heartworm - dirofilaria immitis - requires mosquito vector for L1->L3 development, takes 1-2 weeks depending on temperature. heartworm is a parasite of the pulmonary artery as opposed to the heart, mainly. mosquito (infected) is sole source of dirofilarial infxn PPP (between introduction of L3 and appearance of mmf) is about 190 days/6 mos. canids, wolves, dogs - primary definitive hosts also seen in felids, wild/domestic cats mustelids some other spp in dogs, there is another filarial infxn which causes circulation of mmf - dipetalonema reconditum, transmitted by fleas, benign, but confuses people at time of dx. you will see mmf in circulation so you need to speciate. think back to pertinent facts about pathogenesis of HW infxn. these worms in pulmonary arteries are the primary cause of dz. primary lesion is damage to pulmonary arteries by adult HW living there. grossly you see vessels appear sacculated or dilated. at necropsy you see the intimal lining of the vessels is disrupted, with villi or plaques projecting into lumen - response to damage. these plaques can narrow the lumen significantly or occlude smaller branches. result: pulmonary hypertension, excersise intolerance. platelets have a role in plaque production. once initial injury occurs to intima, platelets are recruited, and PDGF contributes to outgrowth of endothelial cells into the lumen of the artery. the severity of the lesions is due to a couple of things - number of adult worms relative to size of host, and the fact that the lesions are cumulative. the longer the worms are there, the worse the plaques get. the excersise level is also important. sedentary dogs have less severe clinical illness than sporting dogs. so - worm load, chronicity, and exercise level are all factors that come into play in determining clinical outcome of HW dz in dogs. normal PA - smooth vascular intima, affected intima leathery and villous. last time we wondered what happens if one or more HW dies during the infection? dogs can outlive their HW. when worms die (spontaneously or due to tx) there are thromboembolic complications. living HW ->villous lesions. dead HW -> thromboemboli in smaller branches of PAs. clots, edema, vascular leakage into surrounding lung parenchyma. these are complications that result from the death of adult HW, either from natural or drug related causes. this phase of the dz is more severe in cats b/c higher spontaneous death rates of HW. another clinical manifestation which is infrequent is acute liver and heart failure due to retrograde migration of adult worms in dogs with very high worm burden, back into right heart and vena cava. these dogs have weak right heart, decreased CO, due to PA effects, allowing worms to move back -> causing caval syndrome. unlike most features of k9 hw disease, the effects of the caval syndrome are acute - acute wt loss, weakness, anorexia, collapse, another aspect is hemoglobinuria, which is practically pathognomonic for caval syndrome. this is due to cholesterol esterification being blocked, causing increased cholesterol to accumulate in rbc membranes, resulting in hemolytic disease. these dogs can die overnight if this happens. takes heroic intervention to save them, and is a surgical emergency. slide: some guy doing jugular venotomy to reach into RH to grab HW with this little flexible instrument - cut down to jugular, insert probe, get it into RH under fluoroscopic imaging, open instrument, close jaws, and hopefully bring out a group of adult HW. do this a few times - 2-3 times usually gets 80-90% of the HW. in this country, this is done only for dogs with caval syndrome, generally. most adult HW infxns are treated with adulticides. in japan, they do this a lot more, rather than drug tx. clinical improvement post op is remarkable. 12 hrs postop urine will be clear of hemoglobin. microfilarial hypersensitivity: a certain percent of infections are occult - there are adults present, but due to host immune response, mmf are removed from peripheral circulation, and generally adhere to reticuloendothelial cells in microvasculature, esp in lung, which may cause allergic pneumonitis due to infiltration by eosinophils etc. dx of HW infection: clinical: chronic coughing, labored breathing, increased excercise intolerance , also possibly wt loss, anorexia, heart failure, debilitation in more advanced cases. mainly cough, labored breathing and exercise intol. lab: nothing really specific. some dogs are anemic, with decreased HCT, but not pathognomonic. general lab findings are generally nonspecific. radiology: frequently used to confirm or support dx - on angiogram we see saccular dilation of PAs, dendrification of PAs, look like old gnarled oak tree branches. in occult infections, we expect a higher rate of interstitial lung dz, which may also show up as cloudy patches on chest rads. parasitologic dx- detection of mmf in peripheral circulation - direct smear 'quick and dirty' but insensitive; knotts concentration technique; micropore filtration method; concentration tests have increased sensitivity. don't want to say they aren't there until you've done knotts or micropore test. a significant number of cases will be occult for various reasons. no mmf despite presence of adults. until the advent of modern serologic tests these were very difficult to dx. in late 70s, tests were developed for HW Ab. now