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Association of Wolbachia with Heartworm Disease in Cats and Dogs

Permanent Link: http://ufdc.ufl.edu/UFE0024590/00001

Material Information

Title: Association of Wolbachia with Heartworm Disease in Cats and Dogs
Physical Description: 1 online resource (51 p.)
Language: english
Creator: Dingman, Patricia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dirofilaria, heartworm, wolbachia
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous research has determined the presence of Wolbachia and an immunological response against the bacteria in dogs and cats in an experimental environment. But little is known about the role Wolbachia plays on the lung pathology of animals naturally infected with Dirofilaria immitis. The present study investigated the presence of Wolbachia in lung tissue and antibody responses against Wolbachia (anti-WSP) in heartworm (HW)-defined groups of dogs and cats naturally infected or exposed to D. immitis. The objective was to evaluate the severity of pulmonary lesions characteristic of heartworm disease in response to the bacteria. Lung lesions were scored on a grading scale from 0-3 and statistical analyses were performed to compare lesion scores to the detection of Wolbachia DNA, protein, or anti-WSP. There was a positive correlation between heartworm antibody titers and Wolbachia antibody titers in cats (p > 0.0001) and in dogs (p > 0.0001). Cats were more likely to display occlusive hypertrophy compared to dogs (p > 0.0001). Our results were unable to detect an additive effect of Wolbachia in heartworm disease and Wolbachia (DNA, protein, anti-WSP) presence and severity of pulmonary lesions were not significantly associated. The relationship between Wolbachia and lung pathology remains to be determined.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Patricia Dingman.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Levy, Julie K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024590:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024590/00001

Material Information

Title: Association of Wolbachia with Heartworm Disease in Cats and Dogs
Physical Description: 1 online resource (51 p.)
Language: english
Creator: Dingman, Patricia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: dirofilaria, heartworm, wolbachia
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Previous research has determined the presence of Wolbachia and an immunological response against the bacteria in dogs and cats in an experimental environment. But little is known about the role Wolbachia plays on the lung pathology of animals naturally infected with Dirofilaria immitis. The present study investigated the presence of Wolbachia in lung tissue and antibody responses against Wolbachia (anti-WSP) in heartworm (HW)-defined groups of dogs and cats naturally infected or exposed to D. immitis. The objective was to evaluate the severity of pulmonary lesions characteristic of heartworm disease in response to the bacteria. Lung lesions were scored on a grading scale from 0-3 and statistical analyses were performed to compare lesion scores to the detection of Wolbachia DNA, protein, or anti-WSP. There was a positive correlation between heartworm antibody titers and Wolbachia antibody titers in cats (p > 0.0001) and in dogs (p > 0.0001). Cats were more likely to display occlusive hypertrophy compared to dogs (p > 0.0001). Our results were unable to detect an additive effect of Wolbachia in heartworm disease and Wolbachia (DNA, protein, anti-WSP) presence and severity of pulmonary lesions were not significantly associated. The relationship between Wolbachia and lung pathology remains to be determined.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Patricia Dingman.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Levy, Julie K.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024590:00001


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1 ASSOCIATION OF WOLBACHIA WITH HEARTWORM DISEASE IN CATS AND DOGS By PATRICIA ANN DINGMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Patricia Ann Dingman

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3 To my cats, Sara and CC and my dog, Ellington Foxworthy

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4 ACKNOWLEDGMENTS I thank Nicholas Ronnquist for being patie nt and understanding. I thank my m entor and inspiration, Dr. Julie Levy. She has give n me so many opportunities to succeed and has greatly contributed to my personal and pr ofessional growth. I woul d like to thank Dr. Calvin Johnson, Dr. Ellis Greiner, and Dr Charles Courtney for their patience and support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT ...................................................................................................................... ..............9 CHAP TER 1 DIROFILARIA IMMITIS ........................................................................................................11 Identification...........................................................................................................................11 Life Cycle/Pathogenesis........................................................................................................ .11 Epidemiology................................................................................................................... .......13 Heartworm Disease.............................................................................................................. ...13 Heartworm-Associated Lung Pathology................................................................................. 14 Diagnosis of Heartworm Infection......................................................................................... 15 Heartworm Treatment............................................................................................................ .15 2 WOLBACHIA ..........................................................................................................................17 Biology...................................................................................................................................17 Endosymbiotic Relationship with D. immitis .........................................................................17 3 EXPERIMENTAL DESIGN.................................................................................................. 20 Materials and Methods...........................................................................................................20 Animals............................................................................................................................20 Sample Collection...........................................................................................................21 Serology...........................................................................................................................21 Parasitology.....................................................................................................................22 Pulmonary Histologic Morphometry............................................................................... 22 Wolbachia DNA Detection.............................................................................................. 23 Wolbachia Immunohistochem istry.................................................................................. 24 Statistical Analyses.......................................................................................................... 24 4 RESULTS...............................................................................................................................32 Statistical Analyses........................................................................................................... ......32 Serology...........................................................................................................................32 Parasitology.....................................................................................................................32 Detection of Wolbachia ...................................................................................................33 Pulmonary Histologic Morphometry............................................................................... 33

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6 5 DISCUSSION.........................................................................................................................44 6 CONCLUSIONS.................................................................................................................... 46 LIST OF REFERENCES...............................................................................................................47 BIOGRAPHICAL SKETCH.........................................................................................................51

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7 LIST OF TABLES Table page 3-1 Pulmonary arteriolar lesion scoring system....................................................................... 25 3-2 Bronchiolar le sion scoring system .....................................................................................26 3-3 Pulmonary interstitial and alve olar smooth m uscle scoring system.................................. 27 4-2 The prevalence of Ancylostoma spp. and ascarids in cats and dogs with pulm onary lesions.............................................................................................................. 36 4-3 Frequency of pulmonary lesions in cats and dogs with and without Wolbachia ...............37

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8 LIST OF FIGURES Figure page 3-1 Representative feline pulmonary tissu es dem onstrating lesion scores in arterioles.............................................................................................................................28 3-2 Representative feline pulmonary tissu es dem onstrating lesion scores in bronchioles.........................................................................................................................29 3-3 Representative feline pulmonary tissu es dem onstrating lesion scores in alveolar smooth muscle...................................................................................................... 30 3-4 Representative feline pulmonary tissu es dem onstrating lesion scores in interstitium................................................................................................................... ......31 4-1 Feline D. im mitis antibody titer versus Wolbachia surface protein (WSP) antibody titer in all cats..................................................................................................... .38 4-2 Canine D. immitis antibody titer versus Wolbachia surface p rotein (WSP) antibody titer in all dogs.................................................................................................... 39 4-3 Severity of pulmonary lesions in cats with or without detectable Wolbachia DNA and/or Wolbachia surface pro tein in lung tissues..................................................... 40 4-4 Severity of pulmonary lesions in dogs with or without detectable Wolbachia DNA and/or Wolbachia surface pro tein in lung tissues..................................................... 41 4-5 Severity of pulmonary lesions in cats with or without detectable circulating Wolbachia surface protein antibod ies (WSP Ab).............................................................. 42 4-6 Severity of pulmonary lesions in dogs with or w ithout detectable circulating Wolbachia surface protein antibodies (WSP Ab).............................................................. 43

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ASSOCIATION OF WOLBACHIA WITH HEARTWORM DISEASE IN CATS AND DOGS By Patricia Ann Dingman May 2009 Chair: Julie Levy Major: Veterinary Medical Sciences Although the presence of adult Dirofilaria immitis in the pulmonary arteries and its associated arteritis and thromboembolic dis ease can explain some of the manifestations of canine and feline heartworm disease, the cause of other findings remains unclear. For example, cats with D. immitis antibodies but lacking adult parasites in the pulmonary arteries frequent develop hi stological lesions of Heartworm-Associated Respiratory Disease. All D. immitis parasites harbor Wolbachia and D. immitis -infected animals can have circulating Wolbachia antibodies and pro-inflammatory Wolbachia antigens deposited in tissues. Little is known about the role Wolbachia plays in the pulmonary pathology of animals naturally infected with D. immitis. The purpose of this study was to determine the contribution of Wolbachia to the pathogenesis of natural heartworm disease in cats and dogs. We hypothesi zed that animals having sufficient Wolbachia burden to be detected in pulmonary ti ssue by immunohistochemistry and/or by PCR would have more severe pulmonary disease than those with bacteria below the limits of detection. We further hypothesized that animals that were immunoreactive to proinflammatory WSP would have more severe pulmonary lesions than those that were seronegative for WSP antibodies. Blood and pulmonary tissue samples were collected

