Retrospective detection of monkeypox virus in the testes of nonhuman primate survivors.

Close contact through sexual activity has been associated with the spread of monkeypox virus (MPXV) in the ongoing, global 2022 epidemic. However, it remains unclear whether MPXV replicates in the testes or is transmitted via semen to produce an active infection. We carried out a retrospective analysis of MPXV-infected crab-eating macaque archival tissue samples from acute and convalescent phases of infection of clade I or clade II MPXV using immunostaining and RNA in situ hybridization. We detected MPXV in interstitial cells and seminiferous tubules of testes as well as epididymal lumina, which are the sites of sperm production and maturation. We also detected inflammation and necrosis during the acute phase of the disease by histological analysis. Finally, we found that MPXV was cleared from most organs during convalescence, including healed skin lesions, but could be detected for up to 37 d post-exposure in the testes of convalescent macaques. Our findings highlight the potential for sexual transmission of MPXV in humans.

Three methods for integration of environmental risk into the benefit-risk assessment of veterinary medicinal products.

Veterinary medicinal products (VMPs) require, as part of the European Union (EU) authorization process, consideration of both risks and benefits. Uses of VMPs have multiple risks (e.g., risks to the animal being treated, to the person administering the VMP) including risks to the environment. Environmental risks are not directly comparable to therapeutic benefits; there is no standardized approach to compare both environmental risks and therapeutic benefits. We have developed three methods for communicating and comparing therapeutic benefits and environmental risks for the benefit-risk assessment that supports the EU authorization process. Two of these methods support independent product evaluation (i.e., a summative classification and a visual scoring matrix classification); the other supports a comparative evaluation between alternative products (i.e., a comparative classification). The methods and the challenges to implementing a benefit-risk assessment including environmental risk are presented herein; how these concepts would work in current policy is discussed. Adaptability to scientific and policy development is considered. This work is an initial step in the development of a standardized methodology for integrated decision-making for VMPs.

Protective effect of two recombinant ricin subunit vaccines in the New Zealand white rabbit subjected to a lethal aerosolized ricin challenge: survival, immunological response, and histopathological findings.

Ricin, isolated from the castor bean plant Ricinus communis, is included on the Centers for Disease Control and Prevention Category B list of bioterrorism agents, indicating that the toxin is moderately easy to disseminate and could result in moderate morbidity rates. This study evaluated two promising recombinant ricin subunit vaccines, one made using an Escherichia coli codon-optimized gene and the other using a yeast codon-optimized gene in E. coli-based fermentations. Rabbits were vaccinated four times over a period of 6 months and challenged with ∼10 to 30 times the median lethal dose of aerosolized ricin. All unvaccinated control rabbits were either found dead or humanely euthanized within 30 h postchallenge, while the rabbits vaccinated with either vaccine survived the exposure without adverse clinical signs. When the protective antibody responses were analyzed, no significant difference was seen between the two vaccines. However, there was a significant difference in the immune response over time for both vaccines tested. Although clinical pathology was unremarkable, significant histological lesions in the control animals included fibrinonecrotic pneumonia, acute necrotizing lesions in the upper respiratory tract, and necrotizing lymphadenitis in the lymph nodes draining the upper and lower respiratory tract. Vaccine-treated rabbits exhibited resolving lesions associated with ricin exposure, namely chronic inflammation in the upper respiratory tract and lungs, fibrosis, type II pneumocyte hyperplasia, and bronchiolitis obliterans. This study confirmed the safety and efficacy of two recombinant ricin subunit vaccines in rabbits, offering potential protection to warfighters and select populations.

The pathogenesis of Rift Valley fever virus in the mouse model.

Detailed studies describing the pathogenesis of Rift Valley fever (RVF) virus (RVFV) in the mouse model are lacking. A fully characterized small animal model of RVF is needed to evaluate potential vaccines and therapeutics. In this study, we characterized the pathogenesis of RVFV throughout the disease course in mice. Infection produced high-titer viremia and demonstrated RVFV tropism for a variety of tissue and individual cell types. Overwhelming infection of hepatocytes, accompanied by apoptosis, was a major consequence of infection. The majority of mice died or were euthanatized between days 3 and 6 postinfection with severe hepatitis. The remaining mice effectively cleared virus from the liver and blood, but exhibited neuroinvasion and developed panencephalitis. In addition, we characterized a number of other virological, clinicopathological, and histopathological features of RVFV infection in mice. The mouse model therefore mimics both the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF.

Experimental infection of cynomolgus macaques (Macaca fascicularis) with aerosolized monkeypox virus.