they have all these snap tests and stuff to test for circulating HW Ab and are being used in cats. current serologic tests for occult and patent infections in dogs test for circulating adult HW Ag, not Ab. these are soluble metabolites produced by adult HW. most of these tests test for products made by female HW, not males! these use an immobilized monoclonal Ab specific for particular antigen, they capture the antigen if it is present in serum or blood, we add a second monoclonal Ab which matches a different epitope on the Ag , and this Ab is tagged with something so we see a color change if it makes the antigen sandwich. eg, if second Ab sticks, we see the color. the advantage of Ag serology - available in easy kits, highly specific and sensitive, and a new development recently is that many have a semiquantitative capability, so we can estimate # of adult worms present. they are also specific to the point where we can tell apart dipetalonema and dirofilaria. so that's great! go back to the mmf tests - one thing to remember is that frequently there is temptation to equate high numbers of mmf with high numbers of adults. but we know these numbers do not correlate. presence of mmf helps to detect infxn, but doesn't provide info re: # of adults. basics of chemical tx and control of HW infection. sad fact - no one drug will kill all life stages of dirofilaria in the host. to manage HW infection in dogs, we require a 3 pronged approach to chemical tx and control. first prong is adulticidal tx for adults in PA. caparsolate - carbamoyl thioarsenites immiticide - melaminyl thioarsenites - drug of choice all these drugs are polyvalent organoarsenical compounds. melarsamine is a drug that is injectable into epaxial muscles in lumbar area - deep into muscles. standard dose of immiticide - 2 inj 24 hrs apart, 2.5 mg/kg in dogs w/severe disease, lots of worms - alternative regimen is 1 injection, then 30 days later follow standard regimen of 2 inj 24 hrs apart. this kills off some of the worms initially, to reduce potential thromboembolic complications. adulticide tx does carry risk of thromboembolic complications, due to killing off adult worm burden. course of immiticide tx done on inpatient basis with dogs on cage rest. this is done b/c the thromboembolic complications are exacerbated by exercise. immiticide is new, licenced a couple of years ago. so after immiticide, there is a lot of resolution over the 1-3 mos following adulticide tx. prognosis very good for resolution of PA dz. second prong: tx of circulating mmf which are not killed by immiticide. ivermectin is active against mmf at 50 mcg/kg (this dose is not licensed - is safe, but not approved) milbemycin at 500 mcg/kg give 3-6 wks post adulticide tx. observe in hospital for at least 8 hrs, b/c dog may have reactions to mass killing of mmf. then screen for mmf in 2-3 wks. 3rd prong: chemoprophylaxis drugs of choice for this are two we already know - ivermectin, 6-12 mcg/kg, in heartgard; milbemycin 500mcg/kg - interceptor, and diethylcarbamazine the stuff in filaribits. ivermectin and milbemycin are once a month tx. they work by targetting L4s, and getting rid of the accumulated larvae from that month. diethylcarbamazine is given daily, and unlike the macrolides it targets early L3 molting to L4. it works more in the sense of classical preventative, preventing establishment of incoming L3s. this drug, unlike the others, can precipitate a fatal shock reaction if given to dogs with circulating mmf. if you tx with the monthly drugs you won't get that rxn. ---break--- dr guerrero feline hw dz the handout has most of the data in it, all the tables. cats with heartworm - in endemic areas, if you are not seeing the infected cats, they are seeing you... Ray Dillon, Auburn U. the parasite is the same, biology similar, location different. not new. first reported in 1921. prevalence parallels canine hw, but to lesser degree. rates similar to FeLV, FIV, FIP. cycle different from in dogs. to have transmission, you need infected dog reservoir,because cats don't have (many) mmf circulating- first of all. mosquito bites dog, gets infected. L1-L3 in mosquito. cat bitten by mosquito, gets infected. consider size of adult worm in respect to size of normal cat PA. mechanical effects are great. molts and migrations similar to in dogs aberrant migrations to CNS more common in cats - blindness or other dramatic signs - but only seen in experimental infections. not frequent. in cats microfilaremia is rare and transient number of adults only 1 - 3, in dogs 10-100s. adult HW lifespan shorter in cats, only about 2 yrs. if you have canine infections in your area, you will also see feline infections at a lower rate. in SE USA in LA there was a 16% prevalence in cats! american HW society study recently found feline HW dz in 36 states including PA and entire east coast. clinical studies NCSU/TAMU study - showed even totally indoor animals can get feline HW infxn. pathophysiology very different in cats than dogs. two very iportant developmental stages in which severe signs and death can occur =- arrival of L5 into PA and death of adults. these times trigger rapid immunological cascade causing death. this happens probably b/c of large numbers of pulmonary intravascular mphages in cats which aren't present in dogs. also we see dz similar to that in dogs - villus