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10 from cats and dogs representing three different D. immitis infection statuses: heartwormfree, heartworm-exposed, heartworm-infecte d. There was a positive but weak correlation between the magnitude of D. immitis antibody titers and WSP antibody titers in cats (r=0.57, p<0.001) and in dogs (r=0.39, p<0.001). Pulmonary lesions were more common in HW-infected animals than in HW-free animals. Pulmonary arteriolar occlusion was more common in HW-infected cats (57%) (p= 0.003) than in HW-infected dogs (17%). Although pulmonary lesions were most common in HW-infected animals, there was no clear additive effect when either Wolbachia DNA/WSP was detected in pulmonary tissue or when circulating WSP antibodies were dete cted. Similarly, there were no significant differences in the magnitude of pulmonary lesi on scores within each HW-infection status group regardless of whether Wolbachia DNA/WSP or WSP antibod ies were detected. The relationship between Wolbachia and pulmonary pathology in heartworm-infected animals remains to be determined. The l ack of clear evidence for a role of Wolbachia in heartworm disease creates a dilemma for veterinarians treating cats and dogs in D. immitis -endemic areas. Although the indiscriminant use of antibiotics should be avoided, many clinicians prescribe doxycycline based on the favorable responses observed in human filarial diseases and on promising re sults from the first published studies of doxycycline use in D. immitis -infected dogs.

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11 CHAPTER 1 DIROFILARIA IMMITIS Identification Dirofila ria immitis (heartworms) are filarial helmin ths that belong to the phylum, Nematoda (roundworms) and superfamily Filaroidea. Other filarial nematodes, including Wuchereria bancrofti and Brugia malayi are known to cause human lymphatic filariasis (elephantiasis), and Onchocerca volvulus is a causative agent for huma n river blindness disease. Microfilariae (mff) are mobile vermiform embryos and are the microscopic immature stage of D. immitis circulating in the blood of the vertebrate host. They are not sheathed and have a straight tapered tail. Their si ze ranges from 286-340 m in length and 6-7 m in diameter. The physical characteristics of D. immitis microfilariae distinguish them from other filarial species such as Dipetalonema reconditum (Yabsley et al., 2004). Dirofilaria immitis show sexual dimorphism; adult males range between 12 and 20 cm and are much shorter in length than the female worm s, which can grow as long as 30 cm in length. Both sexes are thin, measuring less than 5 mm wide and white in color. They are cylindrical in shape and a psuedocoelon is present. Males have coiled (corkscrew) tails, whereas the females tails are straight. Life Cycle/Pathogenesis There are three factors involving D. immitis transm ission. First there needs to be an infected vertebrate with circ ulating microfilariae, most co mmonly a domestic dog, which is a definitive host for D. immitis. Heartworm-infected canids have circulating microfilariae and are considered the natural reservoir for these parasite s. Although cats are naturally more resistant to D. immitis feline infection is likely to occur anyw here the parasite is found in canids. Microfilaremia is uncommon in cats and ther efore for a cat to b ecome infected with D. immitis

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12 there needs to be an infected canid nearby (Gen chi et al., 2008). Environmental conditions must be appropriate for D. immitis larval development because en vironmental temperature affects growth within the mosquito and microfilariae. Fi nally, vector competent mosquitoes such as the Culex spp. must feed on both infected canids and susceptible hosts to effect transmission (Nelson et al., 2005a; Ralston et al., 1998). Almost 70 species belonging to the family Culicidae are considered potential vectors ( Aedes spp ., Anopheles spp., Culex spp., and Mansonia spp .). To begin the cycle, a female mosquito feeds on a microfilaremic dog and the microfilariae are ingested during the blood meal. The microfilariae pass through the he mocoel and into the malpighian tubules of the mosquito. Ther e they mature into first stage larvae (L1s) and molt to second stage larvae (L2s) within 10 days. Ten to fourteen da ys later, they become third stage larvae (L3s), which is the infective stage. The L3s mi grate to the salivary glands and proboscis and when the mosquito feeds, the L3s are deposit ed onto the skin and then enter the wound made by the mosquito. The larvae begin to develop an d molt into the 4th stage larva in 3-4 days. Fourth-stage larvae migrate to the muscle fascia of the thorax and abdomen, molt, and then L5s enter the circulatory system over a period of 3 to 4 months. They are carried to the pulmonary arteries, heart, and lungs via bl ood circulation 4 to 7 months post infection. Once in the heart and lungs they mature into adult worms approximate ly 6 months after infection if both male and female worms are present. Adult female worms begin to produce microfilariae 6 to 9 months (180 to 190 days) after the in itial infection, which is th e infectious period when D. immitis can be acquired by mosquitoes and then transmitted to other cats or dogs. In cats, arrested development of the worm is common because cats are not th e natural host. Fewer worms reach maturity in cats compared to dogs. Only about 20% of D. immitis larvae become adult worms in cats, whereas up to 75% of D. immitis larvae in dogs become adults. Microfilariae were shown to

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13 remain in blood circulation for up to two years in a dog that was experimentally inoculated with microfilaremic blood (Underwood and Harwood, 1939). Adult worms in dogs can live up to 5 to 7 years in dogs (Newton, 1968) and 2 to 3 years in cats. Aberrant migration of the parasite has been documented, resulting in disease associated with dysfunction of the target tissue (Nogami and Sato, 1997). Epidemiology In areas end emic for D. immitis, prevalence in dogs that ar e not on heartworm preventive can exceed 50%. Feline heartworm prevalence is often 5-20% of the positive dog population (Atkins et al., 1998; Bowman et al., 2007; Carl eton and Tolbert, 2004; Hermesmeyer et al., 2000; Miller, 1998; Nogami and Sato, 1997; Patton and McCracken, 1991; Ryan and Newcomb, 1995). Recently a nationwide survey estimated the felin e heartworm prevalence rate, determined by antigen testing, to be 0.6 times the canine pr evalence (Lorentzen and Caola, 2008). Northern Florida is a high endemic area and many dogs are not on preventive. The prevalence in unprotected dogs is 25-50%. In Northern Florida, feline heartworms are found in 5% of the cat population in an animal shelter (Levy et al., 2003; Snyder et al ., 2000). Studies have demonstrated conflicting results when examining the cats gender as a predisposing risk factor for D. immitis Some researchers have found male cats to be at a higher risk for heartworm infection (Kramer and Genchi, 2002; Levy et al., 2003) whereas others reported gender not being a determining factor for infection (Atkins et al., 2000; Genchi et al ., 2008; Liu et al., 2005). Heartworm Disease Heartworm infection can have di fferent effects on cats and dogs Dogs have higher parasite intensities, often reaching greater than 30 adult wo rms compared to cats that have only 1-3 adult worms (Nogami and Sato, 1997; Ryan and Newcomb, 1995). Dogs are often microfilaremic versus cats are not usually microfilaremic and it is much easier to diagnose infection in dogs than

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14 in cats. Clinical signs in dogs include coughing, exercise intolerance, dyspnea, hepatomegaly, syncope, and ascites. Clinical signs in cats include lethargy, coughing, anorexia, chylothorax, and vomiting. Commonly cats have dyspnea or respir atory distress similar to feline bronchial disease (asthma) and dogs experience right-heart failure (Nels on et al., 2005a, b). Often times, there are no signs. When signs do oc cur, they are often the result of the arrival of L5s in the pulmonary arteries which leads to acute pneumonitis. Signs may also occur when the deaths of adult worms cause thromboembolism and anaphyl axis and may result in sudden death (Dillon, 1998). Heartworm-Associated Pulmonary Pathology Although the presence of adult D. immitis in the pulm onary arteries and its associated arteritis and thromboembolic disease can explain some of the manifestations of canine and feline heartworm disease (HWD), the cause of other findings remains unclear. This is particularly true for cats, which frequently develop generali zed severe bronchoint erstitial disease and extrapulmonary signs in response to very low worm intensitites (Atkins et al., 2000). In cats, even a single worm can produce fatal disease. Browne et al., (2005) examined 630 cats from an animal control shel ter and assigned them to three groups (HW-infected, HW-exposed, and HW-free) based on serological tests and necropsy findings. Pulmonary lesions characteri zed by pulmonary arterial occlusive hypertrophy were common in cats with adult worms and in cats that were free of adult worms but with D. immitis antibodies, suggesting that even transien t infection leave cat s with long-lasting pulmonary pathology. Occlusive hypertrophy is a char acteristic of feline heartworm disease and is defined as >95% occlusion of the arteriolar lumen by an increas e in the thickness of the tunica media. Almost 80% of the HW-infected cats had hypertrophy, followed with the HW-exposed

