The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys (Macaca fascicularis) were exposed by fine-particle aerosol to lethal doses of monkeypox virus, Zaire strain. Death, attributable to fibrinonecrotic bronchopneumonia, occurred 9 to 17 days postexposure. Lower airway epithelium served as the principal target for primary infection. The relative degree of involvement among lymphoid tissues suggested that tonsil, mediastinal, and mandibular lymph nodes were also infected early in the course of the disease, and may have served as additional, although subordinate, sites of primary replication. The distribution of lesions was consistent with lymphatogenous spread to the mediastinal lymph nodes and systemic dissemination of the virus through a monocytic cell-associated viremia. This resulted in lesions affecting other lymph nodes, the thymus, spleen, skin, oral mucosa, gastrointestinal tract, and reproductive system. The mononuclear phagocyte/dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites. Hepatic involvement was uncommon. Lesions at all affected sites were characterized morphologically as necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis. Skin and mucosal surfaces of the respiratory and gastrointestinal tracts also exhibited variable proliferation of epithelial cells and subjacent fibroblasts. Epithelial intracytoplasmic inclusion bodies, consistent with Guarnieri bodies, were usually inconspicuous by light microscopy, but when present, were most readily apparent in the stratified squamous epithelium of the oral mucosa and epidermis. Multinucleated syncytial cells were also occasionally observed in the stratified squamous epithelium of the tongue, tonsil, and skin, and in the intestinal mucosa. Monkeypox virus antigen was readily demonstrated by immunohistochemistry using anti-vaccinia mouse polyclonal antibodies as well as anti-monkeypox rabbit polyclonal antibodies. Detectable poxviral antigen was limited to sites exhibiting obvious morphologic involvement and was most prominent within epithelial cells, macrophages, dendritic cells, and fibroblasts of affected tissues. The presence of poxviral antigen, as determined by immunohistochemistry, correlated with ultrastructural identification of replicating virus. Concurrent bacterial septicemia, present in one monkey, was associated with increased dissemination of the virus to the liver, spleen, and bone marrow and resulted in a more rapidly fatal clinical course.

Evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus infections.

A passive immunization strategy for treating Ebola virus infections was evaluated using BALB/ c mice, strain 13 guinea pigs, and cynomolgus monkeys. Guinea pigs were completely protected by injection of hyperimmune equine IgG when treatment was initiated early but not after viremia had developed. In contrast, mice were incompletely protected even when treatment was initiated on day 0, the day of virus inoculation. In monkeys treated with one dose of IgG on day 0, onset of illness and viremia was delayed, but all treated animals died. A second dose of IgG on day 5 had no additional beneficial effect. Pretreatment of monkeys delayed onset of viremia and delayed death several additional days. Interferon-alpha2b (2 x 10(7) IU/kg/day) had a similar effect in monkeys, delaying viremia and death by only several days. Effective treatment of Ebola infections may require a combination of drugs that inhibit viral replication in monocyte/macrophage-like cells while reversing the pathologic effects (e.g., coagulopathy) consequent to this replication.

Passive immunization of Ebola virus-infected cynomolgus monkeys with immunoglobulin from hyperimmune horses.

A commercially available immunoglobulin G (IgG) from horses, hyperimmunized to Ebola virus, was evaluated for its ability to protect cynomolgus monkeys against disease following i.m. inoculation with 1 000 PFU Ebola virus (Zaire '95 strain). Six monkeys were treated immediately after infection by i.m. infection of 6.0 ml IgG; these animals developed passive ELISA titers of 1:160 to 1:320 to Ebola, two days afer inoculation. However, the beneficial effects of IgG treatment were limited to a delay in onset of viremia and clinical signs, in comparison with untreated controls. The six IgG recipients had no detectable viremia day 5, in contrast with three virus infected controls whose viremias exceeded 7.0 log10 PFU/ml that day. The controls died on days 6, 6, and 7, while two IgG recipients died day 7 and the remaining 4 died day 8, all with high viremias. These results document that passively acquired antibody can have a beneficial effect in reducing the viral burden in Ebola-infected primates; however, effective treatment of human patients may require antibodies with higher specific activities and more favorable pharmacokinetic properties than the presently available equine IgG.

