Distinct transcriptional responses to fatal Ebola virus infection in cynomolgus and rhesus macaques suggest species-specific immune responses.

Ebola virus (EBOV) is a negative single-stranded RNA virus within the Filoviridae family and the causative agent of Ebola virus disease (EVD). Nonhuman primates (NHPs), including cynomolgus and rhesus macaques, are considered the gold standard animal model to interrogate mechanisms of EBOV pathogenesis. However, despite significant genetic similarity (>90%), NHP species display different clinical presentation following EBOV infection, notably a ∼1-2 days delay in disease progression. Consequently, evaluation of therapeutics is generally conducted in rhesus macaques, whereas cynomolgus macaques are utilized to determine efficacy of preventative treatments, notably vaccines. This observation is in line with reported differences in disease severity and host responses between these two NHP following infection with simian varicella virus, influenza A and SARS-CoV-2. However, the molecular underpinnings of these differential outcomes following viral infections remain poorly defined. In this study, we compared published transcriptional profiles obtained from cynomolgus and rhesus macaques infected with the EBOV-Makona Guinea C07 using bivariate and regression analyses to elucidate differences in host responses. We report the presence of a shared core of differentially expressed genes (DEGs) reflecting EVD pathology, including aberrant inflammation, lymphopenia, and coagulopathy. However, the magnitudes of change differed between the two macaque species. These findings suggest that the differential clinical presentation of EVD in these two species is mediated by altered transcriptional responses.

Pterygodermatites nycticebi infections in golden lion tamarins (Leontopithecus rosalia rosalia) and aye-ayes (Daubentonia madagascariensis) from a German zoo.

In a golden lion tamarin (Leontopithecus rosalia rosalia) colony kept indoors in a German zoo, two animals presented a sudden onset of reduced general condition, lethargy, and diarrhea. At animal capture for clinical examination, adult nematode stages were observed after stress-induced defecation. Despite treatment, two golden lion tamarins died in the following 2 days. At necropsy, spirurid stages were found in the lungs and intestine. Additionally, adult Pterygodermatites spp. were identified in histopathological samples of intestine and pancreas, confirming the previous diagnosis. Upon diagnosis, all animals were treated with ivermectin (0.2 mg/kg; SC). Thereafter, the general condition of the golden lion tamarins improved, whereby some of them excreted spirurid nematodes over 3 days. Four weeks after treatment, 20 fecal samples from the colony were examined and proved negative for parasitic stages. Given that common German cockroaches (Blattella germanica) are suitable intermediate hosts of Pterygodermatites nycticebi, 30 specimens were collected from seven different locations around the golden lion tamarins housing. Third-stage larvae of Pterygodermatites spp. were recovered from those cockroaches. Regular anthelmintic treatments, coprological screenings, and controls for intermediate hosts were recommended. More than 2 years later, P. nycticebi infection was diagnosed again histopathologically in an aye-aye (Daubentonia madagascariensis) which suddenly died. Coprological analysis confirmed the presence of spirurid eggs. Due to prosimian primates' cockroach-eating habits and given that total cockroach eradication proved impossible, continuous cockroach control strategies and regular treatments of primates are currently performed to prevent further P. nycticebi infections.

Remdesivir (GS-5734) Is Efficacious in Cynomolgus Macaques Infected With Marburg Virus.

Marburg virus (MARV) is a filovirus with documented human case-fatality rates of up to 90%. Here, we evaluated the therapeutic efficacy of remdesivir (GS-5734) in nonhuman primates experimentally infected with MARV. Beginning 4 or 5 days post inoculation, cynomolgus macaques were treated once daily for 12 days with vehicle, 5 mg/kg remdesivir, or a 10-mg/kg loading dose followed by 5 mg/kg remdesivir. All vehicle-control animals died, whereas 83% of animals receiving a 10-mg/kg loading dose of remdesivir survived, as did 50% of animals receiving a 5-mg/kg remdesivir regimen. Remdesivir-treated animals exhibited improved clinical scores, lower plasma viral RNA, and improved markers of kidney function, liver function, and coagulopathy versus vehicle-control animals. The small molecule remdesivir showed therapeutic efficacy in this Marburg virus disease model with treatment initiation 5 days post inoculation, supporting further assessment of remdesivir for the treatment of Marburg virus disease in humans.

