Application of Dirofilaria immitis immunoreactive proteins in serodiagnosis.

Dirofilariasis is a zoonotic global vector-borne disease caused by Dirofilaria immitis. The present study focuses on the somatic and excretory/secretory (E/S) proteins released from adult D. immitis. We aimed to fractionate and identify adult D. immitis immunoreactive proteins. Somatic and E/S extracts were immunoblotted to identify the immunoreactive proteins. In the current study, we used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/MS) to characterize the immunogenic proteins. Additionally, we used fast protein liquid chromatography (FPLC) to fractionate and evaluate the immunogenicity of the D. immitis secretome. The most immunoreactive proteins were between 10 and 48 kDa. Six proteins including polyprotein antigen, P22u, pepsin inhibitor Dit33, neutrophil chemotactic factor (DiNCF) precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat-shock protein 70 (HSP70) were found in both somatic and E/S extracts. Eluting the FPLC column with NaCl resolved two peaks in which the immunoreactivities of the purified proteins were conserved. Characterization of these proteins could provide a novel perspective for understanding the pathogenesis and diagnosing of this disease.

A possible relationship between Thromboxane B2 and Leukotriene B4 and the encapsulation of Dirofilaria repens worms in human subcutaneous dirofilariasis.

Human subcutaneous dirofilariosis has several clinical presentations. Many cases present as subcutaneous nodules, as a consequence of a local inflammatory reaction that encapsulates and destroys the worms. In addition, there are cases in which migrating worms located in the ocular area remain unencapsulated. In the present work, the levels of two pro-inflammatory eicosanoids, thromboxane B2 (TxB2) and leukotriene B4 (LTB4) are analysed by commercial Enzime-Linked immunosorbent assay (ELISA) in serum samples from 43 individuals, 28 diagnosed as having subcutaneous dirofilariasis presenting a subcutaneous nodule, five diagnosed as having dirofilariasis, in which the worms remained unencapsulated in the periphery of the eye, and ten healthy individuals living in a non-endemic area, used as controls. The worms were surgically removed, identifying Dirofilaria repens as the causative agent in all cases, by Polymerase Chain Reaction (PCR). Individuals with nodules showed significantly higher levels of TxB2 and LTB4 than healthy controls, whereas significant differences in LTB4 levels were observed between individuals with unencapsulated worms and healthy controls. It is speculated that the absence of LTB4 may contribute to the fact that worms remain unencapsulated as a part of immune evasion mechanisms.

Seroreactivity to Dirofilaria antigens in people from different areas of Serbia.

BACKGROUND: The Northern part of Serbia is hyperendemic-endemic for canine dirofilarioses. Considering this fact, many human dirofilarial infections could be expected, however only about 30 cases in Serbia have been described until today. Aims of this survey were to assess the people reactivity to the antigens of D. repens and D. immitis and to identify risk factors for the contact exposure. METHODS: Investigation included sera taken from 297 people (179 women and 118 men) living in different areas of Serbia (Pancevo, Novi Sad, Zajecar, Leskovac, Vranje, Nis, Pirot). Sera were analysed by means of two indirect enzyme-linked immunosorbent (ELISA) home-designed that use as antigens adult somatic/metabolic polyproteins of D. repens (DR) and D. immitis (DI), respectively. The results were elaborated using the statistical method of descriptive and quantitative analysis. RESULTS: Significant differences by area in the reactivity of human sera to dirofilarial antigens were not observed (p = 0.056). A high seroreactivity was demonstrated in people from the towns of northern Serbia (Pancevo = 27,1%; Novi Sad = 16,3%), as well as in people from Zajecar (eastern Serbia = 15,8%) and Vranje (southern Serbia = 15,1%). No differences were evidenced between people reactivity to polyproteins of the two dirofilarial species, nor differences related to the gender of examinees. Factor risks evidenced were: i) place of residence; ii) spending work time outdoors during the mosquito season; iii) spending time outdoors and nearby rivers, lakes, swamps or canals; unespectedly, iv) cat owning. CONCLUSION: The findings emerging from this investigation indicate that clinicians and public health authorities should pay greater attention to this zoonosis. Continuing education and training of physicians will greatly contribute to the knowledge of the actual impact of filarial worms on animal and public health, and allow for the planning of suitable measures to prevent the infections.

