Babesia bovis: continuous cultivation in a microaerophilous stationary phase culture.

The protozoan parasite Babesia bovis, a causative agent of bovine babesiosis, has been continuously cultivated in a settled layer of bovine erythrocytes. Lowered oxygen tension within the layer of host erythrocytes results in a darkening of infected cultures and provides a rapid means of evaluating parasite growth. Deprivation of carbon dioxide causes the merozoites to accumulate in the medium rather than involving new erythrocytes. When separated from the culture, these extraerythrocytic parasites retain their infectivity. Parasites produced in vitro are morphologically identical to parasites from the blood of infected cattle and are susceptible to antibabesial drugs.

Cultivation of Rhinosporidium seeberi in vitro: interaction with epithelial cells.

Rhinosporidium seeberi, a fungus that is associated with polyp-like tumors in animals and man, was successfully cultivated. This organism stimulated proliferation of epithelial cells in vitro, producing polyp-like structures. Spores produced in culture required a period of aging or development, or both, before they were capable of reinitiating the growth cycle.

Infectious diseases of dogs and cats on Isabela Island, Galapagos.

BACKGROUND: Vaccination and importation of dogs and cats are prohibited in the Galapagos, resulting in a uniquely isolated population. The purpose of this study was to determine the prevalence of infectious diseases of dogs and cats that impact their health, could spill over to native wildlife, or sentinel diseases of concern to humans. HYPOTHESIS: The isolation of dogs and cats in the Galapagos protects them from diseases common in mainland populations. ANIMALS: Ninety-five dogs and 52 cats presented during a neutering campaign. METHODS: A prospective cross-sectional study was performed. Blood was collected for serological and DNA evaluation of a panel of infectious diseases. RESULTS: Antibodies against parvovirus (100%), parainfluenza virus (100%), adenovirus 1/2 (66-67%), and distemper virus (22%) were present in dogs. Dirofilaria immitis was also common in dogs (34%), with lower prevalences of Wolbachia pipiens (22%), Bartonella sp. (13%), Ehrlichia/Anaplasma spp. (1%), and Mycoplasma haemocanis (1%) observed. Antibodies against panleukopenia virus (67%), Toxoplasma gondii (63%), calicivirus (44%), and herpesvirus 1 (10%) were detected in cats. Feline leukemia virus antigen, feline immunodeficiency virus antibody, or coronavirus antibodies were not detected. Bartonella sp. (44%) infections were common in cats, but only one was infected with M. haemofelis. CONCLUSIONS AND CLINICAL IMPORTANCE: Despite their relative seclusion from the rest of the world, cats and dogs of Isabela were exposed to many pathogens found in mainland South America. Parasite prophylaxis, neutering, and strict enforcement of animal movement restrictions would control a majority of the diseases. In the absence of vaccination, a reservoir of susceptible animals remains vulnerable to new disease introductions.

Spironucleus meleagridis, an enteric diplomonad protozoan of cockatiels (Nymphicus hollandicus): preliminary molecular characterization and association with clinical disease.

A flagellated enteric diplomonad protozoan consistent with Spironucleus meleagridis (formerly Hexamita meleagridis) associated with gastrointestinal disease and mortality in psittacine birds including cockatiels (Nymphicus hollandicus) has been sporadically described in the literature. However, molecular characterization of psittacine protozoal isolates had not yet been performed. The 16S rRNA gene from a protozoan persistently shed in the feces in a small group of cockatiels demonstrated a 98% molecular identity with S. meleagridis isolated from turkeys. Based on these sequence data, a diagnostic PCR assay was developed to detect the presence of S. meleagridis. Nineteen privately owned pet cockatiels from unrelated households were clinically evaluated. All birds microscopically positive for this organism were PCR positive, with several additional birds microscopically negative but PCR positive. Many of the birds identified as positive for S. meleagridis by fecal PCR had signs of gastrointestinal disease such as diarrhea, soft feces, and melena, whereas none of the birds that tested negative had gastrointestinal signs. Examination of feces from two unrelated cockatiel breeding facilities revealed 70% and 86% PCR positive rates. Prevalence of infection and incidence of clinical disease, including factors that lead to clinical manifestation such as viral, bacterial, or mycotic coinfections, are not yet known and warrant further study, but spironucleosis is likely an under-recognized disease in cockatiels.