Neutralization of pertussis toxin by a single antibody prevents clinical pertussis in neonatal baboons.

Pertussis continues to cause considerable infant mortality world-wide, which could be addressed in part by passive immunization strategies. Antibody hu1B7 is a candidate therapeutic that potently neutralizes pertussis toxin in vitro, prevents leukocytosis in mice and treats established disease in weanling baboons as part of an antibody cocktail. Here, we evaluated the potential for hu1B7 and an extended half-life hu1B7 variant to prevent death, leukocytosis and other clinical symptoms in a newborn baboon model that mimics many aspects of human disease. We administered a single antibody dose to newborn baboons five weeks prior to experimental infection. While all animals were heavily colonized with Bordetella pertussis, prophylaxed animals showed significantly greater survival (P < 0.005), delayed and suppressed leukocytosis (P < 0.01) and enhanced clinical outcomes, including coughing (P < 0.01), as compared to controls. Together, this work demonstrates that a single neutralizing anti-PTx antibody is sufficient to prevent clinical pertussis symptoms.

RETROSPECTIVE MORTALITY REVIEW OF SIX CALLITRICHID SPECIES HOUSED AT A SINGLE INSTITUTION (1990-2014).

Callitrichids are commonly exhibited species in zoological institutions. Retrospective mortality studies allow institutions to evaluate health trends in captive populations and have the potential to advance zoological medicine and husbandry practices. In this single institution, retrospective study, histopathologic records were reviewed for 166 callitrichids comprising six species. For all species and age-groups combined, trauma was the leading cause of death followed by enteritis and stillbirth. In adults and juveniles, the leading cause of death was enteritis, with neonates dying primarily from trauma. Neonatal mortality was high for most species, ranging from 12.5% to 60%, with most neonatal loss in Geoffroy's and pied tamarins. Twin births were commonly associated with neonatal mortality in most species. Intestinal adenocarcinoma was a common finding for adult Geoffroy's tamarin, and hemosiderosis was also identified in this species. Renal disease was a common finding in adult callitrichids and was the second most common cause of death in this age-group. Mortality in the juvenile period (>30 days-1 yr) was uncommon, comprising only 8.4% of the data set. Although capable of surviving into advanced age (>20 yr for one animal), the average age at death for all age classes was 3.84 yr and 9.31 yr for adults. This study contributes to the current knowledge regarding trends in callitrichid health and husbandry and can be used to investigate health trends in this group.

Prevalence and spatial distribution of Ixodid tick populations in the forest fringes of Western Ghats reported with human cases of Kyasanur forest disease and monkey deaths in South India.

Kyasanur forest disease (KFD) is a major tick-borne viral haemorrhagic fever caused by KFD virus (KFDV) (Flaviviridae). The disease was reported to be confined to five districts of Karnataka state India until 2011. During 2012-2016, emergence of KFD has been reported in newer areas of Karnataka and adjoining states. Therefore, survey of tick vectors was carried out in these new areas of Karnataka and adjoining states reported with monkey deaths and human cases of KFD. In all selected sites, ticks from the forest floor were collected by lint clothes using flagging method. Tick samples were tested for KFDV nucleic acid by real-time RT-PCR. A total of 4772 ticks, comprising eight species of genus Haemaphysalis and one species each of genus Amblyomma, Ixodes and Rhipicephalus was collected. Haemaphysalis spinigera, the principal vector of KFDV was the predominant tick species (59.5%) collected followed by H. turturis (8.6%). The abundance of H. spinigera ranged from 9.2 to 33.9 per man-hour in the six districts surveyed. Of 214 (4418 tick samples) pools screened by real-time RT-PCR, two pools of H. spinigera were positive for KFDV. High abundance of Haemaphysalis vectors in the six districts indicated that the districts are receptive for KFD outbreaks. KFDV was detected in the tick vectors in the new foci of the KFD. Data on tick distribution will be useful in creating KFD risk map for strengthening the ongoing preventive measures such as vaccination and supply of insect repellents to the high risk groups and intensive health education.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1.