endarteritis, inflammatory infiltrate of intima, hypertrophy of muscular media important point with this is the ffact that all pathological effects wll also reduce the lumen diameter, producing clinical signs. this cat was presented as sudden death case. in fine branches of PA are small pieces of dead HW. this animal died due to sudden immunological rxn to death of adult HW. transversal section showing villus endarteritis and muscle proliferation in media - can almost completely obstruct PA. pulmonary parenchymal effects - lesions mainly here - pulmonary interstitial dz ARDS like reaction. can seem like asthma. eosinophilic accumulation. pulmonary intravascular mphages are activated and are probably underllying cause of problem. common sign - vomiting. having no signs is common. respiratory signs are common. usually confused with asthma exercise intolerance is rare ascites rare; sudden death common. dx: difil test not very useful. mmf rare in cats. Ag tests good - snap, dirochek, petchek. positive tests are conclusive. negative tests are inconclusive, however. Ag detection depends on number of adult females in host. Ab tests are therefore most useful in cats. Ab is made by cat in response to presence of HW. supports dx. negative test inconclusive. pos test indicates exposure. it's a measurement of risk. paired sera useful. radiographic pictures - difficult to evaluate. it's not clearcut. echocardiography - useful if worms are near heart. there are charts in the yellow handouts. treatment options for cats? a very risky situation. option one: caparsolate - not approved, very toxic option two: let worms die on their own -probably most rational. symptomatic tx. rest and corticosteroids. watch and wait. sudden death may still result. start chemoprophylaxis. only alternative is prevention - heartgard only approved product - ivermectin monthly dose at 24 mcg/kg which also gets hooks at L4 stage (ancylostoma spp). this is a chewable formula that cats seem to like. about 98% of them will eat it. bottom line - yogi berra said "we have every chance of not finding that which we are not looking for." ----break---- dr farrell immunity to helminths no slides today. we're talking in very general terms about immunity to helminths, mainly nematodes b/c that's what we know about. we don't know alot of mechanisms of immunity to GI nematodes - haven't been studied much. there are some recent advances in terms of vaccine development which are kind of interesting and explain how to control this, a little bit at least. very basic review of immunology - in terms of T cells, we know that naive T cells can become either TH1 or TH2 cells. TH1 cells are responsible for CMI and they make IL2, IFNg mainly. TH2 cells are more involved in humoral immunity and help B cells - they make IL4, IL5, IL6, IL 9, IL 10. there are some interleukins made by both, like IL3. some of these cytokines are very important. IL4 is absolutely required for production of IgE Ab for allergic response. IL3 is very important in terms of mast cell production and differentiation. IL5 is very important in terms of eosinophil production and differentiation. if we consider in general all helminth infxns, what are the characteristic immune responses that occur? in almost all helminth infxn, whether hemonchus in sheep or ascaris in humans or trichostrongyles in cattle, there is an elevation in IgE, across the board. IgE levels are always increased. there is eosinophilia, so generally there are increased eosinophil numbers in peripheral blood. in terms of GI nematode infections, there is mastocytosis. if you look at site of parasitization, you will see increased mast cells withinthe tissues. eosinophils may also be recruited to site of infection. all these things are characteristic of allergic type response. Eosinophilia and IgE characterize all allergic responses in humans, atopy, asthma, etc, and are the main things in response to helminths. these are detrimental types of responses that we have to control with allergy shots and drugs, in the popular opinion, but they may have evolved to help to control parasites. IgE can bind to mast cells which express the Fc receptors for the epsilon chain of IgE. the FcE receptor will bind IgE, and when an Ag contacts the IgE, it will crosslink the Fc receptors, and the mast cells wiill degranulate, releasing contents including histamine, leukotriene, prostaglandins. these can either lead to vasoconstriction or vasodilation, contraction of smooth msucle in intestinal tract, increased fluid leakage out of tissue, local edema, etc. eosinophils produce toxic factors, enzymes, cationic proteins, that are highly toxic molecules, which if they contact parasitic organisms could kill them. but none of these are specific. there is no evidence that any of this stuff has a specific effector mechanism against parasitic GI nematodes. so when we think of immunity to helminths, we think of general allergic responses which alter the environment and make it generally inhospitable for the worms to grow, reproduce, feed. if we look at immune response for most gi nematodes, we find that the worms are generally still alive when they are eliminated. they are rarely killed. so this is a nonspecific alteration of the environment which leads to actual elimination of parasite. so what do we think of in terms of general immunity to nematodes? in terms of