Haemogregarine specificity in two communities of Florida snakes, with descriptions of six new species of Hepatozoon (Apicomplexa: Hepatozoidae) and a possible species of Haemogregarina (Apicomplexa: Haemogregarinidae).

Five species of snakes in Florida, from Palm Beach County in the south and Alachua County 450 km to the north, occur in similar habitat but have distinctive Hepatozoon species characteristic of each host species. In Palm Beach County, Diadophis punctatus is host to Hepatozoon punctatus n. sp., Thamnophis sauritus sackenii to Hepatozoon sauritus n. sp., and Nerodia fasciata pictiventris to Hepatozoon pictiventris n. sp. In Alachua County, N. fasciata pictiventris is parasitized by Hepatozoon fasciatae n. sp., Seminatrix p. pygaea by Hepatozoon seminatrici n. sp., and Thamnophis s. sirtalis by Hepatozoon sirtalis n. sp. Each Hepatozoon sp. has distinctive gamonts and sporogonic characters and, in the 4 species where known, meronts. Nerodia floridana is host to Haemogregarina floridana n. sp. in both localities, with generic identification tentative, based upon presence of erythrocytic meronts. The presence of sporocysts in the proboscis of 31% of Aedes aegypti infected by H. pictiventris is the first report of infective stages of a reptilian Hepatozoon species within the mouthparts of a dipteran vector. This study suggests that in Florida, at least, the diversity of the Hepatozoon community not only equals but probably exceeds the diversity of the snake communities present, and that host specificity in nature may be much greater than that postulated from previous studies.

Endothelial cell changes are associated with pulmonary edema and respiratory distress in mice infected with the WA1 human Babesia parasite.

A C3H/HeN mouse model was established to study the pathogenesis of the human babesial parasites, WA1 and Babesia microti. To evaluate the course of parasitemia and the associated lesions, mice were inoculated intraperitoneally with either WA1-infected, B. microti-infected, or uninfected hamster red blood cells. WA1-infected mice developed dyspnea and moderate parasitemias, after which death occurred. Babesia microti-infected mice experienced low parasitemias with no apparent morbidity or mortality. WA1-infected mice were thrombocytopenic but not anemic. Hemograms for B. microti-infected mice were similar to controls. Postmortem examination of WA1-infected mice revealed prominent lesions in the lungs, including pulmonary edema and intravascular margination of leukocytes. No pulmonary changes were detected in B. microti-infected mice. Blood gas measurements of WA1-infected mice showed reduced oxygen saturation and pH, and increased carbonic acid compared to controls, indicating hypoxia and respiratory acidosis. Ultrastructure studies of WA1-infected lungs showed hypertrophied endothelial cells containing transcellular channels associated with protein-rich intra-alveolar fluid. Endothelial cell activation was demonstrated by an upregulation of intercellular adhesion molecule-1 in the lungs of WA1-infected mice. The results suggest that recruitment of inflammatory cells to the lungs in WA1-infected mice induces endothelial cell alterations, leading to pulmonary edema and acute respiratory failure.

Evidence of Pasteurella haemolytica linked immune complex disease in natural and experimental models.

The pathogenesis of bovine pneumonic pasteurellosis is not completely understood, and studies have not established that Pasteurella haemolytica A1 (Ph1) virulence is exclusively responsible for the development of acute pulmonary lesions. The purpose of this investigation was to determine if immune complex disease is involved in the pathogenesis of bovine pneumonic pasteurellosis. A retrospective immunohistologic study of lung tissue from natural cases of bovine pneumonic pasteurellosis (44) as performed, and immune complexes were observed in alveloar spaces and walls in 88% of these cases. To study this pathologic mechanism experimentally, groups of mice were immunized with purified Ph1 outer membranes (OMs) or sham immunized on days 0 and 14. Mice were challenged intratracheally on day 24 with either live Ph1 or Ph1 OMs, and pulmonary lesions were assessed 24 h after challenge. Placebo immunized mice developed focal infiltrates of neutrophils and macrophages centered around large caliber bronchi. Mice immunized with Ph1 OMs and challenged with live Ph1 or OMs developed severe bronchointerstitial pneumonia with diffuse neutrophilic infiltration, focal necrosis, hemorrhage and edema, that is histologically similar to bovine pneumonic pasteurellosis. Immunohistology revealed flocculent aggregates of IgG and complement positive material within alveolar spaces and walls from mice challenged with live Ph1, and fine granular deposits of IgG and complement positive material were observed lining the alveolar walls from mice challenged with Ph1 OMs. Immunized mice exhibited high serum IgG antibody titers to Ph1 outer membrane proteins (OMPs). Results of this study suggest that immune complex disease plays a role in the pathogenesis of bovine pneumonic pasteurellosis.