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15 (50%) and HW-free (13%) and there was a sign ificant association be tween the number of occluded vessels and HW status. Diagnosis of Heartworm Infection There are many blood tests for diagnosis of heartwor m infection. A blood concentration test such as the Knotts method or membrane filtr ation can detect circulating microfilariae in the blood stream. Dirofilaria immitis antigen tests detect proteins shed from female adult worms. False-negatives occur if the dog or cat has an a ll male, a low worm burden, or juvenile worms. Dirofilaria immitis antibody tests indicate exposure to he artworms, but not necessarily current infection. Enzyme-linked immunosorbent assays ( ELISA) or non-ELISA latera l flow tests can be used to detect D. immitis antigen or antibodies. Additi onal support for a diagnosis of D. immitis infection includes thoracic radiography and echocardiography, as we ll as combining or repeating antigen and antibody tests (Ber doulay et al., 2004; Nelson, 2 008; Snyder et al., 2000). A necropsy may provide a definitive answer; however D. immitis have been known to migrate to aberrant locations (Nogami a nd Sato, 1997; Oh et al., 2008) and necropsy may not detect immature infections. Heartworm Treatment Although canine heartw orm infection is ve ry common, it is also 100% preventable. Preventives must be administered monthly star ting approximately one month after the beginning of the transmission season and ending approximately one month after the end of the season according to the American Heartworm Society ( AHS) (Nelson et al., 2005a, b). Prevention is most important during the transmission period but to insure client compliance, and to account for transmission variability in microclimate s, treatment year-round is recommended. Currently there are no FDA-approved drugs avai lable for adulticide treatment in cats and because infected cats usually l ack circulating microfilaria, th ey do not need microfilaricide

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16 treatment. Adulticide therapy in cats has been associated with a mortality rate of 20-30%. AHS recommends prednisone and supportive therapy to am eliorate clinical sign s of disease in cats. Surgical removal of the worms can be attempted in cats with uncontrolled signs. Aspirin is no longer recommended as a treatment for heartwor m disease in cats. FDA-approved heartworm preventives for cats include ivermectin, milbem ycin oxime, and selamectin. The preventives should be administered 30 days after transmissi on season begins and 30 days after it ends on a monthly basis (Nelson et al., 2005a). There are more treatment options for dogs than for cats. Dogs should begin preventives at 8 weeks of age. Adult dogs starting preventi ves for the first time should be tested for D. immitis Ivermectin, milbemycin oxime, moxidectin, a nd selamectin are macrocyclic lactones most effective against mff, L3s, and L4s. Daily diethylcarbamazine (DEC) can also be used as a preventive but is rarely used due to the avai lability of monthly preventives. Testing for microfilariae is required before treating with DEC to avoid adve rse reaction in infected dogs. Retesting is recommended for all the preventives 7 months after the end of the transmission season to assure prevention of infection. The only F DA-licensed adulticide treatment for dogs is melarsomine dihydrochloride. Using low-dose ivermec tin as an adulticide can take several years to cure infection and is not generally reco mmended. Microfilaricide treatment should be performed after adulticide thera py. Re-testing for microfilariae and D. immitis antigens are recommended after adulticide therapy (Nelson et al., 2005b).

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17 CHAPTER 2 WOLBACHIA Biology The genus Wolbachia belongs to the order, Rickettsiales. Wolbachia is phylogenetically rela ted to Ehrlichia spp. and Anaplasma spp. The only species identified in the genus is Wolbachia pipientis. These gram-negative bacteria are found in 100% of the D. immitis population; all individuals are inf ected. The bacteria are present in all developmental stages and are located in the hypodermal ce lls of the lateral chords (K ozek, 2005; Kramer et al., 2003). Wolbachia are transmitted maternally (female to o ffspring). After the first week when L3s entered the host, bacteria growth increases unti l L4 development. The amount of bacterial cells remains constant in male worms, but as the fe males begin to mature, the bacteria increase in number and infect the ovaries, oocytes, and embryonic stages insi de the uteri. They have not been reported from the male reproductive system. Researchers have discovered Wolbachia surface proteins (WSP) circulating in heartworminfected dogs, localized in th e lungs and kidneys where micr ofilariae are common (Kozek, 2005; Kramer et al., 2003). Macrophages containing Wolbachia have been identifie d in the lung, liver, and kidneys of dogs with natural D. immitis infection (Kramer et al., 2005b). Dirofilaria immitis infected animals also have circulating WSP antibodies (Ba zzocchi et al., 2000a; Kozek, 2005; Kramer et al., 2008; Kramer et al., 2005b; Morchon et al., 2004). Endosymbiotic Relationship with D. immitis In recent years, it has been recognized th at a large array of helm inths and arthropods are colonized by Wolbachia (Bandi et al., 1998; McCall, 2005) Human and animal filarial nematodes, including the agents of human river blindness ( Onchocerca volvulus ), lymphatic filariasis ( Brugia malayi ), and feline and canine heartworm disease ( Dirofilaria immitis), harbor

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18 Wolbachia in a mutually dependent manner (Bazzocch i et al., 2000b Kozek, 2005; Kramer et al., 2003; McCall, 2005; Taylor, 2003). Wolbachia aid in the parasites survival and reproduction and can also influence the sex of the filarial parasites (Bandi et al., 1998; Bandi et al., 1999).Filariae rendered free of Wolbachia by treatment with tetracycline antibiotics show inhibition of maturation, survival, and reproduction (Bandi et al., 1999; Casiraghi et al., 2002; Genchi et al., 1998; Makepeace et al ., 2006; Pfarr and Hoerauf, 2006). Wolbachia are released in large numbers duri ng parasite molts, during production of microfilariae, and at death of the parasite (Ba ndi et al., 1999; Taylor, 200 3; Taylor et al., 2005). Wolbachia -associated-molecules (WAMs) have recently been shown to be associated with inflammation in the parasitized mammalian hosts (Bazzocchi et al., 2000a; Bazzocchi et al., 2003; Kozek, 2005; Kramer et al., 2005a; Kramer et al., 2005b; McCall 2005; Morchon et al., 2004; Taylor, 2003). These inflammatory respon ses appear to be most profound when the parasite dies naturally or as a result of anti-f ilarial drug treatment. In contrast, the death of parasites rendered free of Wolbachia stimulates less intense inflammation in the host. This observation has led to treatment st rategies for river blindness and ly mphatic filariasis that include pretreatment with tetracycline antibiotics to reduce the burden of Wolbachia followed by ivermectin to eliminate the parasites (Makepeace et al., 2006; Taylor, 2003). Wolbachia surface protein (WSP) stimulates canine neutrophil chemotaxis and IL-8 production (Bazzocchi et al., 2003). Stimulation of canine vascular endot helial cells with WSP resulted in production of cyclooxyge nase-2, 5-lipooxygenase, leukotriene B4, intracellular adhesion molecules, E-cadherin, a nd vascular endothelial growth factor (Simon et al., 2008). In cats, experimental D. immitis infection resulted in production of antibodies against both D. immitis and Wolbachia within two months of exposure to in fective larvae (Morchon et al., 2004).