Human and animal dirofilariasis: the emergence of a zoonotic mosaic.

Dirofilariasis represents a zoonotic mosaic, which includes two main filarial species (Dirofilaria immitis and D. repens) that have adapted to canine, feline, and human hosts with distinct biological and clinical implications. At the same time, both D. immitis and D. repens are themselves hosts to symbiotic bacteria of the genus Wolbachia, the study of which has resulted in a profound shift in the understanding of filarial biology, the mechanisms of the pathologies that they produce in their hosts, and issues related to dirofilariasis treatment. Moreover, because dirofilariasis is a vector-borne transmitted disease, their distribution and infection rates have undergone significant modifications influenced by global climate change. Despite advances in our knowledge of D. immitis and D. repens and the pathologies that they inflict on different hosts, there are still many unknown aspects of dirofilariasis. This review is focused on human and animal dirofilariasis, including the basic morphology, biology, protein composition, and metabolism of Dirofilaria species; the climate and human behavioral factors that influence distribution dynamics; the disease pathology; the host-parasite relationship; the mechanisms involved in parasite survival; the immune response and pathogenesis; and the clinical management of human and animal infections.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Strong expression of TGF-beta in human host tissues around subcutaneous Dirofilaria repens.

Dirofilaria repens and other Dirofilaria species are widely distributed parasitic nematodes of carnivores, which occasionally are transmitted to men, causing subcutaneous nodules. In humans, it usually occurs only as single male or female filariae without production of microfilariae. The non-productive living or dead Dirofilaria worms in subcutaneous biopsies from 15 human patients permitted us to study the role of the pleiotropic and immunoregulatory cytokine transforming growth factor beta (TGF-beta) independent from the influence of microfilariae. Antiserum against latent TGF-beta 1 was used for an immunohistological examination. In the infiltrates around female and male filariae, there occurred strongly TGF-beta-positive macrophages, mast cells, endothelial cells, fibrocytes, and giant cells adjacent to dead worms. In one nodule, secondary lymph follicles were observed with clearly TGF-beta-positive B cells in the mantle zone and weakly positive macrophages and B cells in the germinal centre. A network of CD35-positive follicular dendritic cells was observed in the germinal centre. All Dirofilaria contained Wolbachia endobacteria, which probably had attracted the numerous TGF-beta-negative neutrophils near to the worm. Wolbachia were phagocytosed by neutrophils adjacent to dead filariae. Macrophages and lymphocytes expressed the MHC class II molecule HLA-DR in small accumulations of immune cells in the outer zone of the infiltrate and the mantle zone and germinal centre of secondary lymph follicles. It is concluded that single non-productive Dirofilaria worms elicit a strong expression of TGF-beta. This result is in accordance with observations on Onchocerca volvulus from patients with the hyporeactive (generalised) form.

Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs.

OBJECTIVE: To evaluate the sensitivity and specificity of a commercially available in-clinic ELISA for detection of heartworm infection and tick-borne diseases in dogs. SAMPLE POPULATION: 846 serum, plasma, or blood samples obtained from dogs. PROCEDURES: Samples were evaluated via the in-clinic ELISA to detect antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis (heartworm) antigen. True infection or immunologic status of samples was assessed by use of results of necropsy, an antigen assay for D immitis, and immunofluorescence assay or western blot analysis for antibodies against B burgdorferi, E canis, and A phagocytophilum. RESULTS: Sensitivity and specificity of the in-clinic ELISA for detection of heartworm antigen (99.2% and 100%, respectively), antibodies against B burgdorferi (98.8% and 100%, respectively), and antibodies against E canis (96.2% and 100%, respectively) were similar to results for a similar commercial ELISA. In samples obtained from dogs in the northeast and upper Midwest of the United States, sensitivity and specificity of the in-clinic ELISA for antibodies against Anaplasma spp were 99.1% and 100%, respectively, compared with results for an immunofluorescence assay. Samples from 2 dogs experimentally infected with the NY18 strain of A phagocytophilum were tested by use of the in-clinic ELISA, and antibodies against A phagocytophilum were detected by 8 days after inoculation. Antibodies against Anaplasma platys in experimentally infected dogs cross-reacted with the A phagocytophilum analyte. Coinfections were identified in several of the canine serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available in-clinic ELISA could be used by veterinarians to screen dogs for heartworm infection and for exposure to tick-borne pathogens.