Granulomatous hepatitis associated with Hepatozoon sp. meronts in a southern water snake (Nerodia fasciata pictiventris).

A wild-caught adult female southern water snake (Nerodia fasciata pictiventris) did poorly in captivity. A peripheral blood-film examination demonstrated numerous hemogregarines characterized as fusiform nondividing intraerythrocytic gametocytes. Xenodiagnostic typing in laboratory-reared mosquitoes demonstrated the parasite to be of the genus Hepatozoon. Gross and histopathologic examination of the liver demonstrated numerous granulomas centered on groups of one to six Hepatozoon sp. meronts, an unusual finding in naturally infected wild-caught snakes.

Clinical, anatomic, and immunopathologic characterization of Babesia gibsoni infection in the domestic dog (Canis familiaris).

The pathology associated with acute, chronic, and recrudescent Babesia gibsoni infections was characterized in a group of 6 naturally or experimentally infected, spleen-intact and splenectomized dogs. All experimentally infected dogs became acutely parasitemic, lethargic, anemic, thrombocytopenic, and hemoglobinuric. Anatomic lesions associated, with the disease included diffuse nonsuppurative periportal and centrilobular hepatitis, multifocal necrotizing arteritis, membranoproliferative glomerulonephritis, reactive lymphadenopathy, diffuse erythrophagocytosis, and extramedullary hematopoiesis. The density of CD3+ lymphocytes within the liver sinusoids was markedly increased. Aggregates of large mononuclear cells with immunohistochemical features of activated macrophages were demonstrated in the central veins of the liver. Kupffer cells throughout the hepatic sinusoids appeared hypertrophic and prominent. The density of sinusoidal T lymphocytes, macrophages in central veins, and the degree of Kupffer cell hypertrophy were greatest in the splenectomized dogs. Multifocal deposits of IgM antibody were immunohistochemically demonstrated within the walls of inflamed arteries and renal glomeruli. The results of this study suggest that intense immunostimulation resulting in activation and expansion of T and B lymphocyte populations, macrophage recruitment and activation, vasculitis, glomerulonephritis and anemia contribute to the pathology associated with B. gibsoni infections.

Comparative pathogenesis of human WA1 and Babesia microti isolates in a Syrian hamster model.

The pathogenesis of a newly recognized, molecularly and antigenically distinct human babesial isolate (WA1) and Babesia microti, the common cause of human babesiosis in the United States, were compared in a Syrian hamster model. A group of 33 adult female hamsters were inoculated intraperitoneally with either WA1-infected, B. microti-infected, or uninfected hamster erythrocytes. All WA1-infected animals became parasitemic by postinoculation (PI) day 3 or 4 and were severely lethargic and dyspneic by PI days 6 to 10. Death often occurred spontaneously by PI day 10, with parasitemia of 12 to 90%. Hamsters inoculated with B. microti became parasitemic by PI day 7 and developed peak parasitemia (42 to 60%) by PI day 14 that subsequently decreased to low or undetectable values. Although the B. microti-infected hamsters developed severe anemia, they generally remained asymptomatic. Postmortem examination of WA1-infected hamsters revealed intravascular aggregates of large mononuclear inflammatory cells that occasionally occluded small to medium veins, pulmonary leukoclastic phlebitis, thrombosis, and multifocal coagulative necrosis in the heart, spleen, lung, and liver. No vascular lesions or areas of coagulative necrosis were detected in any B. microti-infected or control hamsters. The results of this study suggest that marked leukocytosis followed by acute necrotizing phlebitis resulting in disseminated intravascular coagulation, thromboembolism, and infarction may be central to the pathogenesis of WA1 infections.

