Characterization of a new densovirus infecting the German cockroach, Blattella germanica.

A new DNA virus (Parvoviridae: Densovirinae, Densovirus) was isolated and purified from descendants of field-collected German cockroaches, Blattella germanica. Viral DNA and cockroach tissues infected with B. germanica densovirus (BgDNV) were examined by electron microscopy. Virus particles, about 20 nm in diameter, were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule of about 1.2 microm in length. BgDNV isolated from infected cockroaches infected successfully and could be maintained in BGE-2, a B. germanica cell line. The complete BgDNV genome was sequenced and analysed. Five open reading frames (ORFs) were detected in the 5335 nt sequence: two ORFS that were on one DNA strand encoded structural capsid proteins (69.7 and 24.8 kDa) and three ORFs that were on the other strand encoded non-structural proteins (60.2, 30.3 and 25.9 kDa). Three putative promoters and polyadenylation signals were identified. Structural analysis of the inverted terminal repeats revealed the presence of extended palindromes. The genome structure of BgDNV was compared with that of other members of the family Parvoviridae; the predicted amino acid sequences were aligned and subjected to phylogenetic analyses.

Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro.

The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.

High mortality in a large-scale zebrafish colony (Brachydanio rerio Hamilton & Buchanan, 1822) associated with Lecythophora mutabilis (van Beyma) W. Gams & McGinnis.

Zebrafish (Brachydanio rerio) have become an important model system for studying vertebrate embryonic development and gene function through manipulation of genotype and characterization of resultant phenotypes. An established research zebrafish colony without substantial disease problems for more than 7 years of operation began experiencing appreciable mortalities in November of 1997. Young fish (fry), from five to 24 days after hatching, spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore, leading workers in the laboratory to describe the infected fish as "bearded." Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal colonization. The disease was associated with progressive lethargy, reduced feeding, and subsequent mortality. From 10 to 100% of the fry in a given tank were affected. Initial examination indicated that the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans. Environmental samples of air and water in the laboratory were obtained to ascertain the source of the infective agent and to isolate and identify the fungus. A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air. Some biofilm beards on fish were found to consist of relatively pure bacterial populations, and beards on occasional fish examined in the later part of the study consisted of hyphae and spores of the oomycete genus Aphanomyces. Lecythophora mutabilis did not invade tissues; however, elimination of the epizootic correlated with reduction in the number of L. mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems. We conclude that the dense hyphal strands of L. mutabilis composing the predominant biofilm type, along with mixed bacteria and protozoa, contributed to the die-off in young fry by occluding the oral cavity and/or gills, leading to starvation and/or asphyxiation.

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro.

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.

Histologic and immunohistochemical characterization of lens capsular plaques in dogs with cataracts.

OBJECTIVE: To determine histologic and immunohistochemical characteristics of the multifocal adherent plaques that commonly develop on the internal surfaces of the anterior and posterior lens capsules in dogs with cataracts. SAMPLE POPULATION: 31 anterior and 4 posterior capsular specimens collected during lens extraction surgery in dogs with cataracts. PROCEDURE: Specimens were evaluated, using light and transmission electron microscopy. Immunohistochemical techniques were used to localize cytokeratin, vimentin, alpha-smooth muscle-specific actin, fibronectin, tenascin, and transforming growth factor-beta (TGF-beta) within plaques. RESULTS: Histologically, plaques comprised elongated spindle-shaped cells that formed a placoid mass. Cells were embedded in an extracellular matrix containing collagen fibrils, often with duplicated or split basement membranes. Immunohistochemically, normal lens epithelial cells and cells within plaques stained for vimentin. Most cells and some areas of the extracellular matrix within plaques stained for TGF-beta and alpha-smooth muscle-specific actin. Fibronectin and tenascin were also detected in the extracellular matrix. CONCLUSIONS AND CLINICAL RELEVANCE: Canine lens capsular plaques are histologically and immunohistochemically similar to posterior capsule opacification and subcapsular cataracts in humans, which suggests that the canine condition, like the human conditions, is associated with fibrous metaplasia of lens epithelial cells. Transforming growth factor-beta may play a role in the genesis of capsular plaques. Because severity of plaques was correlated with stage of cataract development, earlier surgical removal of cataracts may be useful to avoid complications associated with plaque formation.

Etiologies, observations and reporting of estuarine finfish lesions.