A serological study of Dirofilaria immitis in feral cats in Grenada, West Indies.

A study to determine the seroprevalence of Dirofilaria immitis was carried out in feral cats in Grenada. Of the 137 feral cats tested for circulating antibodies (IgG; lateral-flow immunoassay) and circulating antigens (Ag; enzyme-linked immunosorbent assay), 8% (95% confidence interval (CI) 3.5-12.5%) were antibody positive and 5.1% (95% CI 1.4-8.8%) were antigen positive. No significant difference between cats aged>1 to 4 years and cats less than 1 year of age was found (P>0.05, χ²). There was also no significant difference (P>0.05, χ²) between male and female cats. Dirofilaria immitis prevalence is relatively high in the feral cat population of Grenada. Evidence of D. immitis infection in feral cats coupled with the endemic nature of heartworm disease in dogs in Grenada leads us to suggest the introduction of heartworm prophylaxis in cats. To the authors' knowledge, this serological evidence of heartworm infection in feral cats in Grenada is the first report from the Caribbean region.

Assessing the potential cross-reactivity using a commercial heartworm ELISA kits of serum from dogs naturally infected with Onchocerca lupi.

Onchocerca lupi is an emerging zoonotic parasite of dogs, endemic to the southwestern USA and areas of the Old World. Currently, there are no specific serological diagnostic tests able to detect O. lupi infection. Recent literature has demonstrated that commercially available heartworm antigen tests, despite being highly sensitive, may cross-react with infections by other filarid nematodes. There is no information on potential cross-reactivity of such tests in serum of dogs infected with O. lupi. Our objective was to assess serum samples of dogs naturally-infected with O. lupi for potential cross-reactivity before and after heat-treatment using a commercial heartworm ELISA kit. We obtained serum from 23 dogs naturally-infected with O. lupi. These dogs presented with ocular disease, and were consulted to schedule either surgical removal of ocular nodules due to infection or enucleation. Samples were tested in triplicate using the DiroCHEK® Heartworm Antigen Test kit (Synbiotics Corporation, Zoetis, Kalamazoo, MI, USA) following the manufacturers' protocol pre- and post-heat-treatment. Samples were heat-treated using a dry heat block at 103 °C for 10 min and then centrifuged at 1818×g for 20 min. Out of a total of 23 dogs, 19 (82.6 %) had no antigen detected regardless of heat-treatment, three dogs tested positive before and after heat-treatment, and a single dog turned positive after heat-treatment. These three dogs that were positive before and after heat-treatment were confirmedly co-infected with Dirofilaria immitis by the veterinarians responsible for these cases, and we were unable to get the history or follow up with the dog that turned positive post-heat-treatment only. Our data suggest that O. lupi infections should not result in false-positives when using the DiroCHEK® in dog serum, before or after heat-treatment. Dogs with clinical ocular onchocercosis that test antigen-positive in DiroCHEK® are likely co-infected with D. immitis, and should be further tested, including evaluation of microfilariae in blood and diagnostic imaging. If heartworm infection is confirmed, the animals should be enrolled in the recommended treatment protocol in accordance to the guidelines of the American Heartworm Society or other local organizations.

Dirofilaria immitis proteins recognized by antibodies from individuals living with microfilaremic dogs.

INTRODUCTION: Dirofilaria immitis is a nematode that affects human health in several countries of the world. This study was conducted to examine whether serum samples from the owners of microfilaremic dogs present immunoreactivity to parasite proteins. METHODOLOGY: Eight serum samples from the owners of microfilaremic dogs were examined. Total proteins were extracted from adult worms and 12% SDS-PAGE was performed. The gel was electroblotted to a nitrocellulose membrane, and a Western blot (WB) was performed. Reactive bands of 22, 33, 39, 49, and 63 kDa in WB were excised from the gel and analyzed by mass spectrometry (MS). RESULTS: The MS results showed the presence of 10 different proteins of D. immitis recognized by the human serum samples. CONCLUSIONS: These results indicate that in endemic areas of D. immitis, owners of infected dogs recognize specific proteins of the parasite, suggesting a possible infection.