Neurotoxicity in animals due to arteether and artemether.

Several artemisinin (qinghaosu) derivatives have been developed and are in use as antimalarial drugs but scant animal or human toxicity data are available. We noted a progressive syndrome of clinical neurological defects with cardio-respiratory collapse and death in 5/6 dogs dosed daily for 8 d with intramuscular arteether (AE) at 20 mg/kg/d in a pharmacokinetic study. Neurological findings included gait disturbances, loss of spinal reflexes, pain response reflexes and prominent loss of brain-stem and eye reflexes. Electrocardiography showed prolongation of the QT interval corrected for rate (QTc). Prominent neuropathic lesions were sharply limited to the pons and medulla. Neurological injury, graded by a pathologist 'blinded' to dose group, showed a dose-related region-specific injury which was most pronounced in the pons and medulla in all animals. Rats treated with AE and artemether (AM) at 12.5 to 50 mg/kg/d for 28 d confirmed clinical neurological abnormalities with high doses (> 25 mg/kg/d) after 6-14 d. Neuropathological examination of rat brain sections at 5 levels from the rostral cerebrum to the caudal medulla showed a dose-related pattern of injury characterized by hyalinized neuron cell bodies and loss of Nissl substance; changes congruent with those noted in dogs. No significant difference was noted in the extent, type, or distribution of lesions in the brains of rats treated with equivalent doses of AE or AM.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of butorphanol tartrate and buprenorphine hydrochloride on the inflammatory reaction of the Sereny Test.

Invasion of the ocular epithelia of guinea pigs by virulent Shigella organisms, eliciting keratoconjunctivitis, is the basis of the Sereny Test (ST). This test has been used to ascertain the virulence of Shigella strains and more recently to screen candidate Shigella vaccines for efficacy. This test undoubtedly causes pain in test animals; however, recommendation for use of local analgesics/anesthetics has not been accepted because of concern that these topical agents may affect the ability of the Shigella organisms to invade the ocular epithelia or have a physiologic effect on the inflammatory process. Similarly, investigators are hesitant to use systemic analgesics in conjunction with the ST. Two blinded studies were conducted to evaluate the effects of selected systemic analgesics on the ST in outbred Hartley guinea pigs. Study 1 evaluated the recommended dosages for two systemic analgesics; study groups consisted of those receiving butorphanol tartrate (n = 16), those receiving buprenorphine hydrochloride (n = 16), and untreated controls (n = 5). Study 2 evaluated a low-dose buprenorphine hydrochloride group (n = 16) and an untreated control group (n = 5). All animals were inoculated with Shigella flexneri, strain 2a 2457T, onto the cornea and conjunctiva of each eye. At the onset of clinical signs, analgesics were administered to test groups. The degree of keratoconjunctivitis was evaluated per standard procedure; animals were weighed daily. After 7 days, animals were euthanatized and the eyes were removed for histologic morphometric evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)

A surgical technique for bilateral cochleotomy in the Long-Evans rat.

A bilateral cochleotomized surgical rat model, needed for a study involving microwave effects, was developed, standardized, and assessed for reproducibility. After a review of the literature concerning attempts and approaches with various species, a technique involving an approach through the external auditory canal was chosen and modified. Using a stereomicroscope, a cutaneous incision in the intertragic notch was made and extended medially along the ventral aspect of the external auditory canal to the depth of the external auditory meatus. The tympanic membrane was ruptured and the malleus removed with splinter forceps, allowing visualization of the cochlea. The lateral wall of the cochlea was penetrated with a 0.024-in. wire gauge drill bit and endolymph was suctioned from the cochlea. A 5-mm piece of 3-O silk suture, inserted into the cochlear opening, maintained patency. Appraisal of the reliability and standardization of the procedure was performed utilizing startleometry. Histology assessed completeness of the procedure and any evidence of cochlear infection.