arsenical therapy is 116 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 declined, a lengthy course of prophylactic doses of ivermectin will gradually reduce the number of adult heartworms, but in chronic mature infections this may not be as clinically beneficial. The clinical benefit of exercise restriction in dogs treated with prophylactic doses of ivermectin has not been evaluated. 8.3. Pulmonary thromboembolism Pulmonary thromboembolism is an inevitable consequence of successful adulticide therapy and may be severe if infection is heavy and pulmonary arterial disease is extensive. If signs of embolism (low grade fever, cough, hemoptysis, exacerbation of right heart failure) develop, they are usually evident within 7–10 days, but occasionally as late as 4 weeks, after completion of adulticide administration. Mild embolism in relatively healthy areas of lung may be inapparent clinically. A pivotal factor in reducing the risk of thromboembolic complications is exercise restriction during the critical month following treatment, i.e. a total of 2 months for the three-injection protocol. Diminishing anti-inflammatory doses of glucocorticosteroids given to effect help control clinical signs of pulmonary thromboembolism, but their routine use is not advocated in asymptomatic dogs. The empirical use of aspirin for its anti-thrombotic effect or to reduce pulmonary arteritis is not recommended for heartworm-infected dogs. Convincing evidence of clinical benefit is lacking, and there is some research suggesting that aspirin may be contraindicated. 9. Surgical extraction of adult heartworms 9.1. Caval syndrome (dirofilarial hemoglobinuria) Caval syndrome develops acutely in some heavily infected dogs when large numbers of adult heartworms partially obstruct blood flow through the tricuspid valve and also interfere with valve closure. Severe passive congestion of the liver, a coarse systolic murmur of tricuspid regurgitation, and jugular pulsations are characteristic features of the syndrome. The diagnosis is based on a sudden onset of severe lethargy and weakness accompanied by hemoglobinemia and hemoglobinuria. Caval syndrome can be confirmed conclusively by echocardiographic visualization of heartworms within the tricuspid orifice. The clinical course usually ends fatally within days if surgical extraction of the worms is not pursued promptly. Surgical removal of worms from the right atrium and orifice of the tricuspid valve can be accomplished using local anesthesia and either a rigid or flexible alligator forceps or an intravascular retrieval snare, introduced preferentially via the right external jugular vein. With fluoroscopic guidance, if available, the instrument should continue to be passed until worms can no longer be retrieved. Immediately following a successful operation, the murmur should soften or disappear, and within 12–24 h hemoglobinuria should disappear. Fluid therapy may be necessary in critically ill, hypovolemic dogs to restore hemodynamic and renal function. Within a few weeks following recovery from surgery, adulticide chemotherapy is recommended to eliminate any remaining worms, particularly if many are still visible echocardiographically. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 117 9.2. Pulmonary arterial infections The main pulmonary artery and lobar branches can be accessed with flexible alligator forceps, aided by fluoroscopic guidance. Intraoperative mortality with this technique is very low. Overall survival and rate of recovery by dogs at high risk of pulmonary thromboembolism is improved significantly by physically removing as many worms as possible before beginning adulticide therapy. When the facilities are available, worm extraction is the procedure of choice for the most heavily infected and high risk dogs. However, before electing this method of treatment, sonographic visualization of the right heart and pulmonary arteries should be performed to determine that a sufficient number of worms are in accessible locations. 10. Confirmation of adulticide efficacy Clinical improvement is possible without completely eliminating the adult heartworms. Worms that do survive adulticide treatment are invariably the antigen-producing females. Previously microfilaremic dogs with post-adulticide, female unisex infections eventually become occult, with or without microfilaricide treatment. Consequently, clinical improvement and successful clearance of microfilariae from the blood do not verify a complete adulticide effect. Recurrence of microfilaremia months later generally is indicative of reinfection. Heartworm antigen testing is the most reliable method of confirming the efficacy of adulticide therapy. If all of the adult female worms have been destroyed, heartworm antigen should become undetectable by 6 months post-treatment. The follow-up antigen test can also help differentiate between a persistent infection and reinfection, if an antigenemia is detected again at a later date. Since adult worms may continue to die for more than a month following adulticide administration, dogs that are still antigenemic at 4–5 months post-treatment should be allowed more time to seroconvert before retreatment is considered. The health risk of a few residual heartworms should be assessed on an individual case basis, since complete elimination does not assure further clinical improvement. Factors to consider before electing retreatment are the general health of the patient, age in relation to life expectancy, and the performance expectations for the dog. Before committing to retreatment, there should be a strong expectation that additional benefit will be achieved. 