Identification of primitive hematopoietic progenitor cells in the rhesus macaque.

The close phylogenetic relationship of macaques to humans has resulted in their widespread use as a preclinical model for bone marrow transplantation and stem cell gene therapy. To facilitate further use of this model, we undertook analysis of hematopoietic cells using multiparametric flow cytometric analysis. Rhesus CD34+CD38- cells displayed a number of characteristics of primitive hematopoietic cells, including low forward and orthogonal scatter and the lack of expression of lineage-specific markers or human lymphocyte antigen-DR. Four-color flow cytometric analysis demonstrated that rhesus CD34+CD38- cells were heterogenous with respect to Thy-1 expression and were CD59dim. Quantitative limiting dilution long-term culture-initiating cell (LTC-IC) analysis demonstrated that CD34+CD38- cells were approximately 150-fold enriched for LTC-IC as compared with unfractionated bone marrow, and occurred at a frequency similar to that previously reported in humans. Thus, as in humans, the CD34+38- population of rhesus macaque bone marrow is enriched for primitive, multipotent hematopoietic progenitor cells.

Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells.

Genetic modification of hematopoietic stem cells often results in the expression of foreign proteins in pluripotent progenitor cells and their progeny. However, the potential for products of foreign genes introduced into hematopoietic stem cells to induce host immune responses is not well understood. Gene marking and induction of immune responses to enhanced green fluorescent protein (eGFP) were examined in rhesus macaques that underwent nonmyeloablative irradiation followed by infusions of CD34(+) bone marrow cells transduced with a retroviral vector expressing eGFP. CD34(+) cells were obtained from untreated animals or from animals treated with recombinant human granulocyte colony-stimulating factor (G-CSF) alone or G-CSF and recombinant human stem cell factor. Levels of eGFP-expressing cells detected by flow cytometry peaked at 0.1% to 0.5% of all leukocytes 1 to 4 weeks after transplantation. Proviral DNA was detected in 0% to 17% of bone marrow--derived colony-forming units at periods of 5 to 18 weeks after transplantation. However, 5 of 6 animals studied demonstrated a vigorous eGFP-specific cytotoxic T lymphocyte (CTL) response that was associated with a loss of genetically modified cells in peripheral blood, as demonstrated by both flow cytometry and polymerase chain reaction. The eGFP-specific CTL responses were MHC-restricted, mediated by CD8(+) lymphocytes, and directed against multiple epitopes. eGFP-specific CTLs were able to efficiently lyse autologous CD34(+) cells expressing eGFP. Antibody responses to eGFP were detected in 3 of 6 animals. These data document the potential for foreign proteins expressed in CD34(+) hematopoietic cells and their progeny to induce antibody and CTL responses in the setting of a clinically applicable transplantation protocol. (Blood. 2001;97:1951-1959)

Systemic mycosis due to Aspergillus deflectus in a dog.

A 4-year-old, entire female, German Shepherd Dog was referred with a 3-month history of right foreleg lameness that partially responded to nonsteroidal anti-inflammatory and antimicrobial therapy. The bitch lost weight, was polydipsic and had reduced exercise tolerance. On referral, the animal was in poor condition, pyrexic and exhibited moderate pain on full extension of the right shoulder. Blood, urine and joint fluid were obtained and radiographs were taken of the right shoulder and chest. The bitch was lymphopaenic, hyperfibrinogenaemic, hyperglobulinaemic, mildly azotaemic, mildly proteinuric and isosthenuric. Branching fungal hyphae were present in the urine. On radiography, the thorax contained a large ventral mediastinal mass and the humeral head had extensive areas of radiolucency. An aspirate from the right humeroscapular joint exhibited branched fungal hyphae and numerous neutrophils and macrophages. A diagnosis of disseminated mycosis was made and euthanasia was performed. At necropsy, numerous caseating granulomas were present, especially in the kidneys, adrenal glands, heart and lymph nodes. Extensive osteomyelitis involved the head of the right humerus, the sternebrae and the fifth intervertebral disc. Fungal hyphae were detected in sections of granulomas in all affected organs and a diagnosis of disseminated fungal granulomatosis was made. Aspergillus deflectus was readily isolated from affected lymph nodes, but confirming its identity as A deflectus using standard procedures proved difficult. The identity of the fungus was finally confirmed by sequencing part of the 185 rRNA of the isolate. This is the first report in Australia of a disseminated mycosis caused by A deflectus. Previously, the involvement of A deflectus as a cause of disseminated mycosis was limited to 5 cases from the West Coast of the USA, four of which occurred in German Shepherd Dogs.