Lesions in estuarine finfish are associated with a variety of organisms including parasites and bacterial, viral, and fungal infectious agents. In addition, trauma, suboptimal water quality, and other abiotic stress factors may result in the loss of homeostasis. We have observed solitary ulcerative lesions on menhaden sampled from the Chesapeake Bay, Maryland, the Pimlico River, North Carolina, and the St. Johns River, Florida. Histologically, the lesions demonstrated a marked chronic inflammatory infiltrate and granulomas in response to fungal hyphae throughout large areas of exposed necrotic muscle. Gram-negative rod-shaped bacteria were also observed in the lesions, a common finding in ulcers of aquatic organisms. Similar observations in menhaden and other species have been described previously in the literature as ulcerative mycosis, mycotic granulomatosis, red spot disease, and epizootic ulcerative syndrome. Despite the many different known causes of fish lesions, the popular press and the scientific literature have recently emphasized Pfiesteria piscicida and other Pfiesteria-like dinoflagellates (and their bioactive compounds) as the primary causative agent for finfish lesions, particularly mycotic granulomatous ulcers in Atlantic menhaden. While some laboratory data suggest that Pfiesteria may play a role in field-observed lesions, much more cause-and-effect evidence is needed to determine the importance of other risk factors, both alone or and in combination with Pfiesteria. In order to better understand the etiology of lesion initiation and progression in estuarine finfish, accurate assessments of environmental conditions collected on appropriate temporal and spatial scales, and fish morphological indicators consistent with gross and histological pathologic terminology, should be used for reporting fish lesion observations and kills. Further, this outlook will help to avoid bias and may foster a broader perspective for examining the health of estuarine systems in general.

A three-year study of viable airborne fungi in the North Carolina Zoological Park R.J.R. Nabisco Rocky Coast Alcid Exhibit.

Aspergillosis is a common cause of mortality in captive birds, particularly in recently imported birds or captive chicks and their parents. Use of the Andersen N-6 single-stage viable air sampler in the North Carolina Zoological Park (NCZP) R.J.R. Nabisco Rocky Coast Alcid Exhibit before and after the introduction of birds allowed a unique study of the mycological content of the air in a developing self-contained ecosystem. The Alcid Exhibit had a median count of 17 colony-forming-units (CFU)/m3 of air in comparison to 200-500 CFU/m3 and 1,000-3,500 CFU/m3 reported in human dwellings and the NCZP R.J. Reynolds Forest Aviary, respectively. Cladosporium and Penicillium represented 21.3% and Aspergillus 1.08% of the fungi collected. During the study, no respiratory mycoses were reported in any of the alcids. Continuous high-efficiency particulate air filtration, maintenance of low exhibit air temperatures, and an environment with little residual organic material capable of supporting fungal growth were important factors contributing to low colony counts. All colony counts >100 CFU/m3 in the exhibit were related to the apparent introduction of fungi from outside the facility. A reduction in the number of fungi transported from an external source into enclosed cool-temperature aviaries may be sufficient to avoid outbreaks of aspergillosis.

A description of cutaneous-subcutaneous pythiosis in fifteen dogs.

Information regarding signalment, duration of clinical signs, history of swimming, results of CBC and serum biochemical analyses, biopsy findings and mycological results, together with treatments and outcome, was retrieved from the medical records of 15 dogs with a diagnosis of pythiosis made between 1985 and 1995 at the Colleges of Veterinary Medicine, North Carolina State University and the University of Florida. Most of the dogs were young (median age 22 months) and represented larger breeds (> 20 kg). Lesions were characteristically chronic, ulcerated, and nodular with multiple draining tracts on the limbs, thoracic wall or perineal regions. The median duration of these lesions was 3 months with a range of 2 weeks-6 months. Seven dogs had a history of swimming. Peripheral eosinophilia was observed in 14 of the dogs. Cytological evaluation of discharge, aspirates, or impression smears made from biopsy specimens revealed hyphae in five of 11 dogs (45%). Histopathological evaluation using the Gomori Methenamine-Silver (GMS) stain was the most useful test for providing presumptive evidence of cutaneous pythiosis. Immunotherapy or antifungal therapy using either amphotericin B, liposomal nystatin, itraconazole, or ketoconazole were all unsuccessful. The only dog to survive underwent amputation of the affected limb; thus, the prognosis for cutaneous pythiosis in the dog is poor.

Effect of alpha-chymotrypsin on breaking strength and ultrastructural morphology of canine ciliary zonules.