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19 Treatment with ivermectin 30 days later to abort D. immitis infection was associated with disappearance of D. immitis antibodies, but persistence of Wolbachia antibodies. Dogs experimentally infected with D. immitis and treated with doxycyc line in combination with ivermectin and melarsomine had less pulmonary pathology than dogs treated with melarsomine alone (Kramer et al., 2008). Taken together, these studies suggest a role for Wolbachia in the pathogenesis of heartworm disease. If recently reported experiences with antibiotic treatment of filarial diseases prove to be predictive of responses in naturally infected an imals, then treatment with doxycycline prior to adulticide therapy in dogs may sterilize D. immitis of Wolbachia leading to fewer inflammatory side-effects when the adult worms die. Doxycycline therapy may be especially helpful for cats, which suffer substantial and life-threatening e ffects of heartworm disease, but for which adulticide therapy is associat ed with high mortality rates a nd is generally contraindicated. Elimination of inflammatory Wolbachia organisms may allow cats to coexist more comfortably with their parasites, ev en if an actual cure is not feasible. However, it is not currently known what role Wolbachia plays in the pathogenesis of natural heartworm disease. The purpose of this study was to determine the contribution of Wolbachia to the pathogenesis of natural heartworm di sease in cats and dogs. Since all D. immitis parasites harbor Wolbachia it is difficult to di fferentiate the effects of the parasite from those of its endosymbiont bacteria. We hypothesized that animals having sufficient Wolbachia burden to be detected in pulmonary tissue by immunohistochemistry and/or by PCR would have more severe pulmonary disease than those with bacter ia below the limits of detecti on. We further hypothesized that animals that were immunoreactive to pro-in flammatory WSP would have more severe pulmonary lesions than those that were seronegative for WSP antibodies.

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20 CHAPTER 3 EXPERIMENTAL DESIGN Materials and Methods Animals Over a two year period (2005-2007), dog (n=90) and cat (n=90) cadavers were collected from Alachua County Animal Services (ACAS) in Gainesville, Florida following euthanasia for reasons unrelated to the study. Ca davers were transported to th e University of Florida for necropsy. For this study, samples were collected from cats and dogs representing three different D. immitis infection statuses. The heartworm-free (HWfree) groups were composed of 30 cats and 30 dogs that were free of any evidence of current or previous D. immitis infection (seronegative for D. immitis antigen and antibodies and negative fo r parasites at necropsy). The heartwormexposed (HW-exposed) groups were composed of 30 cats and 30 dogs with evidence of either larval-stage infection or pa st infection (seropositive for D. immitis antibodies), but no evidence of current adult parasite infection (seronegative for D. immitis antigen and negative for parasites at necropsy). The heartworm-infected (HW-inf ected) groups were composed of 30 cats and 30 dogs with adult parasites in the heart or pulmonary arteries at necropsy (regardless of serological status). The severity of pulmonary lesions in each group was correlated with the detection of Wolbachia proteins or nucleic acid seque nces and with the presence of Wolbachia antibodies. This study was approved by the University of Florida Institutional An imal Care and Use Committee (IACUC approval number E12).

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21 Sample Collection Blood (4 mL) was collected by transthoracic cardiocentesis into EDTA a nd serum separator tubes from each animal within 2 hour s after death. Aliquots of EDTA whole blood and serum were stored at -20C pending analysis. The heart and lungs were removed a nd dissected for the detection of D. immitis. Sections of the right caudal lung lobe were preserved in 10% formalin for histomorphometry and immunohistochemistry. Additional sections of fres h lung tissue were stored at -70C for PCR analysis. Any recovered D. immitis were preserved in 70% etha nol at room temperature for immunohistochemistry and PCR. Feces were collected from the large intestin e of each animal and stored at 4C pending flotation analysis. Serology Cat and dog EDTA whole blood sam p les were tested by ELISA for D. immitis antigen (Feline SNAP Heartworm Antigen Test Kit, Canine SNAP Heartworm Antigen Test Kit; IDEXX Laboratories Inc., Westbrook, ME, USA; IDEXX). Feline serum samples were also screened for D. immitis antibodies with a lateral flow immunoassay (HESKA Solo StepTM FH, Heska Corporation, CO, USA). These serological re sults were used to aid in the selection of cadavers for dissection. Dirofilaria immitis antibody titers and Wolbachia surface protein (WSP) antibody titers were determined for both cats and dogs as desc ribed by Morchon et al., (2004). Cat serum samples were also tested for FIV antibodies and FeLV antigen by ELISA (PetChek ELISA, IDEXX Laboratories Inc.)

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22 Parasitology Dirofila ria immitis collected at necropsy were counted and examined with a dissecting microscope to determine sex and age (adult, j uvenile, or unknown) of recovered worms. Male adult worms were identified by their cork-like tails. Several common parasites of cats and dogs migr ate through the lungs as part of their life cycle and can induce damage that is indistingui shable from that induced by HW infection. To control for this, feces were examined for ova and larvae. Feces (approximately 1 g) were mixed with sodium nitrate solution (1.200 specific grav ity; Fecasol Solution, Evsco Pharmaceuticals, Buena, NJ, USA) in a standardized flota tion device (Fecalyzer, Evsco Pharmaceuticals), incubated with a cover slip for 15 minutes, and then evaluated qualitatively by light microscopy at 100X for the presence of any ova or larvae. Pulmonary Histologic Morphometry Form alin-fixed and paraffin-embedded 5 m sections of tissue collected from the right caudal lung lobe were stained with hematoxylin and eosin. Digital images were acquired at 100400X magnification (Q-Capture software, Q-Im aging Corp, Burnaby, BC, Canada). For each animal, 10 arterioles, 10 bronchioles five areas of interstitium, a nd five areas of alveoli were assessed. Arteriolar and bronchi olar wall areas were quantifie d in two-dimensional crosssections of the structures as a measure of wall thickness using computer software (Image J, National Institutes of Health, Bethesda, Maryla nd, USA). Wall area was calculated by digitally tracing the entire external and lu minal surfaces of the structures and then subtracting the luminal area from the total area as described in Browne et al. (2005). Pulmonary arteriolar occlusive hypertrophy was defined as an arteriolar wall th at exceeded 95% of the total area, resulting in a reduced luminal area < 5% of the vessel area.

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23 In addition to morphometric evaluation of arte riolar and bronchiolar thickness, pulmonary tissues were scored for severity of histologic lesions on a 0 (normal) to 3 (severely abnormal) scale. Lesions of the arterioles and bronchioles were scored according to the systems described in Tables 3-1 and 3-2 and are shown in Figures 3-1 and 3-2. Lesions of the interstitium were scored based on the frequency of inflammatory ce lls and the presence of edema within alveolar septa, whereas lesions of the alveoli were scor ed according to the relative quantity of smooth muscle nodules within the tips of alveolar septa as described in Table 3-3 and shown in Figures 3-3 and 3-4. When various components of a structur e had different severity grades, the score was based on the predominant lesion. Mean pulmonary lesion scores for each animal were calculated. For determination of lesion prevalences, mean scores 1 were considered to be free of lesions and mean scores > 1 were considered to have lesions. Wolbachia DNA Detection DNA was extracted from lung tissue (40 mg) a nd parasite tissue (up to 52 g) using a commercial kit (DNeasy Tissue Kit, Qiagen In c., Qiagen Inc.). DNA quality was confirmed by spectrophotometry and amplif ication of the ubiquitous housekeeping gene GAPDH. A conventional PCR assay amplifying a region of the 16S-23S intergenic spacer gene (5CTG GGG ACT ACG GTC GCA AGA C 3 forward; 5CTC CAG TTT ATC ACT GGA AGT T 3 reverse) common to Ehrlichia spp., Anaplasma spp., Neorickettsia risticii and Wolbachia was performed as described by Lappin et al. (2004). For amplicons of the appropriate size, genetic sequencing was performed (Macromolecular Resour ces, Colorado State University, Fort Collins, CO), and the resultant sequences analyzed by comparison to sequences in GenBank. BLAST software (National Institutes of Health, NCBI webs ite) was used to confirm the species source of amplification products. Samples were considered positive for Wolbachia only if PCR assay results were confirmed by genetic sequencing.