11. Elimination of microfilariae Prior to the introduction of the macrocyclic lactones, elimination of circulating microfilariae was the second step in the stage-specific sequential treatment (adult, microfilariae, precardiac larvae) of heartworm infection. Today, the broad life-cycle filaricidal activity of the macrocyclic lactones has generally reduced microfilaricide treatment to a by-product of chemoprophylaxis. Controlling the spread of heartworms entails decreasing the reservoirs of infection in the dog population, and the benefits of doing so have been cited 118 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 (see Section 4). However, the rapidity with which this is accomplished is less important than eventually achieving the goal. Attempts to clear circulating microfilariae prior to completion of adulticide therapy usually are not immediately successful. Treatment specifically targeting circulating microfilariae may be started as early as 3–4 weeks following adulticide administration. However, more commonly, microfilariae are eliminated eventually, even from non-adulticide-treated dogs, after several months of treatment with prophylactic doses of the macrocyclic lactones or 1 month following a moxidectin SR injection (see Section 5.1). No drugs are approved currently as microfilaricides by the US Food and Drug Administration. However, under the Animal Medicinal Drug Use Clarification Act of 1994, licensed veterinarians are permitted extra-label use of certain drugs having an established clinical application, if a valid veterinarian-client–patient relationship exists. The dispensing veterinarian is personally responsible for ensuring administration of the proper dose and providing appropriate aftercare when products are used in an extra-label application. The use of monthly administered heartworm chemoprophylactics as microfilaricides is governed by this regulation. The macrocyclic lactones are the safest and most effective microfilaricidal drugs to become available to date. All are effective at the prescribed prophylactic doses. It is both unnecessary and dangerous to use livestock preparations of these drugs to achieve higher doses for the purpose of achieving more rapid results. Of the products formulated for dogs, milbemycin oxime and moxidectin SR are the most potent microfilaricides at their label doses and produce the most rapid rate of clearance. If prompt termination of a dog’s reservoir potential following adulticide treatment is considered important, this can be achieved most rapidly with milbemycin oxime, but such use of this drug should be done only with much caution. The monthly administered macrocyclic lactones allow the flexibility of shortening the customary intervals between treatments (perhaps to 2 weeks) in order to accelerate removal of microfilariae. The rapid death of large numbers of microfilariae during the early elimination phase, 4–8 h following the first dose, can cause systemic side effects such as lethargy, inappetence, salivation, retching, defecation, pale mucous membranes, and tachycardia. If reactions occur, most are transient and the signs usually are too innocuous to be appreciated. Occasionally, however, a dog with microfilaremia as low as 5000 mf/ml develops acute circulatory collapse. Prompt treatment with parenteral fluids and one or two shock therapy doses of glucocorticosteroids is an effective antidote. Close observation of higher risk dogs is advised for the first 8–12 h following administration of microfilaricidal drugs at doses that produce a rapid reduction in circulating microfilariae. This precaution becomes unnecessary for subsequent doses, since the pool of microfilariae will have been depleted below the critical level. When elimination of microfilariae is accomplished in the course of heartworm chemoprophylaxis, a microfilaria test should be performed in adulticide-treated dogs at the time the antigen test is repeated 6 months post-treatment. If an accelerated rate of clearance is sought, earlier microfilaria testing after two to three macrocyclic lactone doses (repeated as deemed appropriate) is reasonable. For dogs with patent infections administered only chemoprophylaxis, microfilaria testing prior to beginning the next seasonal treatment cycle is recommended. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 119 These guidelines are based on the latest information on heartworm disease. In keeping with the objective of the Society to encourage adoption of standardized procedures for the diagnosis, treatment and prevention of heartworm disease, they will continue to be updated as new knowledge becomes available. Updated guidelines appear in their entirety on our website (http://www.heartwormsociety.org). Comparison of the “safety-net” and “soft-kill” effects of macrocyclic lactone products used for heartworm prevention John W. McCall Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA, USA 1. Introduction The most commonly used heartworm preventive drugs are the macrocyclic lactones (ML), which are administered monthly as oral (ivermectin, IVM; milbemycin oxime, MBO; moxidectin, MOX) or topical (selamectin, SEL) preparations or every 6 months as injectable (moxidectin) formulations (Knight, 2001). These drugs have exceptionally high therapeutic/toxic ratios and are virtually 100% effective in preventing heartworm