OBJECTIVE: To determine the effect of alpha-chymotrypsin treatment on breaking strength and ultrastructural morphology of canine ciliary zonules. SAMPLE POPULATION: Eyes from young random-source dogs from an animal shelter. PROCEDURE: Eyes were obtained immediately after euthanasia of dogs. The enzyme alpha-chymotrypsin was applied to the ciliary zonules of 1 eye of each dog; the other eye was treated with saline solution as a control. The breaking strength of ciliary zonules was measured, using a linear actuator and force transducer. The lenses and ciliary bodies were then analyzed by scanning and transmission electron microscopy. RESULTS: alpha-Chymotrypsin reduced the breaking strength of ciliary zonules by a mean +/- SD 44 (+/- 20)%, compared with that for saline-treated control eyes. Increasing the volume of enzyme further decreased the breaking strength of the zonules. Differences in the appearance of the ciliary body by electron microscopy were not apparent between enzyme- and saline-treated specimens. CONCLUSIONS AND CLINICAL RELEVANCE: Application of alpha-chymotrypsin to enucleated canine eyes at a concentration used in people significantly reduces the breaking strength of canine ciliary zonules without any apparent damage to the ciliary body. alpha-Chymotrypsin may be useful in the removal of subluxated canine lenses and in removal of cataractous lenses in young dogs, in which phacoemulsification often results in appreciable post operative capsular opacification.

A comparison of sampling methods for airborne fungal spores during an outbreak of aspergillosis in the forest aviary of the North Carolina Zoological Park.

An outbreak of aspergillosis with the death of six birds in the North Carolina Zoological Park R. J. Reynolds Forest Aviary in the spring of 1993 led to an investigation of the concentration of Aspergillus fumigatus spores in the air. No Aspergillus sp. was found in the facility through use of the drop plate method (gravitometric sampling) along with swab-sampling of selected surfaces within the exhibit and plating of food samples and nesting material onto petri dishes of nutrient media. A number factors that could stress the avian population were identified. These included excessive heat in the upper portion of the aviary due to the failure of an air handling system, a malfunctioning cooling tower, and large numbers of visitors to the facility (an average of 3,500/day). In addition, the outbreak occurred during a period of increased nesting behavior. Sampling of the fungal population of the air was conducted 1 year later, when no disease was noted, to compare the sensitivity of the commonly used drop plate method (open plates of nutrient media) with a volumetric impaction method (Andersen N-6 Air Sampler). The volumetric method delivered quantitative as well as qualitative data and exhibited more sensitivity for fungal spores of size similar to those of Aspergillus sp.

The experimental antipsychotic agent 1192U90 targets tapetum lucidum in canine eyes.

To assess the potential adverse effects in people of the antipsychotic agent 1192U90, we dosed mice, rats, beagles, and cynomolgus monkeys for up to 3 mo. In dogs, but not the other species, 1192U90 caused ocular changes detectable ophthalmoscopically as loss of tapetal reflectivity, altered tapetal color, and the appearance of black pigmentation on the tapetal fundus. Eyes from affected dogs had atrophic tapeta lucidum due to cell loss. Rodlets in remaining tapetal cells were separated by electron-lucent spaces or finely granular material, varied in size and shape, and often contained irregularly shaped electron-dense inclusions. Nontapetal ocular structures were unaffected. Because 1192U90 caused no ocular changes in nontapetal species, we hypothesized that it targeted only tapetum lucidum and spared other ocular structures. We tried to test this hypothesis by dosing congenitally atapetal dogs; however, although these dogs were ophthalmoscopically "atapetal," they had scattered tapetal cells visible by electron microscopy, and these tapetal cells had ultrastructural changes indistinguishable from those that occurred in treated normal-eyed dogs. Tapetal degeneration caused by 1192U90 resembled that described in hereditary tapetal degeneration in beagles. That 1192U90 caused no ocular changes in nontapetal species suggests that the ocular changes in dogs do not imply a risk for humans, whose eyes also lack a tapetum lucidum.

Lens capsule opacification in aphakic and pseudophakic eyes.

BACKGROUND: Posterior capsule opacification (PCO) is the most common complication of lens extraction. Although intraocular lenses (IOLs) are thought to inhibit capsule opacification, the mechanisms by which they do this are poorly understood. This study was done to determine the effects of pseudophakia on secondary cataract and PCO in experimentally lentectomized dogs. METHODS: Twenty-four normal dogs were bilaterally lentectomized by phacoemulsification and unilaterally implanted with a plano-convex polymethylmethacrylate IOL. Secondary cataracts and capsule opacification were evaluated at weeks 1, 2, 4, 10, 14, and 20 after surgery by retrolillumination photography, light microscopy, and scanning and transmission electron microscopy. RESULTS: The pattern of secondary cataract and PCO in dogs was found to be similar to that in other animal species. Production of new lens material was prominent in the equatorial region, and PCO resulted from fibrous metaplasia of lens epithelium and subsequent capsular fibrosis and wrinkling. The presence of an IOL did not prevent the posterior migration of epithelium, nor did it prevent fibrous metaplasia. The presence of an IOL did, however, minimize the capsule-wrinkling effects of fibroplasia and limit the space available for lentoid formation. CONCLUSION: In pseudophakic eyes, IOLs influence secondary cataract formation by limiting the space available for lentoid formation and by maintaining a linear scaffolding for lens epithelial fibrous metaplasia.