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24 Wolbachia Immunohistochemistry Lung tissues and D. immitis p arasites were assessed for Wolbachia antigen (WSP) by immunohistochemistry as descri bed by Kramer et al. (2003). Fo rmalin-fixed paraffin-embedded 5 m lung sections and heartworm sections were affixed to polylysinated slides. The sections were incubated in 3% H2O2 for 10 minutes at room temperatur e. The slides were then treated with bovine serum albumin and fetal calf seru m with phosphate buffere d saline (PBS) for 15 minutes. Wolbachia antigens were detected by incuba tion with rabbit anti-WSP polyclonal antibody diluted at 1:200 in PBS for 20 minut es followed by anti-rabbit IgG-streptavidin complex (LSAB2 kit, DAKO, Italy). Color was developed with biotin-horseradish peroxidase, and sections were counterstained with hemat oxylin (LSAB2 kit, DAKO, Italy). Slides were reviewed microscopically at 100X and were scored as positive or negative for specific staining. Statistical Analyses Ani mals were grouped by heartworm-infecti on status (HW-free, HW-exposed, HW-infected) and by Wolbachia status (DNA-, protein-, or antibody-positive or ne gative) for statistical analyses. Correlation between the magnitude of D. immitis antibody titers and WSP antibody titers was measured with the Pearsons r test (S igmaStat 3.5 statistical analysis software, SPSS Inc., Chicago, IL, USA). Chi-square analysis was used to compare prevalences of parasitism and pulmonary lesions when n 5 and the Fisher exact test was used to compare prevalences when any group n < 5 (Epi InfoTM 3.5.1, Centers for Disease Control, Atlanta, Georgia). The mean values of the pulmonary lesion scores were compared using the Wilcoxon rank rum test. Differences were considered significant when p<0.05.

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25 Table 3-1. Pulmonary arteriolar lesion scoring system. Lumen Intima Muscularis Adventitia Contents Endothelial and Subendothelial Changes Thickness of Smooth Muscle Thickness and Contents Grade 0 Blood Flattened endothelium and thin intima 2-4 cells; no occlusion of lumen Thin and indistinct Grade 1 Mild margination of eosinophils Cuboidal endothelium and edema of subendothelial zone 5-7 cells; muscle thickness and lumen diameter similar Mild thickening; edema Grade 2 Margination of eosinophils with mild fibrin deposition Folded endothelial surface with edema and eosinophils in subendothelial zone Partial occlusion; wall thickness 50-95% of lumen diameter Marked thickening; edema; mild increase in inflammation Grade 3 Fibrinous thrombosis with many eosinophils Severe edema and eosinophil infiltration of subendothelial zone; dissolution of internal elastic lamina Occlusive hypertrophy; wall thickness >95% of lumen diameter Marked thickening; edema; marked increase in inflammation; fibrosis

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26 Table 3-2. Bronchiolar lesion scoring system. Lumen Mucosa Submucosa Muscularis Contents Epithelial Changes Edema Inflammation Serous Glands Fibrosis Thickness of Smooth Muscle Grade 0 None Cuboidal Minimal Minimal Rare None 1-2 cells Grade 1 Mild seromucoid secretion Low columnar with mild crowding and folding Mild Mild Mild increase Mild 3-4 cells Grade 2 Moderate seromucoid secretion with inflammation Tall columnar with moderate crowding and folding Moderate Moderate Moderate increase (nesting) Moderate 5-6 cells Grade 3 Severe seromucoid secretion with inflammation Tall columnar with severe crowding and folding Severe Severe Severe increase (circumferential) Severe >6 cells

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27 Table 3-3. Pulmonary inters titial and alveolar smooth muscle lesion scoring system. Interstitium Alveolar Smooth Muscle Grade 0 Normal Normal Grade 1 Increased inflammatory cells in capillaries/mild thickening within alveolar septa Nodules Present Grade 2 Moderate thickening Increase in nodules and size; mild bands Grade 3 Severe thickening, edema, increased eosinophils within alveolar septa Large nodules; bands and branching

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28 Figure 3-1. Representative feline pulmonary tissues demonstrating lesion scores in arterioles. Scores were assigned based on descriptions in Table 3-1. Grade 0 represented normal tissue (arrow pointing to red blood cells in open lumen area) and Grade 3 was considered the most severe lesion. There is a slight increase in smooth muscle in the Grade 1 arteriole (arrow). Edema (arrow) and the hypertrophied smooth muscle are present in the Grade 2 arteriole. The Gr ade 3 arteriole is displaying occlusive hypertrophy with no visible lumen (wall area >95% of total area) (arrow) (H&E stain, bar=100 m). Grade 0 Grade 1 Grade 2 Grade 3

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29 Figure 3-2. Representative feline pulmonary tissues demonstrating lesion scores in bronchioles. Scores were assigned based on descriptions in Table 3-2. Grade 0 represented normal tissue and Grade 3 was considered the most severe lesion. The muscularis in a normal bronchiole has one to two smooth muscle layers (arrow pointing to smooth muscle cell). The epithelium is cuboidal and not crowded. In a Grade 1 bronchiole, the epithelium begins to crowd (arrow), whereas Grade 2 and 3 have compressed nuclei. Serous glands become dominant in Grade 2 and are located in the submucosa and in a Grade 3 the glands become circumfere ntial (arrow) (H&E stain, bar=100 m). Grade 0 Grade 1 Grade 2 Grade 3

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30 Figure 3-3. Representative feline pulmonary tissues demonstrating lesion scores in alveolar smooth muscle. Scores were assigned base d on descriptions in Table 3-3. Grade 0 represented normal tissue and Grade 3 was c onsidered the most severe lesion. Smooth muscle nodule (arrow A) size varies with grades. Banding develops in Grade 2, and nodules and bands become undistinguishable in Grade 3 (arrow B) (H&E stain, bar=100 m). Grade 0 Grade 1 Grade 2 Grade 3 A B

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31 Figure 3-4. Representative feline pulmonary tissues demonstrating lesion scores in interstitium. Scores were assigned based on descriptions in Table 3-3. Grade 0 represented normal tissue and Grade 3 was considered the most severe lesion. The arrow is pointing to the thickening within alveolar septa and in flammatory cells within the capillaries. (H&E stain, bar=100 m). Grade 0 Grade 1 Grade 2 Grade 3

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32 CHAPTER 4 RESULTS Statistical Analyses Serology This study was not designed to report the se nsitivity or specific ity of the antibody or antigen tests because serology resu lts were used to select specimens for necropsy. There was a positive but weak correlation between the magnitude of D. immitis antibody titers and WSP titers in cats (r=0.57, p<0.001) an d in dogs (r=0.39, p<0.001) (Figures 4-1, 4-2). Antibodies against WSP were detected in a ma jority of HW-infected cats and dogs and in variable proportions of animals in other HW-s tatus groups (Table 4-1). Of the 90 cats in the study, 10 had FIV antibodies (five from the HW-infected group and five from the HW-exposed group) and one was infected with FeLV (from the HW-exposed group). Parasitology Low D. immitis intensities were com mon in cats in the HW-infected group (mean=1.7.2 worms, range=1-6). Of the 51 D. immitis collected from the 30 HW-infected cats, most were female (35/51, 69%). Only five cats had both male and female worms. Higher worm intensities were common in dogs in the HW-infected group (mean=36.3.9 worms, range=3-133). Of the 1,090 D. immitis collected from the 30 HW-infected dogs, most were female (606/1090, 56%). All dogs had both female and male worms. Ancylostoma spp. were common in both cats (5 2%) and dogs (61%). Other gastrointestinal parasites identified included wh ipworms (0% of cats, 27% of dogs) and ascarids (13% of cats, 9% of dogs). There were no signifi cant associations between the overall presence of pulmonary lesions in cats or dogs and the presence of Ancylostoma spp. or ascarids, two parasites with a pulmonary migratory phase in their life-cycle (p>0.09) (Table 4-2). When