Immune recognition of Ornithodoros tick (Acari: Argasidae) salivary antigens by anti-Psoroptes cuniculi antibodies.

Rabbits infested with either Ornithodoros sp. ticks or Psoroptes cuniculi mites were assayed for anti-tick antibody by enzyme-linked immunosorbent assay. Titration of rabbit serum against Ornithodoros sp. salivary gland extract (SGE) demonstrated both mite- and tick-infested animals to have elevated anti-tick antibody titers. Western blot analysis demonstrated the anti-mite and anti-tick antisera to contain antibodies with affinities for both common and unique subsets of Ornithodoros SGE proteins.

Molecular differentiation of two new world saurian Plasmodium species in different host species using the polymerase chain reaction.

Several isolates of Plasmodium floridense obtained from naturally infected Anolis carolinensis and Anolis sagrei, and 2 isolates of Plasmodium chiricahuae obtained from Sceloporus jarrovi were characterized at the ribosomal DNA (rDNA) locus using the polymerase chain reaction and agarose gel electrophoresis. Enzymatic amplification of the rDNA locus from both Plasmodium species resulted in the generation of a 590-base pair (bp) DNA fragment. The results obtained with all isolates of P. floridense appeared as a doublet, with the second fragment being approximately 630 bp in size. Isolates of P. floridense obtained from A. carolinensis from ecologically different northern and southeastern Florida, and from A. sagrei a the same southeastern Florida site, were demonstrated to be molecularly similar. Plasmodium floridense and P. chiricahuae were molecularly distinct at the 18s rDNA locus, thus confirming their morphological and morphometrical distinction as taxonomic species. Anolis sagrei is a third natural host species for P. floridense in Florida.

Isolation and characterization of mouse monoclonal antibodies reactive with soft tick (Acari: Argasidae) salivary antigens of high molecular weight.

The purpose of this study was to immunologically characterize soft tick salivary antigens. BALB/c mice hyperimmunized with salivary gland extract prepared from Ornithodoros talaje (Guérin-Méneville) were observed to develop high titers of antitick salivary antigen antibodies. Subsequent fusion of splenic lymphocytes from the hyperimmunized mice with SP-2/0-AG14 myeloma cells resulted in the production of 10 antitick IgM-producing hybridoma clones. Partial characterization of the respective tick antigens by gel filtration and SDS-PAGE demonstrated all 10 monoclonal antitick antibodies to be reactive with a salivary gland extract fraction containing proteins 50-110 kDa in molecular weight. Cross-reactivity assays and electrophoretic comparison of salivary gland extract specimens demonstrated similar proteins in several ixodid tick genera and species.

Detection and quantification of Ornithodoros-specific anti-tick antibody by competitive inhibition ELISA.

The objective of this study was to develop a highly specific enzyme-linked immunosorbent assay (ELISA) for the serological detection of anti-Ornithodoros tick antibodies in animals. Affinity-purified rabbit anti-Ornithodoros IgG antibodies were employed in indirect competitive inhibition ELISA assays designed to measure the anti-Ornithodoros antibody titers in other animal species using the domestic goat (Capra hircus) as a large animal model. Repeated infestation of goats with Ornithodoros coriaceus was found to elicit the formation of antibodies capable of inhibiting the binding of the Ornithodoros-specific rabbit IgG. Western blot analysis of goat and rabbit anti-tick antisera demonstrated both animal species to respond immunologically to a set of 9 major protein bands in O. coriaceus salivary gland extracts. The results of these experiments demonstrate that a history of animal exposure to O. coriaceus may be detected serologically by competitive inhibition ELISA.

Characterization of the clinical and anatomical pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts.

