Major histocompatibility complex class II polymorphisms in Macaca mulatta: factors influencing comprehensive genotyping of Macaca mulatta (Mamu)-DQA1 alleles by PCR-RFLP in archival samples.

In the last 5 years, HLA class II genotyping methods have been adapted for genotyping of class II loci in rhesus macaques. Since previously published typing protocols were used on samples that were collected and stored under ideal conditions, it was of interest to determine if these methods were adequate for genotyping a large collection of archival samples from which DNA had been isolated and stored under various conditions. Established macaque DQA1 typing protocols were modified to optimize the typing procedure and enhance the ability to successfully genotype DNA from samples that were of poor quality and/or quantity. Long-term storage of whole-blood buffy coats or stored DNA extracted from whole-blood buffy coats did not affect typing success; however, amplification and typing of DNA extracted from archival samples of plasma were difficult and resulted in a low success rate. This suggests that amplification and DQA1-genotyping of archival samples is possible with a modified protocol, but is influenced by the age and source of the sample, and to a lesser extent, the method used to extract DNA from sample substrates.

Evidence of infection with simian type D retrovirus in persons occupationally exposed to nonhuman primates.

Simian type D retrovirus (SRV) is enzootic in many populations of Asian monkeys of the genus Macaca and is associated with immunodeficiency diseases. However, the zoonotic potential of this agent has not been well defined. Screening for antibodies to SRV was performed as part of an ongoing study looking for evidence of infection with simian retroviruses among persons occupationally exposed to nonhuman primates (NHPs). Of 231 persons tested, 2 (0.9%) were found to be strongly seropositive, showing reactivity against multiple SRV antigens representing gag, pol, and env gene products by Western immunoblotting. Persistent long-standing seropositivity, as well as neutralizing antibody specific to SRV type 2, was documented in one individual (subject 1), while waning antibody with eventual seroreversion was observed in a second (subject 2). Repeated attempts to detect SRV by isolation in tissue culture and by using sensitive PCR assays for amplification of two SRV gene regions (gag and pol) were negative. Both individuals remain apparently healthy. We were also unable to transmit this seropositivity to an SRV-negative macaque by using inoculation of whole blood from subject 1. The results of this study provide evidence that occupational exposure to NHPs may increase the risk of infection with SRV and underscore the importance of both occupational safety practices and efforts to eliminate this virus from established macaque colonies.

Mamu-DQA1 allele and genotype frequencies in a randomly sampled breeding colony of rhesus macaques (Macaca mulatta).

We studied the allelic and genotypic distribution of the major histocompatibility class-II locus DQA1 observed in a random sample of Indian rhesus macaques (Macaca mulatta) from a major breeding facility in the United States. The DNA was isolated from whole blood samples collected between 1991 and 1994 from 65 Indian rhesus monkeys. Polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), which involves use of specific amplification of DQA1 exon 2 and subsequent restriction digestion of the 242-base pair fragment, was used to genotype the animals for the 20 known macaque (Mamu)-DQA1 alleles. Frequencies for four alleles (DQA1*240x, *2502, *2503 and *0102) differed significantly from those reported in a smaller sample of rhesus macaques from the German Primate Center. The modest genetic survey of Mamu-DQA1 genotypes presented here will be particularly useful in designing epidemiologic studies that investigate associations between immunogenetic background and disease susceptibility in macaque models of human disease.

Simian foamy virus infections in a baboon breeding colony.

The prevalence, transmission, and variation of simian foamy viruses (SFVs) in baboons was investigated. Over 95% of adult baboons in the breeding colony as well as recently imported adult animals had high titers of anti-SFV serum IgG. Maternal antibody was detectable in infants' serum up to 6 months of age. Approximately 30% of infants in breeding harems experienced SFV infections by 1 year of age. Shedding of SFV in oral secretions was common, with 13% of samples from normal adult animals and 35% from immunosuppressed animals containing infectious SFV. SFV was isolated from three baboon subspecies (olive, yellow, and chacma baboons) and sequences from both the pol and the LTR regions of the provirus were amplified by PCR and sequenced. Phylogenetic analysis indicated that all baboon isolates formed a single lineage distinct from SFVs of other African monkey species. Within the baboon SFV lineage, two distinct clades were apparent, which consisted of isolates from yellow and olive baboons and isolates from chacma baboons. Competition ELISAs indicated that, while SFV isolates of these two groups were very closely related, antigenic differences do exist between them. SFV isolates from a drill and a mandrill were distinct from baboon SFV isolates, both genetically and antigenically.

Eradication of simian retrovirus type D from a colony of cynomolgus, rhesus, and stump-tailed macaques by using serial testing and removal.