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33 animals were grouped according to subtype of le sion (arteriolar, bronch iolar, alveolar smooth muscle, or interstitial), cats with alveolar lesi ons were less likely to have ascarids than those without lesions (p=0.02) and dogs with inters titial lesions were more likely to have Ancylostoma spp. than dogs without lesions ( p=0.02). Detection of Wolbachia Wolbachia DNA was amplified from adult D. immitis collected from 26 of 30 HWinfected cats, and from D. immitis collected from all of the HW -infected dogs. Three out of the four Wolbachianegative D. immitis samples from the HW-infected cats were GAPDH negative, indicating inadequate DNA quality. WSP was detected by immunohistochemistry in D. immitis specimens from all cats and dogs. In general, Wolbachia DNA or WSP was identified most commonly in the lungs of HW-infected animals, but was also present in va riable proportions of lung samples in the other HW-infec tion status groups (Table 4-1). Pulmonary Histologic Morphometry As expected, pulm onary lesions were more co mmon in HW-infected animals than in HW-free animals (Table 4-1). Pulmonary arteriolar occlusion was more common in HW-infected cats (57%) (p=0.003) than in HW-inf ected dogs (17%). In addition, HW -infected cats with occlusion had more affected arterioles (43%) than did HW-infected dogs with occlusion (12 2%) (p=0.007). In cats, occlusive hypertrophy was id entified most commonly in the HW-infected group, but was also present in the HW-free a nd HW-exposed groups. In contrast, arteriolar occlusion in dogs was present on ly in the HW-infected group. Although there were differences in the proportions of cats affected by arteriolar occlusion, there were no significant differences in the severity of occlusion among the groups. The percentage of occlude d vessels in the HWexposed group (44%) was similar to that of the HW-infected group, and the HW-free group (28%) (p=0.31). Although pulmonary lesions we re most common in HW-infected animals,

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34 there was no clear additive effect on the prevalence of lesions when either Wolbachia DNA/WSP was detected in pulmonary tissue or when circulating Wolbachia antibodies were detected (Table 4-3). Similarly, there were no si gnificant differences in the magnitude of pulmonary lesion scores within each HW-infection status group regardless of whether Wolbachia DNA/WSP or antibodies were detected (Figures 4-3 to 4-6).

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35 Table 4-1. Prevalence of Wolbachia surface protein (WSP) antibodies, Wolbachia DNA, WSP, and pulmonary lesions in cats and dogs with no evidence of D. immitis infection (HW-free), evidence of either larval-stage infection or past infection (HW-exposed), or with adult D. immitis in the heart or pulmonary arteries (HW-infected). Values with different superscript letters within a row are significantly different (p<0.05). Cats HW-free HW-exposed HW-infected WSP antibodies 3 (10%)a 10 (33%)b 18 (60%)c Wolbachia DNA in lung 7 (23%) 12 (40%) 12 (40%) WSP in lung 4 (13%) 5 (16%) 10 (33%) Arteriolar lesions 10 (33%)a 16 (53%) 19 (63%)b Arteriolar occlusive hypertrophy 8 (27%)a 9 (30%) a 17 (57%)b Bronchiolar lesions 8 (27%) 7 (23%) 10 (33%) Interstitial lesions 14 (47%)a 27 (90%)b 27 (90%)b Alveolar smooth muscle lesions 6 (20%)a 8 (27%)a 18 (60%)b Dogs WSP antibodies 14 (46%)a 23 (76%)b 20 (66%) Wolbachia DNA in lung 0 (0%)a 1 (3.4%)a 14(46%)b WSP in lung 7 (23%) 4 (13%)a 12 (40%)b Arteriolar lesions 3 (10%)a 12 (40%)b 18 (60%)b Arteriolar occlusive hypertrophy 0 (0%)a 0 (0%)a 5 (17%)b Bronchiolar lesions 7 (23%)a 24 (80%)b 30 (100%)c Interstitial lesions 12 (40%)a 20 (67%)b 30 (100%)c Alveolar smooth muscle lesions 5 (17%)a 13 (43%)b 16 (53%)b

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36 Table 4-2. The prevalence of Ancylostoma spp. and ascarids in cats and dogs with pulmonary lesions. Values with differ superscript letters within a row are significantly different (p<0.05). Cats (n=90) Lesions No Lesions Ancylostoma spp. 40/81 (49%) 7/9 (78%) Ascarids 9/81 (11%) 3/9 (33%) Dogs (n=90) Ancylostoma spp. 48/75 (64%) 7/15 (47%) Ascarids 7/76 (9.2%) 1/13 (7.7%)

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37 Table 4-3. Frequency of pulm onary lesions in cats and dogs with and without Wolbachia. Wolbachia-negative animals were free of detectable Wolbachia DNA, WSP, and WSP antibodies. Wolbachia-positive animals we re positive for at least one of these tests for Wolbachia. There were no significan t differences in the frequency of lesions in the Wolbachia-negative groups vs. the Wolbachia-positive groups (p>0.05). Cats Wolbachianegative (n=37) Wolbachiapositive (n=53) Arteriolar lesions (n=45, 50%) HW-free (n=10) 7 (37%) 3 (27%) HW-exposed (n=16) 4 (33%) 12 (66%) HW-infected (n=19) 3 (50%) 16 (66%) Arteriolar occlusive hypertrophy (n=34, 38%) HW-free (n=8) 5 (26%) 3 (27%) HW-exposed (n=9) 4 (33%) 5 (27%) HW-infected (n=17) 3 (50%) 14 (58%) Bronchiolar lesions (n=25, 28%) HW-free (n=8) 6 (32%) 2 (18%) HW-exposed (n=7) 3 (25%) 4 (22%) HW-infected (n=10) 2 (33%) 8 (33%) Alveolar smooth muscle lesions (n=32, 36%) HW-free (n=6) 4 (21%) 2 (18%) HW-exposed (n=8) 5 (42%) 3 (16%) HW-infected (n=18) 4 (67%) 14 (58%) Interstitial lesions (n=68, 76%) HW-free (n=14) 8 (42%) 6 (54%) HW-exposed (n=27) 11 (92%) 16 (88%) HW-infected (n=27) 6 (100%) 21 (87%) Dogs Wolbachianegative (n=18) Wolbachiapositive (n=72) Arteriolar lesions (n=33, 37%) HW-free (n=3) 1 (8%) 2 (11%) HW-exposed (n=12) 1 (100%) 11 (44%) HW-infected (n=18) 0 (0%) 18 (62%) Arteriolar occlusive hypertrophy (n=5, 5.6%) HW-free (n=0) 0 (0%) 0 (0%) HW-exposed (n=0) 0 (0%) 0 (0%) HW-infected (n=5) 1 (100%) 4 (13%) Bronchiolar lesions (n=61, 68%) HW-free (n=7) 3 (25%) 4 (22%) HW-exposed (n=24) 5 (100%) 19 (76%) HW-infected (n=30) 1 (100%) 29 (100%) Alveolar smooth muscle lesions (n=34, 38%) HW-free (n=5) 4 (33%) 1 (5.5%) HW-exposed (n=13) 3 (60%) 10 (40%) HW-infected (16) 0 (0%) 16 (55%) Interstitial lesions (n=62, 69%) HW-free (n=12) 5 (42%) 7 (38%) HW-exposed (n=20) 2 (40%) 18 (72%) HW-infected (n=30) 1 (100%) 29 (100%)

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38 R2 = 0.181 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 020406080100 Wolbachia surface protein absorbanceHeartworm antibody absorbance Figure 4-1. Feline D. immitis antibody titer versus Wolbachia surface protein (WSP) antibody titer in all cats (n=90). There was a positive but weak correlation between the magnitude of the titers (p>0.001).

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39 R2 = 0.1591 0.00 0.50 1.00 1.50 2.00 2.50 0.000.200.400.600.801.001.201.401.60 Wolbachia surface protein absorbanceHeartworm antibody absorbance Figure 4-2. Canine D. immitis antibody titer versus Wolbachia surface protein (WSP) antibody titer in all dogs (n=90). There was a positive but weak correlation between the magnitude of the titers (p>0.001).