Survey of Dirofilaria immitis antigen and antibodies to Leishmania infantum and Toxoplasma gondii in cats from Madeira Island, Portugal.

BACKGROUND: Dirofilaria immitis, Leishmania infantum and Toxoplasma gondii are zoonotic parasites which can affect domestic cats. Considering the lack of published data from the local feline population, this study aimed to assess infection with or exposure to these pathogens in cats from Madeira Island, Portugal. METHODS: One hundred and forty-one domestic cats (77 males and 64 females; median age: 2 years) were sampled at a veterinary medical centre in Funchal, from September 2018 to January 2019. Serum samples were tested for D. immitis antigen, with an enzyme-linked immunosorbent assay kit, and for antibodies to Leishmania spp. or to T. gondii, with the direct agglutination test and the modified agglutination test, respectively. RESULTS: Five cats (3.5%; 95% confidence interval, CI: 1.2-8.1) were positive to D. immitis; no cats were seropositive to Leishmania spp. (0%; 95% CI: 0-2.6%); and 43 cats (30.5%; 95% CI: 23.0-38.8%) were seropositive to T. gondii. Prevalence of the D. immitis antigen was significantly different between cats that received ectoparasiticides and those which did not (0 vs 12.2%; P = 0.009). Prevalence of antibodies to T. gondii was significantly different between juvenile and adult cats (12.8 vs 38.0%; P = 0.007). There were two cats concurrently positive to D. immitis and T. gondii, but no statistical association between these two dependent variables was found (P = 0.641). CONCLUSIONS: To our knowledge, this is the first report of the presence of parasites D. immitis and T. gondii in the feline population of Madeira Island. Knowledge on the epidemiological situation of these and other zoonotic pathogens should raise awareness, both at the veterinary medical and public health levels, and contribute to promoting prevention and control.

Pig-hunting dogs are an at-risk population for canine heartworm (Dirofilaria immitis) infection in eastern Australia.

BACKGROUND: Canine heartworm disease, caused by Dirofilaria immitis, has global veterinary importance. In Australia, the prevalence of canine heartworm infection decreased markedly following the introduction of over-the-counter macrocyclic lactones. We aimed to estimate the prevalence of canine heartworm infection in at-risk populations of dogs in eastern Australia and analyse published prevalence data from Australia. METHODS: In total, 566 dogs from eastern Australia were tested for the presence of D. immitis antigen. Four cohorts were studied: pig-hunting dogs from Queensland (Cohort 1, n = 104), dogs from remote New South Wales (NSW) (Cohort 2, n = 332), urban pets from rural NSW (Cohort 3, n = 45) and ex-racing Greyhounds from Sydney, NSW (Cohort 4, n = 85). Serum samples were screened for D. immitis antigen using a reference laboratory microwell-based assay (DiroChek®) or a point-of-care immunochromatography test kit (Anigen Rapid®). Risk factors associated with the odds of D. immitis antigen seropositivity were identified using binary logistic regression models. Seropositive blood samples were tested for the presence and quantity of D. immitis DNA using a species specific real-time (q)PCR assay. A metanalysis of the Australian canine heartworm literature was conducted. RESULTS: The prevalence of dirofilariasis in pig-hunting dogs from Queensland (Cohort 1) was 12.5% (95% CI: 6.5-18.9%), with a subpopulation of dogs from Central Queensland having a prevalence of 21% (95% CI: 12.3-33.4%). Age was significantly associated with D. immitis antigen seropositivity (increased risk with increased age). The odds of being > 5 years versus ≤ 5 years was 3.7-times (95% CI: 1.1-12.5) greater in antigen positive versus antigen negative dogs. No D. immitis antigen positive dogs were detected in dogs from NSW (Cohorts 2-4). The Australian canine heartworm disease literature includes 98 peer-reviewed publications (1901-2019) with 30 studies reporting on D. immitis prevalence in dogs. Throughout the publication peak period (1980s), the primary antemortem diagnostic test was detection of microfilariae. CONCLUSIONS: Canine heartworm infection in dogs used for pig hunting is a previously unexplored topic in Australia. Pig-hunting dogs are infected with canine heartworm in Queensland, Australia, placing pet dogs and cats at increased risk of infection.