disease when given according to the instructions on the label. However, the high level of compliance failure, evidenced by clinic records of much less product purchased than recommended, anecdotal reports of owners not administering all of the doses they purchased, and mistakes in dosing by owners, has resulted in many dogs becoming infected, even while on a prevention program. During the past two decades, my laboratories have been actively engaged in evaluating the effectiveness of MLs against late larval stages, immatures, young adults, and older adults of D. immitis in dogs (McCall et al., 1996, 1998, 2001; McCall, 2002, 2003). These studies have practical relevance in mimicking situations of compliance failure, which is a much greater problem than generally perceived. Owner compliance failure includes missing one or more doses during the heartworm transmission season or initiating treatment 2 or more months after the season starts. 2. Evaluation of the “safety-net” activity of monthly administered IVM and MBO Following up a pilot experiment, a confirmatory trial in beagles was designed to investigate the clinical prophylactic and reach-back efficacy of IVM (Heartgard-30® , Merck & Co., Whitehouse Station, NJ) and MBO (Interceptor® , Ciba-Gigy Corp., Greensboro, NC), when each was administered at monthly intervals, starting 3 or 4 months after SC injection of 50 infective larvae (L3 ) per dog and continuing for 13 or 12 months, respectively (McCall et al., 1996). Microfilarial (MF) (modified Knott test) and antigen (Ag) (DiroCHEK® , Synbiotics Corp.) tests were performed at monthly intervals. Ag test results were subjectively scored (0 = negative, to 5). At necropsy 16 months PI, adult worm counts, compared with controls, were reduced 95.1% (P < 0.01) in the IVM 4-month group and 41.4% in the 120 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 MBO 4-month group. The difference between the IVM and MBO groups was significant (P < 0.01). Live worms were found in all MBO-treated dogs (range, 8–27), all control dogs (range, 12–39), and three of five IVM-treated dogs (range, 2–4). In the 3-month IVM and MBO groups, worm counts were reduced 97.7% (P < 0.01) and 96.8% (P < 0.01), respectively. Microfilariae were seen in all control dogs, two of eight MBO-treated dogs, and one of eight IVM-treated dogs (one MF). The Ag response of MBO-treated dogs in the 4-month group was virtually the same as for control dogs. In all other treated dogs, antigenemia was delayed, weaker, and gradually decreased with time. 3. Light and electron microscopy studies Adult heartworms from dogs receiving monthly prophylactic doses of IVM, beginning during the growth phase of the worm, were small (stunted) and abnormal in motility and/or appearance. Steffans and McCall (1998) demonstrated, by conventional transmission and scanning electron microscopy, gut epithelium changes in adult heartworms obtained from dogs treated monthly for 1 year, beginning at 5 months PI. The intestines of treated worms were distended and contained dark, dense amorphous material that filled the lumen. The increase in thickness of the epithelial layer resulted in a concomitant decrease in the lumenal diameter. Differences in heartworm intestinal epithelial cells included increased intracellular lipid accumulation, a decrease in the volume of mitochondria, and a marked increase in the number and composition of cytoplasmic dense bodies, with calcium replaced by ferric iron in worms from treated dogs. 4. Evaluation of the adulticidal activity of monthly administered IVM The next trial was with older adult heartworms (McCall et al., 1998). Fifteen beagles were given by IV transplantation seven pairs (M/F) of heartworms obtained from dogs infected 7 months earlier. Beginning 1 month later, two groups received 16 consecutive monthly treatments of Heartgard® Plus (6 mcg/kg of IVM and 5 mg/kg of pyrantel, PYR, Merial Limited) and Interceptor® (500 mcg/kg of MBO), respectively, and one group served as a non-treated control. The dogs were bled prior to infection, just before transplantation, and then monthly for MF and Ag (Assure® /CH, Synbiotics Corp.) tests. All dogs were necropsied 16.33 months after the first treatment. Control dogs generally maintained high MF counts, while no MF were seen in MBO- and IVM/PYR-treated dogs after 6 and 11 treatments, respectively. All control and MBO-treated dogs remained Ag-positive throughout the study (generally, scores of 3.5–4.5), while Ag scores in all IVM/PYR-treated dogs gradually declined to a mean of less than 1. At necropsy, all control dogs had normal heartworms (up to 14/dog). All MBO-treated dogs had worm counts similar to controls; only a few worms in 1 dog were abnormal in appearance. In the IVM/PYR-treated group, all dogs had worms, but counts were substantially lower than those for control and MBO-treated dogs, and all surviving worms were abnormal in motility and/or appearance and appeared to be moribund. Thus, monthly dosing with IVM/PYR for 16 months provided a significant reduction of 56% (P < 0.01) in adult heartworm count, while MBO was ineffective. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 121 5. Evaluation of the “soft-kill” adulticidal activity and “safety-net” activity of monthly administered IVM and the adulticidal activity of a single, low dose of melarsomine A trial was then conducted to further evaluate monthly administration of Heartgard® Plus by label instructions for clinical prophylactic (and/or reach-back) activity against 5-month-old immature heartworms and for adulticidal activity against 7-month-old adults (McCall et al., 2001). Twenty-one beagles were infected (50 L3 /dog) and randomly allocated to one non-treated control group of six dogs and three treated groups of five dogs each. One of the three treated groups was given Heartgard® Plus monthly beginning at 5 months PI, another group received Heartgard® Plus monthly beginning at 7 months PI, and the remaining group was given a single IM injection of 2.25 mg/kg of melarsomine dihydrochloride (Immiticide® , Merial Limited) at 19 months PI. Adult heartworm Ag levels (DiroCHEK® ) and MF counts were monitored monthly beginning at 4 months PI, and the dogs were necropsied at 36.2 months PI. The data confirmed and extended our earlier results. Administration of IVM monthly was 98.7% effective against immature worms and 94.9% effective against adult worms. Melarsomine was 70.1% effective. All six control dogs had heartworms (average 15.7; range, 9–27). The earlier treatment with IVM was started, the earlier dogs became negative for MF and Ag. 6. Summary Our studies have clearly demonstrated that the safety-net (retroactive, reach-back, clinical prophylactic) and “soft-kill” (slow killing) adulticidal activity of IVM is superior to that of MBO, and efficacy studies by Bowman and his collaborators (2001) have confirmed this. The unique drug effects of IVM are related to the age of the heartworms at initiation of monthly treatment. The earlier treatment is started, the more stunted the worms are and the shorter their survival time. Conversely, the later treatment is started, the longer the worms live, the more likely Ag will be detected, the higher the Ag level, and the longer the dog will be Ag-positive. The earlier treatment is started, the less likely a patent infection will develop, the lower the MF count, and the shorter the patent period. Drug effects are not greatly enhanced by increasing the dosage and/or administering the drug at shorter intervals, and it appears that continuous monthly treatment is needed to produce the full effects of the drug. As presented in the table, the levels of such activity of topical SEL and injectable MOX lie between those of IVM and MBO. Although prolonged monthly administration of prophylactic doses of IVM clearly kills adult heartworms, the use of this method as an alternative adulticidal therapy at this time should be done only with caution and adequate monitoring (at least once every 6 months) of the infection and disease status of the patient. Although dead adult heartworms induce more acute clinical disease (which sometimes includes death) than do live adult heartworms, it is generally accepted that the presence of live worms during this protracted monthly treatment will continue to cause disease, the severity of which depends on the health status of the dog at the time of treatment, the worm burden, and the level of activity of the patient. It is yet to be determined whether or not monthly treatment with IVM 122 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 would allow irreversible, unacceptable pathologic changes to occur during this time. For many patients, pathologic changes should be minimal and acceptable, compared with the serious consequences frequently resulting from arsenical therapy. Our observation that no heartworm-positive dogs on monthly IVM have died, while at least two non-treated control dogs have died, strongly suggests that less active dogs (e.g. “lap dogs,” “couch potatoes”) are at virtually no risk to severe thromboembolism and death. On the other hand, we must assume that heartworm-positive working dogs would be more at risk from this monthly dosing with IVM than less active dogs, but this is yet to be determined (McCall, 2003). 7. Conclusion Further work is needed to assess any risks that may be associated with the slow killing effect and disintegration of the heartworms. However, prolonged monthly administrations of prophylactic doses of IVM kill older larvae, immatures, and adult heartworms in dogs, even when no one is aware that the dogs are infected. Summary of safety-net (reach-back, retroactive, clinical prophylactic), and adulticidal activity of macrocyclic lactones on Dirofilaria immitis* Drug Age of heartworms (in months) No. of treatments % Efficacy Appearance and/ or motility of live heartworms Reference Ivermectin (6 mcg/kg, per os, monthly) 2 3 3.5 4 4 4.5 5 5.5 6.5 7 8 1 13 12 14 12 12 31 12 12 29 16 100 97.7 97.8 97.8 95.1 86.2 98.7 52.2 34.0 94.9 56.3 ND Abnormal ND Abnormal Abnormal ND Abnormal ND ND Abnormal Abnormal McCall et al. (1986) McCall et al. (1996) Bowman et al. (2001) McCall et al. (1995) McCall et al. (1996) Bowman et al. (2001) McCall et al. (2001) Bowman et al. (2001) Bowman et al. (2001) McCall et al. (2001) McCall et al. (1998) Milbemycin (500 mcg/kg per os, monthly) 2 2 3 3.5 4 4 4.5 5.5 6.5 8 1 2 13 12 14 12 12 12 12 16 95.1 100 96.7 56.5 49.3 41.4 12.7 1.1 15.9 0 ND ND Normal ND Normal Normal ND ND ND Normal Grieve et al. (1991) Grieve et al. (1991) McCall et al. (1996) Bowman et al. (2001) McCall et al. (1995) McCall et al. (1996) Bowman et al. (2001) Bowman et al. (2001) Bowman et al. (2001) McCall et al. (1998) Selamectin (6 mg/kg topically monthly) 2 3 Adult 1 12 18 100 98.5 39.0 ND ND Abnormal McTier et al. (2000) McCall et al. (2001) Dzimianski et al. (2001) Moxidectin (0.5 mcg/kg per os)a 2 1 100 ND McTier et al. (1992) Moxidectin (0.17 mg/kg SC, every 6 months) 4 4/10 6 6/12/18 1 2 1 3 Abnormal Abnormal Abnormal Abnormal McCall et al. (2001) McCall et al. (2001) McCall et al. (2001) McCall et al. (2001) 85.9 97.2 25 25 Modified from McCall et al., 2001. ND: not done; NA: not applicable. a Recommended monthly dosage: 3 mcg/kg. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 123 References Bowman, D.D., Nuemann, N.R., Rawlings, C., Stansfield, D.G., Legg, W., 2001. Effects of avermectins on microfilariae in dogs with existing and developing heartworm infections. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 2001. American Heartworm Society, Batavia, IL, pp. 173–178. Dzimianski, M.T., McCall, J.W., Steffens, W.L., Supakorndej, N., Mansour, A.E., Ard, M.B., McCall, S.D., Hack, R., 2001. The safety of selamectin in heartworm infected dogs and its effect on adult worms and microfilariae. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 2001. American Heartworm Society, Batavia, IL, pp. 135–140. Grieve, R.B., Frank, G.R., Stewart, V.A., Parsons, J.C., Bela, D.L., Hepler, D.I., 1991. Chemoprophylactic effects of milbemycin oxime against larvae of Dirofilaria immitis during prepatent development. Am. J. Vet. Res. 52, 2040–2042. Knight, D.H., 2001. 2002 Guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in dogs. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 2001. American Heartworm Society, Batavia, IL, pp. 259–266. McCall, J.W., 2002. Heartworm update. In: Convention Notes from the 139th American Veterinary Medical Association Annual Convention, Nashville, Tennessee, 13–17 July 2002, pp. 850–851. McCall, J.W., 2003. Heartworm-positive dog requires tailored treatment. DVM Best Pract. (Parasite Control). March, pp. 16–21. McCall, J.W., Dzimianski, M.T., Plue, R.E., Seward, R.L., Blair, L.S., 1986. Ivermectin in heartworm prophylaxis: studies with experimentally induced and naturally acquired infections. In: Otto, G.H. (Ed.), Proceedings of the Heartworm Symposium 1986. American Heartworm Society, Washington, DC, pp. 9–13. McCall, J.W., McTier, T.L., Supakorndej, P., Ricketts, R., 1995. Clinical prophylactic activity of macrolides on young adult heartworms. In: Soll, M.D., Knight, D.H. (Ed.), Proceedings of the Heartworm Symposium 1995. American Heartworm Society, Batavia, IL, pp. 187–195. McCall, J.W., McTier, T.L., Ryan, W.G., Gross, S.J., Soll, M., 1996. Evaluation of ivermectin and milbemycin oxime efficacy against Dirofilaria immitis infections of three and four months’ duration in dogs. Am. J. Vet. Res. 57, 1189–1192. McCall, J.W., Ryan, W.G., Roberts, R.E., Dzimianski, M.T., 1998. Heartworm adulticidal activity of prophylactic doses of ivermectin (6 mcg/kg) plus pyrantel administered monthly to dogs. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 173–178. McCall, J.W., Guerrero, J., Roberts, R.E., Supakorndej, N., Mansour, A.E, Dzimianski, M.T., McCall, S.D., 2001. Further evidence of clinical prophylactic, retroactive (reach-back) and adulticidal activity of monthly administrations of ivermectin (Heartgard PlusJ) in dogs experimentally infected with heartworms. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 2001. American Heartworm Society, Batavia, IL, pp. 189–200. McTier, T.L., McCall, J.W., Dzimianski, M.T., Aguilar, R., Wood, I., 1992. Prevention of experimental heartworm infection in dogs with single, oral doses of moxidectin. In: Soll, M.D. (Ed.), Proceedings of the Heartworm Symposium 1992. American Heartworm Society, Batavia, IL, pp. 165–168. McTier, T.L., Shanks, D.J., Watson, P., McCall, J.W., Genchi, C., Six, R.H., Thomas, C.A., Dickin, S.K., Pengo, G., Rowan, T.G., Jernigan, A.D., 2000. Prevention of experimentally induced heartworm (Dirofilaria immitis) infections in dogs and cats with a single topical application of selamectin. Vet. Parasitol. 91, 259–268. Steffans, W.L., McCall, J.W., 1998. Fine structural observations of gut epithelial changes in adult heartworms induced by monthly treatment of dogs with ivermectin/pyrantel. In: Seward, R.L. (Ed.), Recent Advances in Heartworm Disease: Symposium 1998. American Heartworm Society, Batavia, IL, pp. 217–224. 124 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 Wolbachia endosymbionts in filarial nematodes: an overview C. Genchi, C. Bazzocchi Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Italy Intracellular bacteria were first observed within filarial nematodes in the early seventies by transmission electron microscopy (McLaren et al., 1975; Kozek, 1977). It was only recently however, that these bacteria were identified by Sironi et al. (1995) as belonging to the genus Wolbachia (Rickettsiales), which also include enosymbionts found in arthropods. These authors were the first to describe Wolbachia within Dirofilaria immitis and this “rediscovery” paved the way for some of the most exciting and ground-breaking research activity in the phylogeny and biology of filarial nematodes and in the pathogenesis of filarial infection. 