most nematodes, infxns are initiated with an L3 which penetrates into skin or is ingested. after multiple exposures to infection, there are many examples where immune response is directed to L3, the infective larval stage. we also know in general terms that adult worms in immune animals grow more slowly, may be stunted, have reduced fecundity, females make fewer eggs than females in naive hosts. in some cases, we know that existing adult populations may be eliminated, so we have multiple mechanisms to control infections - reducing fecundity, reducing worm health, inhibiting growth of new worms via response against L3s all control the size of the adult population, and sometimes we see elimination of part of adult population, even significant parts. factors of resistance - host genetics, very important - one example from small animals - you see we can infect mice with trichinella spiralis, two strains of mice, one being a kind that supports adults in intestines for two weeks, at which time they are all spontaneously eliminated, and another strain will support them for about 6 weeks - probably due to histocompatibility gene differences. host age - young animals much better hosts. esp sheep - young sheep notoriously poor at mounting immune response to nematodes. sheep under 3 mos of age infected with trichostrongylus develop high parasite burdens. if infected over and over and over again they will show almost no immunity. vaccination fails. no response is mounted. not until about 6 mos of age will they gain the capacity to respond to infection, and exhibit some of the control mechanisms we have discussed. this is odd, b/c young animals make Ab the same way older aniamls do. they elicit T cell responses the same way. but defects in inflammatory response in young animals may be present. unfortunately, most animals on pasture are exposed to infxn w/nematodes at a young age. route of infection also influences ability to develop immunity. there is a worm used routinely in studies called nippostrongylus - has skin penetrating L3 which eventually develops in small intestine. if we infect a rat with maybe 100 or 200 L3s, and then look at the time when the worms enter GI tract eg at 5 or 6 days, probably 50 adults will be in small ientestine. then they would be spontaneously eliminated in about 3 weeks. same thing if you give 200 worms - 100 will survive, and then be eliminated. BUT if you innoculate 5 larvae per day, for a period of several weeks, we can show that these animals can build up huge worm populations that won't be eliminated. we call these trickle infections (?) and it's well known with many nematodes that if small doses of worms are taken in over time, the immune response is much less vigorous than that which develops if you ingest many worms all at once. so these then are general factors which affect host immunity. now lets talk about a couple specific worms. haemonchus contortus of sheep. we heard before about the self cure response. basically lambs going onto pasture show buildup of adults in intestine over time. it's been shown that if conditions are right, the ingestion of large numbers of L3 larvae can lead to spontaneous removal of the majority of the adult population. so it's less an immune response directed that will prevent the establishment of new L3s, because they may establish in the intestine, but rather a response which occurs which eliminates most of the adults. we think this involves some kind of allergic response, b/c all the signs of it are there - mast cells, eosinophils, high levels of IgE. we also know that if a sheep is infected not only with haemonchus but with ostertagia and maybe trichostrongylus, ingestion of these L3s may eliminate not only the hemonchus but also the adult ostertagia and adult trichostrongyle worms, all of which live in abomasum. this suggests that actual effector mechanism is some kind of nonspecific process. immunity is specific, but ingestion of large numbers of hemonchus leads to elimination of all kinds of worms. probably allergic phenomenon at site of infection making environment inhospitable to parasites. these responses can also occur down GI tract. if sheep have these parasites, and also have nematodirus in the small intestine, some of the nematodirus will also be eliminated. so the response occuring in abomasum can eliminate worms posterior to that site. it doesn't occur the other way. if sheep were infected with these worms and then ingested large numbers of nematodirus, only nematodirus would be eliminated - not the worms more anterior or cranial in the GI tract. but incoming larvae set off nonspecific process in environment of adults which works it's way down GI tract a little bit. wouldn't get oesophagostomum in large intestine. what about spontaneous self cure/elimination of adults? we talked about the nippostrongylus in the rat. in ruminants, nematodirus will do similar things. this is a dose dependent response. if a lamb ingests low numbers of larvae, say 50,000; it develops a stable adult population. if it ingests maybe 100,000 larvae, it will develop an infection but many of the adults will be lost after about a month. if it ingests even MORE larvae, very high dose - will get very high level of infection but will self cure in about 4-5 weeks. so level of ingestion/infection has consequences on development of immune response. low