Monoclonal gammopathy associated with heartworm disease in a dog.

A 12-year-old, intact female, mixed Yorkshire terrier was evaluated for syncopal episodes, weakness, decreased appetite, and weight loss. Heartworm disease was diagnosed based on evidence of circulating microfilariae of Dirofilaria immitis on direct examination of blood smears and a positive SNAP heartworm antigen test. An immunoglobulin G (IgG) gammopathy, demonstrated by serum protein electrophoresis, was associated with heartworm disease in this dog. Response to treatment with both an adulticide and the microfilaricide ivermectin included remission of clinical signs and a decrease in the monoclonal gammopathy. To our knowledge, this is the first report of an IgG gammopathy associated with heartworm disease in the dog.

Highly modified and immunoactive N-glycans of the canine heartworm.

The canine heartworm (Dirofilaria immitis) is a mosquito-borne parasitic nematode whose range is extending due to climate change. In a four-dimensional analysis involving HPLC, MALDI-TOF-MS and MS/MS in combination with chemical and enzymatic digestions, we here reveal an N-glycome of unprecedented complexity. We detect N-glycans of up to 7000 Da, which contain long fucosylated HexNAc-based repeats, as well as glucuronylated structures. While some modifications including LacdiNAc, chitobiose, α1,3-fucose and phosphorylcholine are familiar, anionic N-glycans have previously not been reported in nematodes. Glycan array data show that the neutral glycans are preferentially recognised by IgM in dog sera or by mannose binding lectin when antennal fucose and phosphorylcholine residues are removed; this pattern of reactivity is reversed for mammalian C-reactive protein, which can in turn be bound by the complement component C1q. Thereby, the N-glycans of D. immitis contain features which may either mediate immunomodulation of the host or confer the ability to avoid immune surveillance.

Galectin and aldolase-like molecules are responsible for the specific IgE response in humans exposed to Dirofilaria immitis.

Dirofilaria immitis is the agent of the heartworm disease in canids and felids, and of pulmonary dirofilariosis in man. Like other filariae, D. immitis harbours endosymbion Wolbachia bacteriae. In this work we analyse the response of specific IgE antibodies against both D. immitis antigens and the Wolbachia surface protein (WSP) in two groups of persons living in an area of canine endemia, one presenting high levels of total IgE (group 1) and other with normal levels (group 2). Infections with D. immitis were demonstrated by the presence of specific IgG in 228 individuals(48.8%) of the group 1 and only in one of the group 2. Specific IgE antibody response against D. immitis antigens was detected only in individuals of the group 1. IgE response against WSP was not detected in any group. The IgE response was directed mainly against two molecules of 33 and 42 kDa of the antigenic extract of D. immitis. These molecules were identified by mass spectrometry as a galectin and an aldolase, respectively. Their possible role in the survival mechanisms of the parasite and their contribution to development of allergic reactions in individuals resident in areas with heartworm disease are discussed.

Acute death in heartworm-infected cats: unraveling the puzzle.