Mechanisms associated with thymocyte apoptosis induced by simian immunodeficiency virus.

Despite considerable research, the mechanisms by which HIV disrupts thymic function remain controversial. We have described the phenotypic changes that occur in the thymus of SIV-infected macaques during acute SIV infection. In this study, we analyzed the effects of SIV infection on apoptotic pathways in thymic tissue from newborn macaques infected with SIV. Thymocyte apoptosis was accompanied by a modest increase in surface Fas expression, a profound decrease in the frequency of bcl-2-positive cells, as well as the amount of bcl-2 per cell. With control of viral replication, levels of bcl-2 and Fas returned to baseline together with a return to basal levels of apoptosis. In the thymus, SIV infection resulted in depletion of CD4+CD8+ thymocytes, an increase in apoptosis of thymocytes, and a down-regulation of MHC class I molecules. These changes peaked 14-21 days after infection at or just after peak viremia. This data further suggests disruption of the antiapoptotic pathway regulated by bcl-2 plays a critical role in SIV-induced apoptosis of thymocytes.

Natural pathogens of laboratory animals and their effects on research.

The natural fungal pathogens of laboratory animals such as rabbits and guinea pigs are mainly dermatophyte species, most commonly Trichophyton mentagrophytes and also, less frequently Microsporum gypseum and M. canis. However, the incidences of infection and clinical disease are low in well-managed animal facilities. Young or immunocompromised rabbits are thought to be most susceptible. Dermatophytes infect the epidermis and adnexal structures, including hair follicles and shafts, usually on or around the head, and cause pruritus, patchy alopecia, erythema and crusting. Histopathological changes in the underlying skin occur and these changes could confound histological studies involving the skin. Yeast infections usually due to Candida spp. have been reported occasionally in laboratory animals. In this paper, the role of rodents in the evaluation of topical antifungal agents, dermatophytosis and two species of Candida, which are natural pathogens of laboratory animals, are discussed in relation to their effects on research. Pneumocystis carinii, an inhabitant of the respiratory tract of laboratory mice and rats, is a pathogen only under conditions of induced or inherent immunodeficiency. Infected mice and rats are likely to develop severe pneumocystosis following immunosuppression and will be rendered unsuitable for most experimental purposes.

Keratomycosis in a Percheron cross horse caused by Cladorrhinum bulbillosum.

This report describes an infection of a horse's cornea caused by Cladorrhinum bulbillosum. Minor surgery and treatment with antibiotics successfully resolved the infection. The only previous reported case involving this fungus was an Argentinian boy who was infected while working with horses.

Rhinocerebral and nasal zygomycosis in sheep caused by Conidiobolus incongruus.

The clinical findings, pathology and mycology of a cluster of 5 ovine cases of rhinocerebral and nasal zygomycosis caused by Conidiobolus incongruus are described. All cases were in Border Leicester or Merino x Border Leicester ewes from a flock pastured in a low-lying paddock adjoining a small tidal river in subtropical Queensland (latitude 28 degrees S). These cases of zygomycosis are believed to be the first infections due to C incongruus recorded in animals other than humans. The disease was subacute in 4 animals with a course of up to several weeks. In these, the primary site of infection was the posterior nasal cavity. The lesions extended to the dorsum of the face between the eyes, to the orbital cavity and to the anterior brain and meninges in the cranial cavity. In one animal, where the anterior nasal cavity was affected and iodine treatment used, the course was longer. The fungal granulomas had numerous foreign body giant cells, neutrophils and eosinophils. Fungal hyphae were thin walled, 6 to 8 microns in diameter, with occasional septa and irregular branching. They were cuffed with a wide zone of necrotic cell coagulum, or with homogeneous eosinophilic Splendore-Hoeppli granules.

Review of animal mycoses in Australia.