Standardization of the diagnostic criteria for canine ehrlichiosis. Towards a universal case definition.

Canine ehrlichiosis is a highly variable syndrome presenting a significant differential diagnostic difficulty. It imitates many metabolic and infectious diseases and lacks standardized diagnostic criteria, common reagents, and database resources. A clinical diagnosis of canine ehrlichiosis may be based on the manifestation of fever, thrombocytopenia, anorexia, nasolacrimal discharge, epistaxis, and exclusion of autoimmune and common canine vector borne diseases. These parameters are not invariably observed especially in the atypical form of the disease often caused by species other than Ehrlichia canis. A definitive diagnosis is based on the presence of specific antibodies to ehrlichial agent(s), the demonstration of the etiologic agent(s) itself, or specific amplicons by a strigently quality controlled PCR protocol. The relationship of the various clinical and laboratory parameters, the status of the currently available tests, and their real or presumed predictive value are discussed in the context of stimulating an effort to formulate an international standard for the diagnosis of this and related diseases of man and animals.

Development of Dot-enzyme immunoassay for diagnosis of canine babesiosis.

A reagent conservative Dot-enzyme immunoassay (Dot-EIA) was developed primarily for canine babesiosis caused by Babesia canis. The technique is simple, specific, and sensitive. All steps were carried out at room temperature. Strong agreement was observed between Dot-EIA and the conventionally used indirect immunofluorescence test. The procedure is adaptable to other protozoal disease, e.g., bovine babesiosis and human malaria.

The use of a polymerase chain reaction as a diagnostic test for Rocky Mountain spotted fever.

A polymerase chain reaction (PCR) for amplifying ribosomal DNA of Rickettsia rickettsii was performed on blood clots and urine samples from 10 patients with suspected Rocky Mountain spotted fever (RMSF) and five controls with nonrickettsial diseases. The results of this PCR-based procedure were positive in four of the five patients with probable RMSF, but reamplification was required in three patients. Rickettsia rickettsii was grown from the blood of two of these four patients. The urine from one patient was also PCR-positive. These results confirm earlier findings that the PCR can detect R. rickettsii, but the need for reamplification indicates that the lack of sensitivity is a serious limitation in the usefulness of the PCR as a clinical diagnostic test.

Attempted Ehrlichia risticii transmission with Dermacentor variabilis (Acari: Ixodidae).

Larval Dermacentor variabilis (Say) (n = 327) were fed on Balb/C mice inoculated with Ehrlichia risticii, the etiologic agent of equine monocytic ehrlichiosis (Potomac horse fever). All mice displayed clinical signs of E. risticii infection at the time of feeding. After molting, resulting nymphs (n = 74) were fed on susceptible mice. No clinical signs were observed, and the mice remained seronegative for 6 wk after feeding.

Attempted mechanical transfer of Ehrlichia risticii by tabanids (Diptera: Tabanidae).

The ability of tabanid mouthparts to retain and to transfer mechanically Ehrlichia risticii Holland, Weiss, Burgdorfer, Cole & Kakoma was evaluated by feeding flies on infected and noninfected mice and on capillary tubes containing infected cells and cell-free medium. Flies representing two genera and 29 species were collected at equine boarding stables, farms, and along riding trails in Wake, Johnston, and Duplin counties in North Carolina for the feeding trials. Two species, Tabanus fulvulus Wiedemann and T. pallidescens Philip, fed on mice but failed to transfer the pathogen from infected to susceptible mice. Chrysops vittatus Wiedemann, Tabanus americanus Forster, and T. sulcifrons Macquart transferred E. risticii-infected cells from capillary tubes containing infected cells in medium to tubes containing medium. These studies document that E. risticii-infected cells can be retained on mouthparts and potentially transferred by tabanids.