1. Wolbachia phylogeny Six supergroups of Wolbachia (A–F) have thus far been described, on the basis of branching and clustering patterns in unrooted phylogenetic trees derived from 16S rDNA and ftsZ gene sequences (Lo et al., 2002). The majority of nematode wolbachiae have been assigned to supergoups C and D. However, there is one species of filaria (Mansonella ozzardi) whose Wolbachia has not been precisely assigned to any of the six supergroups (Lo et al., 2002). Supergroups A, B, E and F encompass the wolbachiae of arthropods. The branching order of these six supergroups of Wolbachia is not clear, and some of these groups might be paraphyletic. It is clear however that all the wolbachiae thus far found in nematodes are phylogenetically very distant from any of the wolbachiae thus far found in arthropods. There is therefore no evidence for current transmission of Wolbachia from arthropods to nematodes, or vice versa. Wolbachia is present in many of the filarial nematodes that cause disease in humans and animals, including Onchocerca volvulus, Wuchereria bancrofti, Brugia malayi and D. immitis. In species harbouring Wolbachia, the prevalence of infection appears to be 100%. Moreover, the infection appears stable along evolutionary times: main branches of filarial evolution are composed of species harbouring Wolbachia (Casiraghi et al., 2001). In adult nematodes, Wolbachia is present in the hypodermal cells of the lateral chords; the cytoplasm of some of these cells is filled with Wolbachia and resemble insect bacteriocytes. In females, Wolbachia is also present in the ovaries and in developing embryos, but has not been demonstrated in the male reproductive apparatus (Sacchi et al., 2002). The bacterium is vertically transmitted through the cytoplasm of the egg, and there is no evidence for horizontal transmission or for paternal transmission. Indeed, the phylogeny of Wolbachia matches that of the host filariae (Casiraghi et al., 2001). 2. Wolbachia: arthropods versus filarial nematodes It is now widely accepted that maternal transmission of symbiotic microorganisms (from host mother to offspring) can lead to the establishment of two different types of J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 125 host–symbiont relationships: (1) mutualism, where the symbiont increases its own fitness by increasing the fitness of host females; and (2) reproductive parasitism, which encompass those associations where the symbiont manipulates the reproduction of its host in ways that enhance its spread in the population (Werren and O’Neill, 1997; Bandi et al., 2001a). In mutualism, although males are not involved in the transmission of the symbiont they often also benefit from the infection. The main difference between mutualism and reproductive parasitism is the consequence on host evolution. In mutualism, selective pressures acting on the host and the symbiont are convergent and can lead to stable infection, co-adaptation and reciprocal dependence between partners (i.e. obligatory symbiosis) (Lipsitch et al., 1995). Inversely, in reproductive parasitism the symbiont can spread even though it has a detrimental effect on infected females. Interestingly, the strictly intracellular Wolbachia bacteria have adopted both strategies in their invertebrate hosts. In filarial nematodes, the hosts seem to be dependent on the infection. Indeed, the removal of bacteria has detrimental effects on development, survival and reproduction (Bandi et al., 2001b). Inversely in arthropods, Wolbachia infection most often induces reproductive alterations, including cytoplasmic incompatibility (CI), male killing, feminization or parthenogenesis (Werren and O’Neill, 1997). Assuming that Wolbachia in nematodes and arthropods share a common symbiotic ancestor, the evolutionary interpretation of such diversity in Wolbachia lifestyles is not clearly understood. Moreover, obligatory symbiosis was recently found in insects, suggesting that host differences between arthropods and nematodes are not sufficient to explain this diversity. 3. Wolbachia-filarial nematodes: obligatory symbiosis? Current information on the distribution and phylogeny of Wolbachia in filarial nematodes suggests that Wolbachia is necessary for the host nematode (100% prevalence in infected species; whole branches of filaria evolution composed by infected species; consistency of host and symbiont phylogenies). The results of experiments with tetracycline (and derivates) on animal hosts infected by filariae are in agreement with the idea that the Wolbachia-filaria association is obligatory (McCall et al., 1999). Tetracyclines have indeed been shown to inhibit the development from the infective larva (L3 ) to the adult, to inhibit embryogenesis and microfilaria (L1 ) production and to interfere with the long-term survival of adult nematodes. In addition, tetracycline has also been shown to interfere with L1 –L3 development of the filaria in mosquito hosts (reviewed in Bandi et al., 2001b). Similar results have been obtained in in vitro experiments, and other antibiotics have been shown to cause attrition in filariae harbouring Wolbachia. It still must be demonstrated that there is a cause-and-effect relationship between the activity of antibiotics on Wolbachia and the deleterious effects on the nematodes. That tetracycline treatments do not interfere with L3 -adult development or with microfilaria production in a filarial species which is free of Wolbachia (Acanthocheilonema viteae) is strongly suggestive of such a relationship. The fact that tetracycline and other antibiotics are detrimental to filarial species harbouring Wolbachia has opened the way to new therapeutic strategies for the control of filariasis. For example, the combination of a “traditional” antifilarial drug (ivermectin) associated with doxycycline (a derivate of tetracycline) appears very 126 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 promising for the therapy of human onchocerciasis (Taylor et al., 2000; Hoerauf et al., 2001). 