This review covers the available literature on the mycoses of animals in Australia since the last review published in 1967. Of the cutaneous infections, dermatophytoses have been recorded in a wide range of animals: cattle, horses, goats, pigs, sheep, cats, dogs, mice, guinea-pigs, rabbits, a lion, kangaroos, a camel, koalas and wallabies. These infections were caused by several species and varieties of the genera, Microsporum and Trichophyton. Eight agents of ringworms have been recorded in the horse. Two subcutaneous mycoses, phaeohyphomycosis and sporotrichosis have been reported. Phaeohyphomycosis is becoming more common but sporotrichosis is rare having been recorded only once in a cat. The following systemic mycoses have been recorded: adiaspiromycosis, aspergillosis, candidiasis, cryptococcosis, dactylariosis, fusariomycosis, histoplasmosis, miscellaneous mycoses, mycotic abortion and related conditions, zygomycosis, pythiosis, protothecosis and green algal infections. Cryptococcosis has affected 11 different animal species. Mycotic abortion is a serious disease in Victoria. Pythiosis of horses has been extensively studied in northern Australia.

Subcutaneous phaeohyphomycosis caused by Moniliella suaveolens in two cats.

Moniliella suaveolens was isolated in pure culture from histologically typical phaeohyphomycotic granulomas containing dematiaceous fungi in two cats. One cat had several slow-growing black lesions up to 2 cm in diameter in the abdominal subcutis. These lesions recurred after surgical excision was attempted. The second cat had a single black subcutaneous 0.5 X 1.5-cm lesion near one dewclaw. This lesion was successfully removed surgically without recurrence. M. suaveolens has not been isolated previously from lesions in animals including man.

Mycotoxins in animal feeds and toxic fungi in Queensland 1971-80.

During the years 1971 to 1980, 355 samples of animal feeds submitted to this laboratory in connection with suspected mycotoxicoses were analysed for aflatoxins. Most major Queensland agricultural crops were represented. Aflatoxins were detected in 67 samples, 45 of which were peanut meals and by-products (55% of all peanut materials analysed). Of 200 Aspergillus flavus isolates cultured on maize meal, aflatoxins were produced by 49%. Ochratoxin A was detected in one sample of another 25 feeds analysed for the compound. Ochratoxin A was produced by 28% of 47 A. ochraceus isolates, and two Fusarium equiseti isolates produced zearalenone. The present situation with regard to mycotoxins and mycotoxicosis in Queensland is briefly reviewed.

Acute aflatoxicosis in calves fed peanut hay.

Acute aflatoxicosis was believed to be the cause of death of 12 to 90 drought-stricken Hereford calves fed peanut hay on the southern Darling Downs in Queensland. Jaundice, photosensitisation, diarrhoea, anorexia and depression were seen before death. Serum levels of enzymes of hepatic origin and bilirubin were elevated. After death haemorrhage, hepatocyte damage, bile ductule proliferation were found. Total aflatoxin levels up to 2230 microgram/kg were detected in the peanut hay with most toxin concentrated in nut-in-shell.

Mycotic nasal granuloma in cattle.

Seven cases of eosinophilic granulomas containing fungal chlamydospores and short septate hyphae in the submucosa of the anterior nasal cavity were described from Queensland beef cattle during 1966-75. Drechslera rostrata was isolated from 2 cases and unidentified dematiaceous fungi from 2 others.

Eumycotic mycetoma due to Drechslera rostrata infection in a cow.

A case of eumycotic mycetoma with lesions in the skin, nasal cavity and lymph nodes of a cow is reported. The case history, clinical course, macroscopic and microscopic appearance of lesions, and fungal isolation are described. Drechslera rostrata was isolated from pathological tissue and Koch's postulates were satisfied on mouse inoculation. This is the first infection of its type recorded in Australia and features contrasting with cases reported in other countries are discussed.

Canine aflatoxicosis.

Poisoning with aflatoxin derived from mouldy bread was confirmed as the cause of death of one dog and was suspected as the cause of death of two other dogs on the same ration. A jaundiced carcass, firm bile-stained liver and haemorrhage into the gastro-intestinal tract were seen at autopsy. Swelling and foamy vacuolation of hepatocytes due to fatty infiltration, marked perioportal proliferation of bile ductules and some periacinar necrosis were the microscopic changes seen in the liver. Aspergillus flavus was isolated from the mouldy bread and also from a sample of vomitus. Aflatoxin B1, 6.7 ppm, was detected in the mouldy bread and extremely high levels of 100 ppm of aflatoxin B1 and 40 ppm of aflatoxin G1 were present in a sample of vomitus.