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Intratracheal exposure of common marmosets to MERS-CoV Jordan-n3/2012 or MERS-CoV EMC/2012 isolates does not result in lethal disease.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT), with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication.

Seasonal electrocution fatalities in free-range rhesus macaques (Macaca mulatta) of Shivalik hills area in northern India.

BACKGROUND: The arboreal nature of rhesus macaques makes them vulnerable to electrocution from high-voltage industrial supply as well as low-tension domestic wires. The data on pattern of electrocution injuries and electrocution mortalities in free-ranging rhesus macaques are rarely reported. METHODS: From January 2011 to 2013, the cases of accidental electrocution injuries were recorded in difference to sex, age group, season, body region distribution, contact details, and extent of injuries in free-ranging rhesus macaques (Macaca mulatta) of Shivalik hill areas. RESULTS: electrocution injuries were highest in juveniles (52.05%), followed by adults (30.13%) and old (10.95%) rhesus macaques. Of the 73 cases recorded, 23 (31.50%) succumbed to death following severe electrocution and associated injuries. The intensity and body region distribution of electrocution injuries was recorded. CONCLUSION: High-voltage current is the most common factor for fatal injuries. Strategies to avoid such electrocutions include burying all lines and transformers, shielding and insulating all high-tension lines, installing bridges that monkeys, and other arboreal species can use to move around.

Apparent mortality as a result of an elevated parasite infection in Presbytis rubicunda.

We provide the first parasite assessment of wild red langurs (Presbytis rubicunda). Seven helminth taxa and 4 protozoa were recovered from faecal samples. We report the details of the death of an adult female red langur with potential association of an elevated parasite infection. There is a paucity of published literature on red langurs and nothing is known of the factors affecting interspecific parasite transmission and disease spread between other primate species. Parasites may be substantial determinants of host health and may present a significant influence on the survival and reproduction of individuals and the subsequent fecundity of populations. Further research into the connection of parasite burden with individual deaths and potential population declines is recommended for monitoring population health.

Mortality in captive baboons (Papio spp.): a-23-year study.

BACKGROUND: We report the causes of mortality for 4350 captive baboons that died or were euthanized due to natural causes during a 23 year period at the Southwest National Primate Research Center. METHODS: Necropsy records were retrieved and reviewed to determine a primary cause of death or indication for euthanasia. Data was evaluated for morphological diagnosis, organ system, and etiology. RESULTS: The 20 most common morphologic diagnoses accounted for 76% of the cases, including stillborn (10.8%); colitis (8.6%); hemorrhage (8.4%); ulcer (5.2%); seizures (4.7%); pneumonia (4.2%); inanition (4.1%); dermatitis (3.8%); spondylosis (3.3%); and amyloidosis (3.0%). The digestive system was most frequently involved (21.3%), followed by the urogenital (20.3%), cardiovascular (12.2%), and multisystem disease (10.3%). An etiology was not identified in approximately one-third of cases. The most common etiologies were trauma (14.8%), degenerative (9.5%), viral (8.7%), and neoplastic/proliferative (7.0%). CONCLUSION: This information should be useful for individuals working with baboons.

Differential effects of unusual climatic stress on capuchin (Cebus capucinus) and howler monkey (Alouatta palliata) populations on Barro Colorado Island, Panama.