4. Conclusion Wolbachia endosymbionts have provided numerous new insights into the biology of filarial nematodes and have offered a unique opportunity to develop new control strategies against those filariae that cause disease. Several questions still remain to be answered (what are the main conditions required for a Wolbachia infection to evolve toward obligatory symbiosis? What are the evolutionary mechanisms capable of generating such evolutionary transitions?), but research is continuing into this fascinating microorganism and its relationship to filarial worms, including D. immitis. References Bandi, C., Dunn, A.M., Hurst, G.D.D., Rigaud, T., 2001a. Inherited microorganisms, sex-specific virulence and reproductive parasitism. Trends Parasitol. 17, 88–94. Bandi, C., Trees, A.J., Brattig, N.W., 2001b. Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases. Vet. Parasitol. 98, 215–238. Casiraghi, M., Anderson, T.J.C., Bandi, C., Bazzocchi, C., Genchi, C., 2001. A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122, 93–103. Hoerauf, A., Mand, S., Adjei, O., Fleischer, B., Buttner, D.W., 2001. Depletion of Wolbachia in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357, 1415–1416. Kozek, W.J., 1977. Transovarially-transmitted intracellular microorganisms in adult and larval stages of Brugia malayi. J. Parasitol. 63, 992–1000. Lipsitch, M., Nowak, M.A., Ebert, D., May, R.M., 1995. The population dynamics of vertically and horizontally transmitted parasites. Proc. R. Soc. Lond.: Ser. B Biol. Sci. 260, 321–327. Lo, N., Casiraghi, M., Salati, E., Bazzocchi, C., Bandi, C., 2002. How many Wolbachia supergroups exist? Mol. Biol. Evol. 19, 341–346. McCall, J.W., Jun, J.J., Bandi, C., 1999. Wolbachia and the antifilarial properties of tetracycline. An untold story. Ital. J. Zool. 66, 7–10. McLaren, D.J., Worms, M.J., Laurence, B.R., Simpson, M.G., 1975. Micro-organisms in filarial larvae (Nematoda). Transact. R. Soc. Trop. Med. Hyg. 69, 509–514. Sacchi, L., Corona, S., Casiraghi, M., Bandi, C., 2002. Does fertilization in the filarial nematode Dirofilaria immitis occur through endocytosis of spermatozoa? Parasitology 124, 87–95. Sironi, M., Bandi, C., Sacchi, L., Di Sacco, B., Damiani, G., Genchi, C., 1995. A close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Mol. Biochem. Parasitol. 74, 223–227. Taylor, M.J., Bandi, C., Hoerauf, A.M., Lazdins, J., 2000. Wolbachia bacteria of filarial nematodes: a target for control? Parasitol. Today 16, 179–180. Werren, J.H., O’Neill, S.L., 1997. The evolution of heritable symbionts. In: O’Neill, S.L., Werren, J.H., Hoffmann, A.A. (Eds.), Influencial Passengers. Oxford University Press, New York, pp. 1–41. J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 127 Wolbachia endosymbionts and the immunopathogenesis of filarial disease L. Kramera , F. Simònb , M. Martarinoc , C. Bazzocchic a Dipartimento di Produzioni Animali, Universià di Parma, Italy; b University of Salamanca Medical School, Spain; c Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Italy Human and animal parasitic filarial nematodes, often the cause of severe disease, harbour intracellular bacteria of the genus Wolbachia (Rickettsiales). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. The immunopathology of filarial disease is extremely complex and the clinical manifestations of infection are strongly dependent on the type of immune response elicited by the parasite. Furthermore, the fact that adult parasites can survive for years in otherwise immunecompetent hosts is likely due to the parasite’s ability to avoid/modulate the immune system of the host. It is therefore extremely important to identify which components of the parasite are responsible for modulation of the host’s immune system, particularly those involved in stimulating the production of pro- and anti-inflammatory cytokines. Research aimed at elucidating these mechanisms would have important implications not only in filarial disease, but also in the fields of immunology and immunopharmacology. 1. How does the filarial worm-infected host come into contact with Wolbachia? So far, all experimental evidence indicates that infected hosts come into contact with Wolbachia following the death of the parasite (natural attrition, microfilarial turnover, pharmacological intervention). Keiser et al. (2002) reported Wolbachia DNA in the blood of patients following DEC treatment for O. volvulus. Brattig et al. (2001) described the presence of Wolbachia in macrophages in skin nodules from O. volvulus-infected humans. Inflammatory cells staining positive for the Wolbachia surface protein (WSP) surrounded dead and degenerated microfilariae. We have recently seen similar staining in D. immitis-infected dogs both in the lung and in circulating monocytes (Kramer et al., 2003). 2. Wolbachia-derived molecules (WAMs): pathogen-associated molecular patterns? The role of Wolbachia in the host response to filarial infection may include interaction between bacterial molecules and the innate and adaptive immune system. The innate immune system represents a defense mechanism against molecular structures that are conserved among a wide range of organisms. It consists in the recognition of specific “markers” (pathogen-associated molecular patterns, PAMPs) that signal the presence of “generic” pathogens. The consequent recognition of these PAMPs by Toll-like receptors (TLR4) on the surface of antigen-presenting cells leads to the production of reactive oxygen species, pro-inflammatory cytokines and to the up-regulation of co-stimulatory molecules that assist in development of an adaptive immune response. Bacterial molecules that are able to induce an innate immune response include GroEL protein (HSP60), lipopolysaccharides (LPS), formylmethionine groups and non-methylated CpG motifs (Abbas and Janeway, 2000). The 128 J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 potential PAMPs that have been identified from different filarial Wolbachia include LPS, Wolbachia Surface Protein and heat shock protein 60. 