Although the acute death syndrome in feline heartworm disease is widely recognized, its pathogenesis remains a mystery. The most widely held hypothesis is that an acute anaphylactic reaction, perhaps precipitated by the death of the parasite, is the underlying cause. This study investigated the role of the physical form of antigen (Ag) in the ensuing reaction when Dirofilaria immitis-sensitized cats are challenged by intravenous (IV) administration of heartworm Ag. Healthy D. immitis-naive cats (n = 23) were sensitized using subcutaneous injections of adjuvanted D. immitis Ag administered weekly for 6 weeks. After sensitization, cats (n = 20) were anaesthetized and challenged with IV D. immitis Ag in various forms or with IV sterile 0.9% saline (n = 3). Systolic blood pressure, respiratory rate, degree of dyspnea, blood oxygen saturation, and heart rate were measured immediately before and at 10-15 min intervals after challenge until terminal apnea occurred or until euthanasia at 140 min after challenge. Blood samples were collected for complete blood count and measurements of serum serotonin immediately before and at 10, 20, and 35 min after challenge. Clinical observations were recorded as they occurred, or at 10-15 minute intervals, whichever was the more frequent. The most severe post-challenge reactions occurred in cats challenged with Ag from dead worms, live worms, and 20 ng/mL Ag. Dyspnea increased significantly after challenge in all three groups (p < 0.001; p = 0.04, and p = 0.002, respectively), and blood oxygen saturation dropped post-challenge in the Dead Worm (p < 0.001) and the 20 ng/mL Ag (p = 0.002) groups. In the 20 ng/mL Ag group, systolic blood pressure decreased (p <0.05) and respiratory rate increased (p < 0.05) post-challenge. Clinical observations included dyspnea, gastrointestinal signs (retching, defecation, or flatulence), urination, and less commonly, hemorrhage from the nostrils or anus, or cutaneous swelling (general or specifically facial). The 20 ng/mL Ag group had the highest rate of clinical signs, followed by the Dead Worm group. The most common and reliable hematologic change associated with severe clinical effects of D. immitis Ag challenge was increased hematocrit, which was statistically higher after challenge than at baseline in the Dead Worm group (p = 0.012). The model demonstrated that the physical form of heartworm Ag used for IV challenge in D. immitis-sensitized cats is an important factor for determining the characteristics of the post-challenge reaction, and the amount of exposed internal filarial Ag presented to the feline immune system may influence the severity of the response to challenge.

Activity of pulmonary intravascular macrophages in cats and dogs with and without adult Dirofilaria immitis.

Pulmonary intravascular macrophages (PIMs), large (20-80 microm diameter) monocytes are present in sheep, pigs, and horses, but not in dogs, rats, rabbits, or primates. The present study evaluated the phagocytic activity of various organs in cats and dogs and determined the influence of Dirofilaria immitis infections on PIM activity. Live or dead adult heartworm (HW) was transplanted via jugular venotomy into cats and dogs. Cats (four per group) were allocated to five groups: surgical controls--no HW, dead HW for 1 week, live HW for 1 week, dead HW for 3 weeks, or live HW for 3 weeks. Radioactive technetium (Tc-99m, 1.2mCi in 0.3ml) sulfa-colloid was injected intravenously. All cats with HW were clinically asymptomatic and developed radiographic pulmonary parenchymal changes. No gross changes were visible at necropsy for cats with HW; inflammatory changes were less severe in cats with live HW. In cats with dead HW for 3 weeks, worms were present but folded, flattened, and located in distal pulmonary arteries. Uninfected control dogs and those with dead HW did not demonstrate any PIM activity. In control cats, lungs were the primary phagocytic organ after systemic IV colloid injection (72.5% of the total recovered radioactive dose). The lung and liver together represented over 95% of the recovered Tc-99m colloid in all cats. In each group of cats with HW, phagocytic activity of the lung was significantly less (p < 0.001) than the PIM activity of controls. Cats with dead HW at 1 week (50.1%) had a significant (p < 0.019) decrease in PIM activity compared with cats with dead HW at 3 weeks (59.5%). The PIM activity in cats with live HW was significantly decreased (p < 0.001) from that in groups with dead HW, but there was no significant difference between the two groups infected with live worms. There were no significant differences in recovery between any groups in pairwise analysis of the spleen, heart, skeletal muscle, kidney, bone marrow, or blood. Significant increases (p < 0.001) in liver activity for each group inversely reflected the decreased lung activity; consistent with increased hepatic uptake of Tc colloid "escaping" a relatively suppressed lung macrophage system. Transmission electron microscopy confirmed PIM glycocalyx changes and vacuolization, moderate Type 1 cell damage and Type II cell hypertrophy in cats with dead HW. There was no evidence of PIM death. The significant decrease in PIM activity in groups with dead HW and a greater decrease in groups with live HW are consistent with a down-regulation of PIM function in cats with live HW.

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection.

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.