Laboratory-reared Aedes aegypti mosquitoes were employed in the successful transmission of Hepatozoon mocassini from a cotton-mouth moccasin (Agkistrodon piscivorus leucostoma) to 3 lizard species (Sceloporus undulatus, Eumeces obsoletus and Sceloporus poinsetti). Marked to severe lethargy and anorexia developed in the S. undulatus, E. obsoletus and S. poinsetti at 15, 38, and 96 days postinfection (PI), respectively. All 3 lizards developed a leukocytosis and had increased plasma aspartate aminotransferase activity (AST) by 14 days PI. Multifocal random hepatocellular necrosis and intrahepatic aggregates of heterophils centered on mature H. mocassini meronts were demonstrated in all 3 lizards. The pulmonary interstitium was multifocally thickened by aggregates of heterophils centered on meronts. No comparable clinical or anatomical pathological changes were demonstrated in naturally infected snakes. The results of this study suggest that H. mocassini is capable of inducing necrotizing inflammatory by lesions in unnatural reptilian hosts.

Demonstration of common and stage-specific anti-Hepatozoon mocassini antibodies in experimentally infected unnatural lizard hosts.

This study examines the immunological response to Hepatozoon mocassini in lizards. Three lizard species were infected experimentally with H. mocassini. Baseline and post-infection (PI) sera were assayed for anti-H. mocassini meront and gametocyte antibody by immunohistochemistry and IFA. Seroconversion occurred at 38 d PI with endpoint IFA titers of 1:64. Antisera from non-parasitaemic and parisitaemic lizards exhibited similar affinities for merozite and gametocyte antigens. Antibody specific for the membranes of gametocyte-infected erythrocytes was demonstrated exclusively in parasitaemic lizards. The results demonstrate that lizards infected with snake haemogregarines mount an antibody response with common and stage-specific components.

Evidence of common and genus-specific epitopes on Ornithodoros spp. Tick (Acari: Argasidae) salivary proteins.

New Zealand White rabbits were repeatedly infested with Ornithodoros turicata (Duges), Ornithodoros talaje (Guérin-Méneville), and Ornithodoros coriaceus (Koch) at 2-wk intervals. Blood samples were taken from each animal 10 d after each infestation and the titer of anti-tick antibody was determined by enzyme-linked immunosorbent assay. Subsequent cross-reactivity studies demonstrated that the antitick antisera nonspecifically bound to salivary gland extract proteins prepared from several other tick genera and species' Amblyomma maculatum (Koch), Dermacentor andersoni (Stiles), Dermacentor variabilis (Say), and Ornithodoros moubata (Murray). Absorption of the antisera against an immobilized extract of A. maculatum substantially increased specificity at the genus level. Western blots of electrophoretically separated Ornithodoros salivary gland extract samples were used to further compare the specificity of absorbed and nonabsorbed antitick antisera. The blots demonstrated that many of the Ornithodoros salivary gland extract proteins bear genus specific epitopes. Some differences were noted among the Ornithodoros species examined with respect to the degree of antigenic relatedness with the ixodid ticks.

Diagnosis and management of Baylisascaris procyonis infection in an infant with nonfatal meningoencephalitis.

Baylisacaris procyonis, the common raccoon ascarid, is known to cause life-threatening visceral, neural, and ocular larva migrans in mammals and birds. Two human fatalities have been previously described; however, little is known about the spectrum of human disease caused by B. procyonis. In this report, the case of a 13-month-old child who had nonfatal meningoencephalitis secondary to B. procyonis infection is presented. The suspected diagnosis was confirmed with use of newly developed enzyme immunoassay and immunoblot techniques. The diagnosis, management, and prevention of B. procyonis infection in humans is discussed. Clinical, serological, and epidemiological evaluations established B. procyonis as the etiologic agent. The child survived his infection but continued to have severe neurological sequelae. The potential for human contact and infection with B. procyonis is great. There is no effective therapy; therefore, prevention is paramount.

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.

Antibody responses of laboratory mice to sequential feedings by two species of argasid ticks (Acari: Argasidae).

The host antibody response of CD1 mice resulting from sequential exposures to two species of Ornithodoros ticks was isotyped (ELISA) in an effort to determine the mechanism for the development of acquired resistance. In addition, salivary gland proteins from two argasid and one ixodid species were examined (RIA) for cross-reactivity. Significant antibody responses, detectable for at least 90 d after last exposure, were shown to occur. Isotyping showed that the initial response was of the IgM class with a secondary class switch to the IgG1 subclass occurring. Evidence that cross-reactive proteins exist between argasid and ixodid ticks is also discussed.