Whole blood assay for examining lymphocyte blastogenesis of percichthyid bass (Morone) [corrected].

A simple and reproducible method was developed for the measurement of blastogenesis of peripheral blood lymphocytes using whole blood of hybrid bass (striped bass [Morone saxatilis] female x white bass [M. chrysops] male) stimulated with Concanavalin A, phytohemagglutinin-P, lipopolysaccharide or pokeweed mitogen. Compared to traditional methods which use leucocyte separation procedures, whole blood culture is faster and less expensive. Only small aliquots of blood (10 microliters per culture well) were needed, which would be beneficial for sampling small fish as well as for taking multiple samples from single animals. Optimal culture conditions for hybrid bass, including mitogen concentration, incubation temperature and incubation period, were determined. This is the first report to demonstrate a blastogenic response of whole blood cells in fish.

Immunoblot analysis of the immunoglobulin G response to Ehrlichia canis in dogs: an international survey.

Historically, considerable variation has been reported in the type and severity of clinical and hematologic abnormalities associated with canine ehrlichiosis. Because of difficulties associated with the isolation of intracellular monocytic Ehrlichia species in tissue culture systems, few E. canis isolates are available for comparative microbiologic studies. To address the issue of potential E. canis antigenic diversity in different regions of the world, dog sera reactive by indirect fluorescent antibody testing to E. canis (Florida) antigen were obtained from France, Israel, Italy, the United States, the Virgin Islands, and Zimbabwe. Ehrlichia canis proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and at least 5 sera from each region were stained by western immunoblotting. Antibody immunodominance was scored based upon staining intensity. There was relative homogeneity in the immunogenic protein reactions to E. canis antigens. Of the 58 E. canis reactive sera, 54 samples resulted in immunoblot patterns indicative of chronic ehrlichiosis. Four reactive sera (reciprocal titers of 160-2,560) did not recognize any genus-specific antigens resulting in protein bands between 22 and 29 kD, indicating serologic cross-reactivity with other microorganisms. Relatively homogenous immunoblot patterns, consistent with the reported immunoblot response of dogs with experimental chronic ehrlichiosis, were observed with sera from Arizona, France, Israel, North Carolina, Texas, and the Virgin Islands. In contrast, unique major proteins were observed in dog sera from Italy and Zimbabwe. Our results indicate that although relatively homogeneous, antigenic diversity may exist among E. canis organisms in different regions of the world.

Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to the parasitic dinoflagellate Amyloodinium ocellatum in Oreochromis aureus.

An enzyme-linked immunosorbent assay (ELISA) using antibody to affinity-purified Oreochromis aureus immunoglobulin and antigens from the parasitic dinoflagellate amyloodinium ocellatum was developed. The ELISA was then used to evaluate the immune response of the tilapine fish to immunization with the parasite. Fish immunized with antigens of the dinospore stage, either live or sonicated, produced a specific immune response that was detectable by this ELISA. Combinations of serial dilutions of A. ocellatum antigen and fish anti-A. ocellatum serum were examined to determine which dilutions provided optimal differentiation of seropositive from seronegative fish. Fresh and heat-inactivated serum from both seropositive and seronegative fish produced similar results.

Detection and molecular characterization of a novel large Babesia species in a dog.

Babesia canis has generally been considered the only large Babesia to infect dogs. Here we describe the molecular characterization of a large Babesia species that was detected in the blood and bone marrow of a dog with clinical and hematological abnormalities consistent with babesiosis. Analysis of the 18S rRNA genes revealed a unique sequence that shared 93.9% sequence identity with B. bigemina and 93.5% sequence identity with B. caballi, compared to 91.2-91.6% identity with B. canis canis, B. c. vogeli, and B. c. rossi. Cross-reactive antibodies against B. canis, B. gibsoni (Asian genotype), or B. gibsoni (California genotype) antigens were not detected in acute or convalescent serum samples. The dog was treated with imidocarb diproprionate, which resulted in the resolution of clinical signs, and subsequently Babesia DNA was not detectable by PCR in post-treatment samples. The organism described in this report represents a genetically unique large Babesia sp. and is the eighth genetically distinct piroplasm capable of infecting the domestic dog.