Though the harmful effects anthropogenic disturbances pose to wild primates are well appreciated, comparatively little is known about the effects of natural disturbances. From December 2010 to January 2011, different mortality patterns were observed for two primate species, capuchins and howler monkeys, on Barro Colorado Island (BCI), Panama. Unusually high rainfall in 2010 was associated with census and cadaver data indicating the rapid loss of >70% of the capuchin population in late 2010 to early 2011. In contrast, over this same period, no decline was documented for howler monkeys and cadaver data for howlers was unexceptional. The high mortality experienced by the capuchin population was unexpected and its extent was not fully appreciated until the event was largely over. Explanations proposed for it included effects of hypothermia, disease or a shortage of some essential nutrient(s). Of these, the dietary explanation seems most probable. BCI capuchins depend most heavily on arthropod foods in December, when few higher quality ripe fruits are available. The unprecedented high rainfall in December 2010 is hypothesized to have largely eliminated the arthropod peak expected on BCI each December. A lack of protein-rich arthropods, when coupled with the climatic and nutritional stress capuchins generally experience at this time of year, appears to have precipitated the rapid die-off of most of the island's capuchin population. As howler monkeys obtain dietary protein primarily from leaves, a shortage of edible arthropods would not affect howler numbers. Comparison of our 2010 data with similar data on earlier primate/mammalian mortality events reported for BCI and for Corcovado, Costa Rica indicates that our understanding of the effects of natural disturbances on wild primate populations is not profound. We suggest that more research be devoted to this increasingly timely topic, so important to conservation policy.

Fatal atypical O:3 Yersinia pseudotuberculosis infection in cynomolgus macaques.

Fatal Yersinia pseudotuberculosis infection in cynomolgus macaques was diagnosed based upon pathology, microbiology and PCR for this study. Pathological findings included acute, erosive to ulcerative, necrohemorrhagic enterocolitis. Genotyping by PCR showed an O:3 pattern (gmd-fcl(+), ddhC-prt(+), manB(+), ddhA-B(+)), but an additional gene, wbyK, was detected. This is the second report to identify wbyK+ O:3 genotype as the cause of fatal yersiniosis. The first case was reported in 2008, and involved farm deer in the U.S. As the frequency of wbyK+ O:3 genotype is found more often in different carriers, O:3 genotype is proposed to be divided into two subtypes: O:3a without wbyK and O:3b with wbyK. Virulence gene analysis showed the presence of inv, ypmC, irp1, ybtP-ybtQ, yadA, yopB, yopH, lcrF, and suggested that this O:3b isolate could be a highly pathogenic strain to cynomolgus macaques.

Canine distemper virus associated with a lethal outbreak in monkeys can readily adapt to use human receptors.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Specific-pathogen-free status is associated with lower infant mortality rate in rhesus macaque (Macaca mulatta) colonies at the California National Primate Research Center.

BACKGROUND: Specific-pathogen-free (SPF) rhesus macaques, Macaca mulatta, are a valuable resource in biomedical research, and demographic analysis plays a significant role in colony management. METHODS: Data collection included SPF levels, gender, birth year, season of birth, birth location, rearing condition, maternal pregnancy history, and maternal age. Infant mortality in SPF rhesus macaques was compared with that in non-SPF rhesus macaques at the California National Primate Research Center over a six-year period, using Cox proportional regression analysis. RESULTS: In infants born to multiparous dams, the SPF infants had a significantly lower rate of mortality than non-SPF infants. There was no statistically significant difference in infant mortality between different SPF levels. CONCLUSIONS: Elimination of selected endemic viruses from breeding populations of rhesus macaques for the purpose of SPF colony development is associated with a significant reduction in the infant mortality rate.

Mother-to-infant transmission of simian immunodeficiency virus is rare in sooty mangabeys and is associated with low viremia.