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40 Figure 4-3. Severity of pulmonary lesions in cats with or without detectable Wolbachia DNA and/or Wolbachia surface protein in pulmonary tissues. Each D. immitis infection status group included 30 cats, and histologic lesions were scored on a 0-3 scale. Boxes represent the middle 50th percentile, bars represent the 25th and 75th percentiles, and dots represent outlier data points. There were no significant differences in median pulmonary lesion scores between groups (p>0.05). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosArteriolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosBronchiolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosAlveolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosInterstitial lesion score HW-free HW-exposed HW-infected

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41 Figure 4-4. Severity of pulmonary lesions in dogs with or without detectable Wolbachia DNA and/or Wolbachia surface protein in pulmonary tissues. Each D. immitis infection status group included 30 dogs, and histologic lesions were scored on a 0-3 scale. Boxes represent the middle 50th percentile, bars represent the 25th and 75th percentiles, and dots represent outlier data points. There were no significant differences in median pulmonary lesion scores between groups (p>0.05). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosArteriolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosBronchiolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia Pos Alveolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Wolbachia NegWolbachia PosWolbachia NegWolbachia PosWolbachia NegWolbachia PosInterstitial lesion score HW-free HW-exposed HW-infected

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42 Figure 4-5. Severity of pulmonary lesions in cats with or without detectable circulating Wolbachia surface protein antibod ies (WSP Ab). Each D. immitis infection status group included 30 cats, and hi stologic lesions were scor ed on a 0-3 scale. Boxes represent the middle 50th percentile, bars represent the 25th and 75th percentiles, and dots represent outlier data points. There were no significant differences in median pulmonary lesion scores between groups (p>0.05). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab PosArteriolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab PosAlveolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab PosBronchiolar lesion score HW-free HW-exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab Pos WSP Ab Neg WSP Ab PosInterstitial lesion score HW-fee HW-exposed HW-infected

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43 Figure 4-6. Severity of pulmonary lesions in do gs with or without detectable circulating Wolbachia surface protein antibod ies (WSP Ab). Each D. immitis infection status group included 30 dogs, and histologic lesions were scored on a 0-3 scale. Boxes represent the middle 50th percentile, bars represent the 25th and 75th percentiles, and dots represent outlier data points. There were no significant differences in median pulmonary lesion scores between groups (p>0.05). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosArteriolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosBrochiolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosAlveolar lesion score HW-free HW-Exposed HW-infected 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 WSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosWSP Ab NegWSP Ab PosInterstitial lesion score HW-free HW-Exposed HW-infected

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44 CHAPTER 5 DISCUSSION In this study, we identified the presence of Wolbachia in H W-defined groups of cats and dogs to determine if there was an associati on between the bacterium and the severity of pulmonary lesions characteristic of heartworm disease. Although pulmonary lesions were most severe in animals with adult D. immitis there was no clear additive effect when Wolbachia was present in sufficient amounts for detection in the lungs or when immunoreactivity against Wolbachia was detected. Since all D. immitis carry Wolbachia it is difficult to separate the effect of the parasite from the effect of its endosymbiont. In both cats and dogs, the freque ncy of detection of Wolbachia in the lung of HW-exposed animals was intermediate between that of HW-free animals and HW-infected animals. Similarly, the fr equency and severity of pulmonary lesions in HW-exposed animals also tended to be intermedia te between that of HW -free animals and HWinfected animals. Although these findings could suggest that the presence of Wolbachia in amounts sufficient for detection in th e lungs or the immune responses to Wolbachia antibodies are associated with increased pulmonary disease, it is not possible to completely rule out an effect of the parasite as well. Wolbachia was detected in animals with no evidence of adult D. immitis Studies have demonstrated bacteria are released in large amou nts when the parasite dies (Kramer et al., 2008; Saint Andre et al., 2002). Therefore it is possibl e the HW-exposed group of animals had previous D immitis infection that deposited WSP or that had initiated an anti-WSP response (Morchon et. al., 2004). When interpreting the pathological findings of the pulmonary tissues, it was important to account for the potential contri bution by migration of immature intestinal parasites such

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45 Ancylostoma spp. and ascarids. A Baermanns test was not conducted and it is possible other parasites, such as lungworms, could also cont ribute to the pulmonary disease but escaped detection (Patton and McCracken, 1991). The lack of clear evidence for a role of Wolbachia in heartworm disease creates a dilemma for veterinarians treating cats and dogs in D. immitis -endemic areas. Although the indiscriminant use of antibiotics should be avoided, many clinicians prescribe doxycycline based on the favorable responses observed in hu man filarial diseases and on pr omising results from the first published studies of doxycycline use in D. immitis -infected dogs.

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46 CHAPTER 6 CONCLUSIONS Our study design was did not dem onstrate that the presence of Wolbachia or WSP antibodies was associated with increase d pulmonary disease associated with D. immitis infection. Future studies could further address this pot ential association by trea ting HW-exposed and HWinfected animals with the anti-Wolbachia antibiotic doxycycline and ob serving the evolution of pulmonary disease. Alternatively, studies could be performed to compare disease development in animals experimentally infected with natural D. immitis versus D. immitis rendered free of Wolbachia by doxycycline treatment.

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47 LIST OF REFERENCES Atkins, C.E., DeFrancesco, T.C., Coats, J.R ., Sidley, J.A., Keene, B.W ., 2000, Heartworm infection in cats: 50 cases ( 1985-1997). J Am Vet Med Assoc 217, 355-358. Atkins, C.E., DeFrancesco, T.C., Miller, M.W ., Meurs, K.M., Keene, B., 1998, Prevalence of heartworm infection in cats with signs of cardiorespiratory abnormalities. J Am Vet Med Assoc 212, 517-520. Bandi, C., Anderson, T.J., Genchi, C., Blaxter, M.L., 1998, Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci 265, 2407-2413. Bandi, C., McCall, J.W., Genchi, C., Corona, S., Venco, L., Sacchi, L., 1999, Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. Int J Parasitol 29, 357-364. Bazzocchi, C., Ceciliani, F., McCall, J.W., Ricci, I., Genchi, C., Bandi, C., 2000a, Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis. Proc Biol Sci 267, 2511-2516. Bazzocchi, C., Genchi, C., Paltrinieri, S ., Lecchi, C., Mortarino, M., Bandi, C., 2003, Immunological role of the endosymbionts of Dirofilaria immitis: th e Wolbachia surface protein activates canine neutrophils with production of IL-8. Vet Parasitol 117, 73-83. Bazzocchi, C., Jamnongluk, W., O'Neill, S.L., Anderson, T.J., Genchi, C., Bandi, C., 2000b, wsp gene sequences from the Wolbachia of filarial nematodes. Curr Microbiol 41, 96-100. Berdoulay, P., Levy, J.K., Snyder, P.S., Pegelo w, M.J., Hooks, J.L., Tavares, L.M., Gibson, N.M., Salute, M.E., 2004, Comparison of serol ogical tests for the de tection of natural heartworm infection in cats. J Am Anim Hosp Assoc 40, 376-384. Bowman, D.D., Torre, C.J., Mannella, C., 2007, Su rvey of 11 western states for heartworm (Dirofilaria immitis) infe ction, heartworm diagnostic and pr evention protocols, and fecal examination protocols for gastro intestinal parasites. Vet Ther 8, 293-304. Browne, L.E., Carter, T.D., Levy, J.K., Snyder, P.S., Johnson, C.M., 2005, Pulmonary arterial disease in cats seropositive for Dirofilaria immitis but lacking adult heartworms in the heart and lungs. Am J Vet Res 66, 1544-1549. Carleton, R.E., Tolbert, M.K., 2004, Prevalence of Dirofilaria immitis and gastrointestinal helminths in cats euthanized at animal c ontrol agencies in northwest Georgia. Vet Parasitol 119, 319-326. Casiraghi, M., McCall, J.W., Simoncini, L., Kr amer, L.H., Sacchi, L., Genchi, C., Werren, J.H., Bandi, C., 2002, Tetracycline treatment and sex-ra tio distortion: a role for Wolbachia in the moulting of filarial nematodes? Int J Parasitol 32, 1457-1468.