Notexin-induced muscle injury in the dog.

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is a spontaneous, X-linked, progressively fatal disease of dogs and is also a homologue of Duchenne muscular dystrophy (DMD). Two-thirds of DMD patients carry detectable deletions in their dystrophin gene. The defect underlying the remaining one-third of DMD patients is undetermined. Analysis of the canine dystrophin gene in normal and GRMD dogs has failed to demonstrate any detectable loss of exons. Here, we have demonstrated a RNA processing error in GRMD that results from a single base change in the 3' consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. This is the first example of dystrophin deficiency caused by a splice-site mutation.

An epizootic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiobolus ranarum.

Morbidity and mortality approaching 100% occurred in dwarf African clawed frogs (Hymenochirus curtipes) from a culture facility in central California. Moribund frogs exhibited preference for a terrestrial environment rather than their normal aquatic environment. Affected animals had a slight pallor of the integument but were otherwise grossly unremarkable. Microscopic examination revealed a fungal infection of the integument primarily characterized by the presence of surface and intra-epidermal conidia. Skin cultures of the infected animals yielded an organism identified as Basidiobolus ranarum, based on the formation of conidia in culture with internal cleavage to form sporangiospores. The organism was transmitted to healthy frogs by co-habitation with infected frogs but not by short-term immersion exposure of healthy frogs to homogenized broth cultures of the fungus. Benzalkonium chloride at 2.0 mg l-1 was efficacious in controlling the infection. Although Basidiobolus species are normally found in the intestinal tract of amphibians, the severity of this epizootic indicates that B. ranarum may be an important pathogen of amphibians reared in culture facilities.

Attenuation of Borrelia anserina by serial passage in liquid medium.

Borrelia anserina (Sakharoff) was successfully grown in a liquid medium (Barbour-Stoenner-Kelly) for 39 passages. By the 12th serial passage in medium, infectivity of B anserina for chicks was lost. Electron microscopy did not reveal structural differences between non-infective and infective cultured organisms. Changes in the protein profiles were found by electrophoresis as the organisms were passed in culture.

Chronic inflammatory cells with epithelial cell characteristics in teleost fishes.

Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. "Mature granulomas" of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation.

Scedosporium inflatum, an emerging pathogen.

The salient morphologic and physiologic characteristics of 18 isolates of Scedosporium inflatum, a newly reported human pathogen, were compared with those of the morphologically similar fungi Scedosporium apiospermum, Scopulariopsis brevicaulis, and Scopulariopsis brumptii. The formation by S. inflatum of annelloconidia in wet clumps at the apices of annellides with swollen bases was found to be the most useful characteristic in differentiating this potential pathogen.

A quantitative study of single and mixed infection of the chicken trachea by Mycoplasma gallisepticum.

The interaction between Mycoplasma gallisepticum (MG) and the tracheal mucosa of the young chicken was studied. The use of a selective plating method permitted differentiation between a pathogenic tylosin-resistant strain (227) and a less pathogenic tylosin-sensitive vaccine strain (F). Both MG strains adhered to the tracheal mucosa and colonized equally well. In mixed infection, the presence or absence of the second strain did not change the efficiency of colonization by either strain. When chickens were exposed to the vaccine strain 24 hr or 2 weeks before superinfection by the pathogen, there was no significant reduction in the efficiency of superinfection, despite the presence of 10(6) colony-forming units of MG strain F in the trachea. However, chickens had an increased ability to resist superinfection 5 weeks after exposure via the air sac. These results suggest that the biological mechanism underlying protection of F-strain-vaccinated chickens against adventitious infection by the homologous species does not involve competition for adherence sites or blockage by prior colonization.

Comparison of guinea pig and protozoan models for determining virulence of Legionella species.

Legionella pneumophila organisms are able to infect and multiply within the ciliated protozoan Tetrahymena pyriformis. This ability may be associated with virulence, because an attenuated strain of L. pneumophila fails to multiply within this protozoan, whereas a virulent strain increases 10,000-fold in number when coincubated with T. pyriformis. Seventeen strains (11 species) of legionellae were evaluated for virulence by intraperitoneal injection of guinea pigs and inoculation of protozoan cultures. Analysis of the data indicates that there are four categories of legionellae with respect to virulence as follows: organisms that infect and kill guinea pigs and multiply in T. pyriformis; organisms that infect but do not kill guinea pigs and multiply in T. pyriformis; organisms that do not infect guinea pigs but are lethal at high concentrations and multiply in T. pyriformis; and organisms that neither infect nor kill guinea pigs and fail to multiply in T. pyriformis. Evidence suggests that these distinctions are based on two virulence factors: intracellular multiplication in a host and toxic activity.