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) occurs in utero, intrapartum, and through breastfeeding, with a cumulative rate of transmission of 35 to 40%. As a result, ∼ 400,000 children become infected each year. Little is known about mother-to-infant transmission (MTIT) during natural simian immunodeficiency virus (SIV) infection of sooty mangabeys (SMs) that typically is nonpathogenic despite high viral loads. In this study, we retrospectively investigated the rates of MTIT in a large colony of naturally SIV-infected SMs using serological (anti-SIV antibody by enzyme-linked immunosorbent assay [ELISA] and Western blot analysis) and virological (SIV(smm) real-time reverse transcription-PCR) methods. We examined 161 SM infants born to SIV-infected mothers and found that 150 (93.2%) were infected by non-MTIT (n = 120) or remained uninfected (n = 30). The remaining 11 SM infants (6.8%) were defined as acquiring SIV by presumptive MTIT based on (i) the presence of anti-SIV antibodies without seroreversion and (ii) a viral load of >500 copies/ml of serum in the first year of life. SM infants infected with SIV by presumptive MTIT did not show any increased morbidity or mortality, indicating that the infection is nonpathogenic even when acquired early in life. Interestingly, viral loads of SIV-infected SM infants with presumptive MTIT were 2-log lower than those of SIV-infected adult SMs living in the same colony (i.e., ∼ 1,000 and 100,000 copies/ml, respectively). These results indicate that MTIT is substantially less frequent in naturally SIV-infected SMs than in HIV-1-infected humans and results in nonpathogenic infection associated with low SIV viremia. Evolutionary pressure to reduce MTIT may have contributed to the restriction of SIV pathogenesis in natural hosts.

Pathological changes in captive monkeys with spontaneous yersiniosis due to infection by Yersinia enterocolitica serovar O8.

An outbreak of fatal yersiniosis due to infection with Yersinia enterocolitica serovar O8 is documented in two species of captive monkey. Five of 50 squirrel monkeys (Saimiri sciureus) and one of two agile gibbons (Hylobates agilis) died following several days of diarrhoea. Necropsy examination revealed necrotizing enterocolitis and multifocal necrosis or abscesses in various organs. Microscopically, these lesions comprised multifocal necrosis with bacterial colonies, neutrophils and accumulation of nuclear debris. Occasional lesions included macrophages and abscess formation. Immunohistochemically, the bacteria were identified as Y. enterocolitica O8. In addition, Y. enterocolitica serotype O8 was isolated from animal organs in pure culture. This is the first report of fatal cases of infection with Y. enterocolitica serovar O8 in animals.

Distinct physiologic and inflammatory responses elicited in baboons after challenge with Shiga toxin type 1 or 2 from enterohemorrhagic Escherichia coli.

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-alpha]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-alpha. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.

Generation of a specific-pathogen-free baboon colony.

We undertook establishing an SPF baboon colony in response to requests from researchers. To enable the widest possible future use of SPF baboons, our aim was to develop an SPF colony of baboons (Papio hamadryas anubis) free of 12 target viruses: 5 herpesviruses, 4 retroviruses, simian virus 40, measles, and monkeypox. Infant baboons were removed from their mothers within 24 h of birth and nursery-reared. Groups of 3 to 8 age-matched conspecifics were isolated in separate rooms for 1 y while undergoing repeated testing for target viruses. During the initial 7 y of the SPF program, 171 infants were enrolled, of which 76 (44.4%) subsequently were removed from the program. Of those removed, 54 (71.0%) were culled due to breaks in virus-free status, 12 (15.8%) died of various causes, 4 (5.3%) developed seizures, and 6 (7.9%) were removed for other reasons. The most problematic viruses were baboon cytomegalovirus (25.9% of culls), Herpesvirus papio 1 (51.9%), and simian foamy virus (7.4%). Using conspecific groups of 3 to 4 infants reduced first-year program losses as compared with groups of 6 to 8. There have been 17 births in the SPF colony, and all these infants have been free of all target viruses since birth. On the basis of these results, early removal of infants from their dams, housing in small peer groups, frequent virus testing, and aggressive culling of virus-positive animals is an effective approach for development of a baboon colony free of multiple viruses.