Cryptosporidia-positive rates of avian necropsy accessions determined by examination of auramine O-stained fecal smears.

Fecal smears from 112 avian necropsy accessions representing 431 birds were stained with auramine O and examined for Cryptosporidium oocysts by fluorescence microscopy. Stained Cryptosporidium oocysts fluoresced bright yellow-green and were easily differentiated from extraneous material by their uniform small size (approx. 5 micron) and morphology. The rates of cryptosporidia-positive accessions were 27.3% (9/33) of broilers, 10% (3/30) of broiler breeders, and 5.9% (1/17) of layers. Further analyses of available data for various risk factors that may have influenced rates of cryptosporidia-positive samples in broilers, broiler breeders, and layers failed to show significant relationships. However, it was apparent that positive samples were clustered within accessions and not scattered throughout the population sampled. This survey also resulted in the first reported identification of Cryptosporidium oocysts from a budgerigar, macaw, and tundra swan.

Experimental reproduction of enteritis in bobwhite quail (Colinus virginianus) with Cryptosporidium and reovirus.

Five-day-old bobwhite quails were inoculated with reovirus and Cryptosporidium previously isolated from the intestinal contents of young, commercially raised bobwhite quails experiencing severe enteritis. Quails inoculated with reovirus alone did not develop clinically apparent disease, infection was localized principally in the intestinal tract, and no lesions were detected. Quails inoculated with Cryptosporidium, alone or with reovirus, developed severe enteritis with high mortality and marked growth depression. Cryptosporidia caused blunting of intestinal villi and provoked a mononuclear cell response in the lamina propria. The severity of intestinal lesions correlated with numbers of parasites. An apparent synergistic effect in dually infected quails was indicated by enhanced Cryptosporidium oocyst shedding, greater numbers of cryptosporidia in the intestinal tracts, and systemic reovirus infection. In addition, multifocal liver necrosis was detected in dually infected quails but was absent in quails infected with only reovirus or Cryptosporidium. The results suggest that Cryptosporidium promoted systemic spread of reovirus, and reovirus intensified Cryptosporidium infection, but no significant synergistic effect on mortality or weight gain was detected. The most important agent in the naturally occurring acute enteritis of bobwhite quails was Cryptosporidium.

Interaction of reovirus and Cryptosporidium baileyi in experimentally infected chickens.

No clinical signs, gross lesions, or increased mortality were observed in specific-pathogen-free chickens orally inoculated at 5 days of age with Cryptosporidium baileyi, reovirus 2035, reovirus 2408, or combinations of these agents. Weight gain of chickens inoculated with only reovirus 2408 was depressed 0-8 days postinoculation (PI) (P less than 0.01) but not for the 21-day period PI. Weight gain of chickens inoculated with only reovirus 2035 was not affected. Cryptosporidium baileyi infection significantly depressed weight gain 8-14 days PI but not for the entire 21-day period PI. Weight gain of chickens infected with both C. baileyi and reovirus 2035 was significantly depressed 0-14 days PI and for the entire 21-day period PI. Dual infection with C. baileyi and either reovirus appeared to promote shedding of both agents. Cryptosporidia were found principally in the rectum 2-10 days PI and in the bursa of Fabricius 6-10 days PI. Reovirus infection did not cause any microscopic lesions and did not modify lesions caused by C. baileyi infection.

Experimental cryptosporidiosis and infectious bursal disease virus infection of specific-pathogen-free chickens.