3. Onchocerca and WAMs In a murine model of river blindness (caused by the inflammatory response to dead microfilariae in the eye of O. volvulus-infected humans), Saint Andre et al. (2002) showed that intracorneal injection of O. volvulus extracts caused an increase in stromal haze and thickness, associated with neutrophil infiltration. When O. volvulus was depleted of Wolbachia through antibiotic treatment of infected hosts, the inflammatory response was notably decreased following injection. Furthermore, the same decrease in stromal haze and neutrophil infiltration was observed when extracts from Wolbachia-positive worms were injected into the cornea of TLR4 knock-out mice, indicating a dependence on this receptor for Wolbachia activity. Brattig et al. (2000) have also reported CD14-dependency on cytokine production in monocytes exposed to O. volvulus extracts. Wolbachia from O. volvulus also stimulates an adaptive immune response as shown by the presence of circulating anti-WSP antibodies in human patients with onchocerciasis (Brattig et al., 2001). Interestingly, antibodies were of the IgG1 class, suggesting a possible polarization of the acquired response. 4. Brugia and WAMs Taylor et al. (2000) showed that extracts of B. malayi stimulate the production of TNFalpha, Il-1beta and NO by CD14+ macrophages, cytokines normally produced after exposure to bacterial LPS. The presence of LPS in filarial extracts was also suggested by positive results of the Limulus amoebocyte lysis test (LAL). Studies with extracts of A. viteae, that lack Wolbachia, showed no cytokine production by macrophages, confirming the direct effect of Wolbachia in this innate inflammatory response. Anti-WSP antibodies have also been observed in B. malayi infected humans and monkeys, again indicating a role for Wolbachia in the humoral response to filarial infection (Punkosdy et al., 2001, 2003). 5. D. immitis and WAMs Bazzocchi et al. (2003) have recently studied the effect of Wolbachia surface protein from D. immitis on canine neutrophils. The authors showed that WSP stimulates neutrophil chemokinesis and IL-8 production. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP. Several studies have also been carried out on the humoral response to D. immitis WSP. Infected cats have circulating anti-WSP antibodies, as shown by Western blot analysis (Bazzocchi et al., 2000). Humans living in D. immitis-endemic areas have circulating anti-WSP antibodies and, more interestingly, patients diagnosed with J.W. McCall et al. / Veterinary Parasitology 125 (2004) 105–130 129 pulmonary nodules caused by D. immitis have much higher titres (Simòn et al., 2003). Human infection with D. immitis is often aborted; however, in certain cases, the worm may develop and migrate, often reaching the lungs, where it is subsequently sequestered in an inflammatory nodule. The high level of circulating antibodies against a Wolbachia protein in these patients confirms the hypothesis that contact between Wolbachia and the host immune system is more likely following worm death and degeneration. Recently, cytokine production and antibody response in BALB/c mice inoculated with soluble antigens from third-stage larvae or from adult worms of D. immitis were reported (Marcos-Atxutegi et al., 2003). Inoculated mice first produced IFN-␥ followed by a peak in IL-4. Specific antibodies to the Wolbachia protein WSPr were exclusively IgG2a, while antibodies against peptides derived from antigens of D. immitis were in the IgG1 and IgE subclasses. The results of this study suggest not only a role for Wolbachia in the immune response to filarial infection, but also a potential skewing in favor of a Th1 response. These results have important implications for immunoprotection against infection and merit further study. 6. Conclusion There is compelling evidence that the bacterial endosymbiont Wolbachia may play a role in the immunopathogenesis of filarial disease. This of course could lead to further implications, not only in the control of infection (see Genchi et al., this volume), but to the possible use of antibiotics to decrease Wolbachia populations in the worms, thus reducing the inflammatory effect due to WAMs. References Abbas, A.K., Janeway Jr., C.A., 2000. Immunology: improving on nature in the twenty-first century. Cell 100 (1), 129–138. Bazzocchi, C., Ceciliani, F., McCall, J.W., Ricci, I., Genchi, C., Bandi, C., 2000. Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis. Proc. R. Soc. Lond. B 267, 2511–2516. Bazzocchi, C., Genchi, C., Paltrinieri, S., Lecchi, C., Mortarino, M., Bandi, C., 2003. Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8. Vet. Parasit. 117, 73–83. Brattig, N.W., Rathjens, U., Ernst, M., Geisinger, F., Renz, A., Tischendorf, F.W., 2000. Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes. Microbes Infect. 2, 1147–1157. Brattig, N.W., Buttner, D.W., Hoerauf, A., 2001. Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria. Microb. Infect. 3 (6), 439–446. 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