Monkeypox virus (MPXV) infection in humans results in clinical symptoms very similar to ordinary smallpox. Aerosol is a route of secondary transmission for monkeypox, and a primary route of smallpox transmission in humans. Therefore, an animal model for aerosol exposure to MPXV is needed to test medical countermeasures. To characterize the pathogenesis in cynomolgus macaques (Macaca fascicularis), groups of macaques were exposed to four different doses of aerosolized MPXV. Blood was collected the day before, and every other day after exposure and assessed for complete blood count (CBC), clinical chemistry analysis, and quantitative PCR. Macaques showed mild anorexia, depression, and fever on day 6 post-exposure. Lymphadenopathy, which differentiates monkeypox from smallpox, was observed in exposed macaques around day 6 post-exposure. CBC and clinical chemistries showed abnormalities similar to human monkeypox cases. Whole blood and throat swab viral loads peaked around day 10, and in survivors, gradually decreased until day 28 post-exposure. Survival was not dose dependent. As such, doses of 4 × 10(4) PFU, 1 × 10(5) PFU, or 1 × 10(6) PFU resulted in lethality for 70% of the animals, whereas a dose of 4 × 10(5) PFU resulted in 85% lethality. Overall, cynomolgus macaques exposed to aerosolized MPXV develop a clinical disease that resembles that of human monkeypox. These findings provide a strong foundation for the use of aerosolized MPXV exposure of cynomolgus macaques as an animal model to test medical countermeasures against orthopoxviruses.

Beyond-use dating of extemporaneously compounded ketamine, acepromazine, and xylazine: safety, stability, and efficacy over time.

Ketamine-acepromazine-xylazine (KAX) has long been a popular combination of injectable anesthetics for use in laboratory rodents. These drugs are compounded extemporaneously at research facilities because a commercial mixture is not available. This study was designed to determine an appropriate period of use for this mixture by examining its safety, stability, and efficacy at 30-d intervals over an aging period of 270 d. For as long as 270 d after compounding, most of the data collected (chemical stability, sterility, pH, particulate formation, times to loss of righting reflex in injected mice and rats, and histopathology from these animals) supported the finding that the component drugs do not change or degrade. However, mice and rats did show significant differences in anesthetic responses after injection with KAX mixtures of different ages. In light of these findings, we suggest that KAX remains safe, stable, and efficacious for at least 180 d after mixing, and that 180 d constitutes an appropriate period of use for this drug combination when stored in a dark, room-temperature environment.

Effect of antibiotic pretreatment on cerebrospinal fluid profiles of children with bacterial meningitis.

OBJECTIVE: The goal of this study was to evaluate the effect of antibiotic administration before lumbar puncture on cerebrospinal fluid profiles in children with bacterial meningitis. METHODS: We reviewed the medical records of all children (1 month to 18 years of age) with bacterial meningitis who presented to 20 pediatric emergency departments between 2001 and 2004. Bacterial meningitis was defined by positive cerebrospinal fluid culture results for a bacterial pathogen or cerebrospinal fluid pleocytosis with positive blood culture and/or cerebrospinal fluid latex agglutination results. Probable bacterial meningitis was defined as positive cerebrospinal fluid Gram stain results with negative results of bacterial cultures of blood and cerebrospinal fluid. Antibiotic pretreatment was defined as any antibiotic administered within 72 hours before the lumbar puncture. RESULTS: We identified 231 patients with bacterial meningitis and another 14 with probable bacterial meningitis. Of those 245 patients, 85 (35%) had received antibiotic pretreatment. After adjustment for patient age, duration and severity of illness at presentation, and bacterial pathogen, longer duration of antibiotic pretreatment was not significantly associated with cerebrospinal fluid white blood cell count, cerebrospinal fluid absolute neutrophil count. However, antibiotic pretreatment was significantly associated with higher cerebrospinal fluid glucose and lower cerebrospinal fluid protein levels. Although these effects became apparent earlier, patients with >or=12 hours of pretreatment, compared with patients who either were not pretreated or were pretreated for <12 hours, had significantly higher median cerebrospinal fluid glucose levels (48 mg/dL vs 29 mg/dL) and lower median cerebrospinal fluid protein levels (121 vs 178 mg/dL). CONCLUSIONS: In patients with bacterial meningitis, antibiotic pretreatment is associated with higher cerebrospinal fluid glucose levels and lower cerebrospinal fluid protein levels, although pretreatment does not modify cerebrospinal fluid white blood cell count or absolute neutrophil count results.

Clinical prediction rule for identifying children with cerebrospinal fluid pleocytosis at very low risk of bacterial meningitis.