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48 Dillon, R., 1998, Clinical significance of feline heartworm disease. Vet Clin North Am Small Anim Pract 28, 1547-1565, x. Genchi, C., Sacchi, L., Bandi, C., Venco, L., 1998, Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion. Parassitologia 40, 247-249. Genchi, C., Venco, L., Ferrari, N., Mortar ino, M., Genchi, M., 2008, Feline heartworm (Dirofilaria immitis) infection: a statistical ela boration of the duration of the infection and life expectancy in asymptomatic cats. Vet Parasitol 158, 177-182. Hermesmeyer, M., Limberg-Child, R.K., Mur phy, A.J., Mansfield, L.S., 2000, Prevalence of Dirofilaria immitis infection among sh elter cats. J Am Vet Med Assoc 217, 211-212. Kozek, W.J., 2005, What is new in the Wolbachia /Dirofilaria interaction? Vet Parasitol 133, 127-132. Kramer, L., Genchi, C., 2002, Feline heartworm infection: serological survey of asymptomatic cats living in northern Italy. Vet Parasitol 104, 43-50. Kramer, L., Grandi, G., Leoni, M ., Passeri, B., McCall, J., Genchi, C., Mortarino, M., Bazzocchi, C., 2008, Wolbachia and its influence on th e pathology and immunol ogy of Dirofilaria immitis infection. Vet Parasitol 158, 191-195. Kramer, L., Simon, F., Tamarozzi, F., Genchi, M., Bazzocchi, C., 2005a, Is Wolbachia complicating the pathological effects of Dirofilaria immitis infections? Vet Parasitol 133, 133-136. Kramer, L.H., Passeri, B., Corona, S ., Simoncini, L., Casiraghi, M., 2003, Immunohistochemical/immunogold detection an d distribution of the endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a polyclonal antiserum raised against WSP (Wolbachia surface protein). Parasitol Res 89, 381-386. Kramer, L.H., Tamarozzi, F., Morchon, R., LopezBelmonte, J., Marcos-Atxutegi, C., MartinPacho, R., Simon, F., 2005b, Immune response to and tissue lo calization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection. Vet Immunol Immunopathol 106, 303-308. Lappin, M.R., Breitschwerdt, E.B., Jensen, W. A., Dunnigan, B., Rha, J.Y., Williams, C.R., Brewer, M., Fall, M., 2004, Molecular a nd serologic evidence of Anaplasma phagocytophilum infection in cats in North America. J Am Vet Med Assoc 225, 893-896, 879. Levy, J.K., Snyder, P.S., Taveres, L.M., Hooks, J. L., Pegelow, M.J., Slater, M.R., Hughes, K.L., Salute, M.E., 2003, Prevalence and risk factors for heartworm infection in cats from northern Florida. J Am Anim Hosp Assoc 39, 533-537.

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49 Liu, J., Song, K.H., Lee, S.E., Lee, J.Y., Lee, J.I., Hayasaki, M., You, M.J., Kim, D.H., 2005, Serological and molecular surv ey of Dirofilaria immitis infection in stray cats in Gyunggi province, South Korea. Vet Parasitol 130, 125-129. Lorentzen, L., Caola, A.E., 2008, Incidence of positive heartworm antibody and antigen tests at IDEXX Laboratories: trends and potential impact on feline heartworm awareness and prevention. Vet Parasitol 158, 183-190. Makepeace, B.L., Rodgers, L., Trees, A.J., 2006, Ra te of elimination of Wolbachia pipientis by doxycycline in vitro increases following drug withdrawal. Antimicrob Agents Chemother 50, 922-927. McCall, J.W., 2005, The safety-net story about macrocyclic lactone heartworm preventives: a review, an update, and reco mmendations. Vet Parasitol 133, 197-206. Miller, M.W., 1998, Feline dirofilariasis. Clin Tech Small Anim Pract 13, 99-108. Morchon, R., Ferreira, A.C., Martin-Pacho, J.R ., Montoya, A., Mortar ino, M., Genchi, C., Simon, F., 2004, Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections. Vet Parasitol 125, 313-321. National Institutes of Health, NCBI. BLAST software. Accessed on-line at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Nelson, C.T., 2008, Dirofilaria immitis in cats: diagnosis and management. Compend Contin Educ Vet 30, 393-400; quiz 400. Nelson, C.T., McCall, J.W., Rubin, S.B., Buzhardt, L.F., Dorion, D.W., Graham, W., Longhofer, S.L., Guerrero, J., Robertson-Plouch, C., Paul, A., 2005a, 2005 Guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in cats. Vet Parasitol 133, 267-275. Nelson, C.T., McCall, J.W., Rubin, S.B., Buzhardt, L.F., Dorion, D.W., Graham, W., Longhofer, S.L., Guerrero, J., Robertson-Plouch, C., Paul, A., 2005b, 2005 Guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria immitis) infection in dogs. Vet Parasitol 133, 255-266. Newton, W.L., 1968, Longevity of an experimental in fection with Dirofilaria immitis in a dog. J. Parasitol 54, 187-188. Nogami, S., Sato, T., 1997, Prevalence of Dirofilari a immitis infection in cats in Saitama, Japan. J Vet Med Sci 59, 869-871. Oh, H., Jun, H., You, M., Hayasaki, M., S ong, K., 2008, Ectopic Migr ation of an Adult Heartworm in a Dog with Dirofilari asis. Korean J Parasitol 46, 171-173.

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50 Patton, S., McCracken, M.D., 1991, Prevalence of Diro filaria immitis in cats and dogs in eastern Tennessee. J Vet Diagn Invest 3, 79-80. Pfarr, K.M., Hoerauf, A.M., 2006, Antibiotics wh ich target the Wolbachia endosymbionts of filarial parasites: a new stra tegy for control of filariasis and amelioration of pathology. Mini Rev Med Chem 6, 203-210. Ralston, S., Stemme, K., Guerrero, J., 1998, Prev enting heartworm disease. Feline Pract. 26, 1822. Ryan, W.G., Newcomb, C.T., 1995, Prevalence of fe line heartworm disease-a global perspective. In: Proceedings of the Heartworm Symposiu m, American Heartworm Society, Batavia, Illinois. Saint Andre, A., Blackwell, N.M., Hall, L.R., Hoerauf, A., Brattig, N.W ., Volkmann, L., Taylor, M.J., Ford, L., Hise, A.G., Lass, J.H., Diaconu, E., Pearlman, E., 2002, The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295, 1892-1895. Simon, F., Morchon, R., Rodriguez-Barbero, A., Lopez-Belmonte, J., Grandi, G., Genchi, C., 2008, Dirofilaria immitis and Wolbachia-deriv ed antigens: its effect on endothelial mammal cells. Vet Parasitol 158, 223-231. Snyder, P.S., Levy, J.K., Salute, M.E., Gorman, S.P., Kubilis, P.S., Smail, P.W., George, L.L., 2000, Performance of serologic tests used to de tect heartworm infection in cats. J Am Vet Med Assoc 216, 693-700. Taylor, M.J., 2003, Wolbachia in the inflammatory pathogenesis of human filariasis. Ann N Y Acad Sci 990, 444-449. Taylor, M.J., Bandi, C., Hoerauf, A., 2005, Wolb achia bacterial endosymbionts of filarial nematodes. Adv Parasitol 60, 245-284. Taylor, M.J., Cross, H.F., Ford, L., Makunde, W.H., Prasad, G.B., Bilo, K., 2001, Wolbachia bacteria in filarial immunity and disease. Parasite Immunol 23, 401-409. Yabsley, M.J., Dresden-Osbourne, C., Pirkle, E.A ., Kirven, J.M., Noblet, G.P., 2004, Filarial worm infections in shelter dogs and cats from northwestern south Carolina, U.S.A. Comp. Parasitol 71, 154-157. Willard, M.D., Roberts, R.E., Allison, N., Grieve, R.B., Escher, K., 1988, Diagnosis of Aelurostrongylus abstrusus and Dirofilaria immitis infections in cats from a humane shelter. J Am Vet Med Assoc 192, 913-916.

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51 BIOGRAPHICAL SKETCH Patricia Ann Dingman was born in 1982, in Angeles City, Philippines. She attended elementary school in Okinawa, Japan, and Plat tsburgh, New York, and finished high school in Coudersport, Pennsylvania. Patric ia graduated from the State Un iversity of New York Oswego with a B.S. in zoology in 2006. She received her M.S. in veterinary medical sciences in 2009 from the University of Florida and plans to continue her edu cation in veterinary medicine. When Patricia volunteered for Dr. Levy, she had the opportunity to investigate disease transmission in cats and dogs fo llowing Hurricane Katrina and attend monthly trap-neuter-return clinics for feral cats (Operation Catnip). Thes e experiences have insp ired her to continue pursuing veterinary-relat ed research in cats.