Specific-pathogen-free chickens orally inoculated at 4 days of age with a moderately pathogenic vaccine strain of infectious bursal disease virus (IBDV) and/or at 5 days of age with Cryptosporidium baileyi oocysts remained free of overt clinical signs throughout a 16-day period postinoculation (PI). The prepatency period for C. baileyi oocyst shedding was shorter in chickens receiving higher numbers of oocysts, but once shedding was detected, there were no obvious differences in shedding patterns among groups receiving 10(3) through 10(6) oocysts. On days 8 and 16 PI, cryptosporidia were located primarily in the bursae of Fabricius. IBDV exposure was associated with bursal follicle atrophy, whereas C. baileyi infection resulted in bursal epithelial hypertrophy and hyperplasia, mild follicle atrophy, and heterophil infiltration of the bursal mucosa. Examination of experimental groups of 30 birds each indicated that concurrent infection with both agents resulted in more severe bursal lesions, more infected birds, and greater numbers of cryptosporidia in infected tissues. At the termination of the trial, 16 days PI, Cryptosporidium infection was associated with a 6% decrease in mean body weight compared with controls.

Intestinal and bursal cryptosporidiosis in turkeys following inoculation with Cryptosporidium sp. isolated from commercial poults.

Cryptosporidium meleagridis oocysts, originally isolated from droppings of commercial turkey poults with increased mortality due to viral (reovirus) hepatitis and enteritis, were treated with peracetic acid to kill companion bacteria and viruses and then propagated by passage in young turkeys. Thirty-eight 5-day-old large white turkey poults were inoculated by crop gavage with 500,000 cryptosporidial oocysts and compared with 40 uninoculated poults. Cryptosporidial oocysts shedding began 3 days postinoculation (PI), peaked on day 4 PI, and persisted at a low level for the duration of the 21-day trial. Low to moderate cryptosporidial infections of the ileal mucosa (days 3, 6, and 15 PI), cecal mucosa (days 3, 6, and 21 PI), and bursa of Fabricius (days 6, 12, 15 and 21 PI) were found on histopathological examination. There were no differences in mean body weights between the inoculated and uninoculated groups, and no mortality or clinical signs of disease were seen in either group.

Acquired immunity to amyloodiniosis is associated with an antibody response.

The dinoflagellate Amyloodinium ocellatum, which causes amyloodiniosis or 'marine velvet disease', is one of the most serious ectoparasitic diseases plaguing warmwater marine fish culture worldwide. We report that tomato clownfish Amphiprion frenatus develop strong immunity to Amyloodinium ocellatum infection following repeated nonlethal challenges and that specific antibodies are associated with this response. Reaction of immune fish antisera against dinospore and trophont-derived antigens in Western blots indicated both shared and stage-specific antibody-antigen reactions. A mannan-binding-protein affinity column was used to isolate IgM-like antibody from A. frenatus serum. The reduced Ig consisted of one 70 kD heavy chain and one 32 kD light chain with an estimated molecular weight of 816 kD for the native molecule. Immunoglobulin (Ig) isolated from immune but not non-immune fish serum significantly inhibited parasite infectivity in vitro. An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal rabbit antibody produced against affinity-purified A. frenatus Ig. Anti-Amyloodinium serum antibody was not always detectable in immune fish, although serum antibody titers in immune fish increased after repeated exposure to the parasite. These results suggest that there may be a localized antibody response in skin/gill epithelial tissue, although antibody was rarely detected in skin mucus.

Use of rDNA polymorphism for identification of Heterophyidae infecting freshwater fishes.

Infections by trematodes are among the most common fish-borne zoonoses. Metacercariae of the Family Heterophyidae in marine and freshwater fishes are nonfastidious in their choice of definitive hosts, and therefore, cause infections in human and domestic animals. In the present study, species-specific polymerase chain reaction (PCR) assays were developed for identifying and differentiating the various species examined. Sequencing and aligning the 18S (SSU) rDNA revealed interspecific variation for which species-specific DNA oligonucleotides were designed and used for the identification of 6 heterophyid species recovered from piscivorous birds. The oligonucleotides were further used to evaluate the various stages (cercariae recovered from snails, metacercariae recovered from fish and adult trematodes) of the digeneans. By applying this method we elucidated for the first time the life cycle of Pygidiopsis genata. The phylogenetic interrelationship among the newly sequenced species of Heterophyidae is outlined.