CONTEXT: Children with cerebrospinal fluid (CSF) pleocytosis are routinely admitted to the hospital and treated with parenteral antibiotics, although few have bacterial meningitis. We previously developed a clinical prediction rule, the Bacterial Meningitis Score, that classifies patients at very low risk of bacterial meningitis if they lack all of the following criteria: positive CSF Gram stain, CSF absolute neutrophil count (ANC) of at least 1000 cells/microL, CSF protein of at least 80 mg/dL, peripheral blood ANC of at least 10,000 cells/microL, and a history of seizure before or at the time of presentation. OBJECTIVE: To validate the Bacterial Meningitis Score in the era of widespread pneumococcal conjugate vaccination. DESIGN, SETTING, AND PATIENTS: A multicenter, retrospective cohort study conducted in emergency departments of 20 US academic medical centers through the Pediatric Emergency Medicine Collaborative Research Committee of the American Academy of Pediatrics. All children aged 29 days to 19 years who presented at participating emergency departments between January 1, 2001, and June 30, 2004, with CSF pleocytosis (CSF white blood cells > or =10 cells/microL) and who had not received antibiotic treatment before lumbar puncture. MAIN OUTCOME MEASURE: The sensitivity and negative predictive value of the Bacterial Meningitis Score. RESULTS: Among 3295 patients with CSF pleocytosis, 121 (3.7%; 95% confidence interval [CI], 3.1%-4.4%) had bacterial meningitis and 3174 (96.3%; 95% CI, 95.5%-96.9%) had aseptic meningitis. Of the 1714 patients categorized as very low risk for bacterial meningitis by the Bacterial Meningitis Score, only 2 had bacterial meningitis (sensitivity, 98.3%; 95% CI, 94.2%-99.8%; negative predictive value, 99.9%; 95% CI, 99.6%-100%), and both were younger than 2 months old. A total of 2518 patients (80%) with aseptic meningitis were hospitalized. CONCLUSIONS: This large multicenter study validates the Bacterial Meningitis Score prediction rule in the era of conjugate pneumococcal vaccine as an accurate decision support tool. The risk of bacterial meningitis is very low (0.1%) in patients with none of the criteria. The Bacterial Meningitis Score may be helpful to guide clinical decision making for the management of children presenting to emergency departments with CSF pleocytosis.

Challenges in biodefense research and the role of US Army veterinary pathologists.

For years the nation's development of medical countermeasures to biowarfare agents has primarily existed as the domain of the United States military, but it has taken on increased urgency in the last few years. The realization that the civilian population is also at risk from biological agents has resulted in the institution of new biodefense programs at a variety of nonmilitary organizations. USAMRIID, a long-time leader in the nation's biodefense effort, will soon be joined by other US government agencies as part of a planned National Interagency Biodefense Campus at Fort Detrick Maryland. US Army veterinary pathologists at USAMRIID have played an important role in the nation's biodefense effort, along with our veterinary colleagues representing other specialties, our military colleagues in other Army Medical Department corps, and our civilian colleagues. Together, we will continue to strive to develop the diagnostics, vaccines, therapeutic agents, and operational practices that are required to meet the great demands posed by the threat of biowarfare agents.

Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum infections in dogs.

Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii, and Neospora caninum are related apicomplexans that can cause systemic illness in many species of animals, including dogs. We investigated one breeder's 25 Basset Hounds for these infections. In addition, tissues from dogs and other non-canine hosts previously reported as S. canis infections were studied retrospectively. Schizonts resembling those of S. neurona, and recognized by polyclonal rabbit anti-S. neurona antibodies, were found in six of eight retrospective cases, as well as in two additional dogs (one Basset Hound, one Springer Spaniel) not previously reported. S. neurona schizonts were found in several tissues including the central nervous system, lungs, and kidneys. Fatal toxoplasmosis was diagnosed in an adult dog, and neosporosis was diagnosed in an adult and a pup related to the one diagnosed with S. neurona. No serological reactivity to S. neurona antibodies occurred when S. canis-like liver schizonts were retrospectively assayed from two dogs, a dolphin, a sea lion, a horse, a chinchilla, a black or either of two polar bears. Sequencing conserved (18S) and variable (ITS-1) portions of nuclear ribosomal DNA isolated from the schizont-laden liver of a polar bear distinguished it from all previously characterized species of Sarcocystis. We take this genetic signature as provisionally representative of S. canis, an assumption that should be tested with future sequencing of similar liver infections in other mammalian hosts. These findings further extend the uncharacteristically broad intermediate host range for S. neurona, which also causes a neurologic disease in cats, mink, raccoons, skunks, Pacific harbor seals, ponies, zebras, lynxes, and sea otters. Further work is necessary to delineate the causative agent(s) of other cases of canine sarcocystosis, and in particular to specify the attributes of S. canis, which corresponds morphologically to infections reported from wide range of terrestrial and marine mammals.

Intrarenal pelvic nephroblastoma in a meerkat (Suricata suricatta).

Nephroblastoma is the most common primary renal tumor in children and has also been reported in domestic and nondomestic animal species. Intrapelvic renal nephroblastoma is a rare variant of this tumor type in human patients. Postmortem examination of a captive meerkat (Suricata suricatta), which was found dead, revealed enlargement of the pelvis of the left kidney by a tumor mass. Gross, histological, and immunohistochemical findings were consistent with a diagnosis of triphasic intrapelvic renal nephroblastoma. This is the first reported spontaneous case of intrapelvic renal nephroblastoma in a nonhuman species.