levels of infection lead to chronic stable adult population; ingestion of huge numbers elicits strong response which eliminates majority or all adults. many examples of immunity to L3. this can ahppen with trichostrongylus, trichinella, etc. this is a situation where infections, multiple infections, with L3, lead to buildup of adult populations in small intestine. eventually, ingestion of L3 will not lead to increase in adult numbers, because over time, animals get immune response to developing L3 worms going, and the new L3s are rapidly eliminated from the intestine and fail to develop further. they call this rapid expulsion, and it is a specific response against L3 worm. if one were to artificially transplant large number of adults into GI tract, those hosts would not have resistance to L3 challenge. but if you gave multiple L3 doses, then treated to eliminate them after a few days, you could show immune response to early developing L3s. L3s have to molt in intestine, and secrete a lot of factors. probably response of host is against the factors the L3 needs to molt. so this mechanism prevents increase of adult population. what about general effector mechanisms? we don't know much. we know allergic phenomena are required. if we infect rodents with trichuris muris, we can find that many strains of mouse develop stable adult populations, other strains spontaneously eliminate the populations. the ones that stay infected have TH1 response, the ones that eliminate have TH2 response. can these responses be manipulated? in lab, yes. in nature? probably yes! we talked about coccidians which infect intestines. in response to those organisms, hosts generate CMI response. animals need high levels of IFNg to fight infection. all the T cells are influenced by the cytokines being generated. if naive T cells are present, which can go either way - in order to get TH2 response you need IL4 and to get TH1 you need IL12 and IFNg. so, high levels of particular cytokines will drive one response and inhibit the other response. so, people are now looking at the fact that most animals in nature ar enot only infected with worms but coccidians andother beasts. since most ruminants maintain large adult populations - it may be due to competition in intestinal tract for immune response against coccidia vs worms or whatever. if infection with coccidia is present, there may be high levels of IFNg present, so animal can't mount vigorous TH2 response because the cytokines are driving towards TH1 response. the opposite may also hold true. finally, what's going to happen with vaccines and so forth? well. as you may know there are no effective vaccines for worms in the GI tract. there was a hookworm irradiated L3 vaccine but it didn't succeed - the immune response inhibited L3 in tissue but didn't affect adults in small intestine. people have been trying to vaccinate against hemonchus, trichostrongylus, and ostertagia for years. nothing works. then, several years ago some australian people tried something new. they took the intestinal tract, dissected it out, ground it up, and vaccinated sheep with that - complex antigens. this elicited some type of protective immunity. this has progressed and now they can show there are enzymes made by intestinal cells of the worms which are necessary for metabolism of adult worm. so there is an H11 vaccine antigen, which is 110 kilodalton enzyme from h.contortus. this enzyme is produced locally by epithelial cells and is an enzyme which is an aminopeptidase. this aminopeptidase has been cloned. it is used to vaccinate against h.contortus with remarkable (98-99%) effectiveness against development of adult populations. they've immunized sheep with this molecule, put the sheep on pasture, and over time, the sheep get very few worms. this is much greater than any irradiated larvae vaccine or any natural protection they might develop. it turns out this enzyme is critical for hemonchus to have to feed. in a naturally infected sheep, no antibodies to it are made - naturally, sheep are not exposed to this enzyme, it's just within the intestinal tract of the worm. but you immunize with it and make high levels of Ab to it, adult worms will ingest the Abs, which inhibit the enzyme, adults can't feed, and they are eliminated from the animal. the Ab is transmitted in colostrum, too, so mom can protect new lambs. not fully protected from colostral Ab, but pretty good. why doesn't this work with other helminths? hemonchus is a blood feeder, so high levels of serum antibodies for an enzyme in the worm will get into the adult worm when it takes in a blood meal. but this strategy won't work for ostertogia or trichostrongyles. it does work for ticks, boophilus, other blood feeding parasites. it's hoped that these vaccines will at least work for these parasites. commercial vaccine should be ready soon. things are slower for the other nonbloodfeeding helminths. people are trying to use things to make high levels of IgA against things the tissue feeders need. remember - allergic responses critical, effector responses nonspecific, almost all responses characterized by eosinophilia, IgE, mast cell accumulations in intestine, effector mechanisms are often nonspecific and act against multiple worm populations in same host/environment. also keep in mind the hemonchus vaccine using molecular technology. --end---