The markedly compromised health of animals in a macaque colony and the problematic interpretation of data from two drug safety assessment studies prompted a review of the effect of simian retrovirus type D on the drug-development process at a Midwest pharmaceutical company. After reviewing relevant literature and consulting with an expert in simian retroviruses, we initiated a program of eradication. During a 16-month period, all cynomolgus (Macaca fascicularis), rhesus (Macaca mulatta), and stump-tailed (Macaca arctoides) macaques housed in the facility were evaluated as many as eight times for the presence of simian retrovirus type D by using serology, virus isolation, and/or polymerase chain reaction tests. All animals with positive test results were removed from the colony immediately. No test results indicative of simian retrovirus type D infection have occurred during the subsequent 2.5 years. We attribute the successful eradication and prevention of re-introduction of the virus to regular testing, purchasing animals from sources free of simian retrovirus type D, and assiduous application of procedures designed to prevent transmission between animals.

Cardiovascular, respiratory, thermoregulatory, sedative, and analgesic effects of intravenous administration of medetomidine in rhesus macaques (Macaca mulatta).

BACKGROUND AND PURPOSE: Medetomidine is a selective, specific, and potent alpha2-adrenergic receptor agonist that has been utilized successfully as a sedative/analgesic agent in a variety of domestic and nondomestic animals. The objective of this study was to document the physiological effects of the intravenous administration of medetomidine in rhesus macaques (Macaca mulatta). METHODS: Fifteen healthy rhesus macaques (Macaca mulatta), 5 to 15 years old and weighing 5.5 to 11.8 kg, were given four dosages of medetomidine (50, 100, 150, and 200 microg/kg of body weight) intravenously, and cardiovascular, respiratory, thermoregulatory, sedative, and analgesic effects were determined. RESULTS: All four doses of medetomidine induced a similar and significant decrease in mean arterial pressure, as well as a transient but significant increase in respiratory rate followed by a longer-lasting significant decrease. Bradycardia, hypotension, and loss of thermoregulatory ability accompanied by a biphasic respiratory response and inconsistent sedation, analgesia, and muscular relaxation were observed. Heart rate decrease was rapid for all doses, but was significantly lower and of shorter duration after administration of the 50 microg/kg dosage. CONCLUSION: The inconsistency of the anesthetic plane induced by intravenous administration of medetomidine precludes it from being used alone to sedate rhesus macaques.

A simian model of human granulocytic ehrlichiosis.

Following intravenous inoculation with horse blood-infected with the agent of human granulocytic ehrlichiosis (HGE) from a human fatality, two rhesus macaques (Macaca mulatta) exhibited pyrexia and lethargy on days 4-12 postinfection (PI). Hematology revealed neutropenia, thrombocytopenia, and anemia, with ehrlichial morulae in monocytes and neutrophils on days 4-12. Blood was polymerase chain reaction (PCR)-positive on days 4-12 and bone marrow was PCR-positive on day 11. There was a minor increase in gamma-glutamyl transpeptidase on day 12 and serum interferon-gamma levels increased by day 18. Seroconversion occurred on day 20 PI to a titer of 100 by day 22. Western blot bands characteristic of HGE included 25-, 44-, 80-, 94-, 105-, and 125-kD bands. There was generalized lymphohistiocytic infiltration in the liver, spleen, lymph nodes, and other tissues. The liver had focal hepatocyte apoptosis. There was HGE DNA (by PCR) only in the spleen. Comparable findings were not observed in a monkey that received uninfected horse blood as a control. This animal model of human disease is important for further studies of HGE diagnosis, management, and pathogenesis.

Effect of weaning time and associated management practices on postweaning chronic diarrhea in captive rhesus monkeys (Macaca mulatta).

OBJECTIVE: Our purpose was to assess the extent to which early weaning and other weaning-management factors affect development of postweaning chronic diarrhea in captive rhesus monkeys at the California Regional Primate Research Center between 1992 and 1995. METHODS: Data for weaning, management, and onset of diarrhea were obtained from daily records. The Cox proportional hazard model was used to assess whether the risk of chronic diarrhea was related to early weaning. RESULTS: Monkeys that were lighter at weaning had a threefold increase in risk of postweaning chronic diarrhea (P = 0.07), compared with that in heavier monkeys. An episode of preweaning diarrhea increased the risk of postweaning chronic diarrhea twofold (P = 0.08). Relocation of monkeys to outdoor facilities in the fall was associated with a fivefold decrease in risk (P < 0.001), compared with that of other seasons, and weaning in 1993 was associated with a twofold decrease in risk, compared with that of other years (P = 0.04). CONCLUSIONS: Multiple factors need to be considered for prevention of postweaning chronic diarrhea, including weaning weight, preweaning diarrhea, season weaned, and weaning conditions that change from year to year.

Social separation, housing relocation, and survival in simian AIDS: a retrospective analysis.

OBJECTIVE: To test the hypothesis that changes in housing, particularly those involving social separations, would have a negative impact on survival in rhesus monkeys experimentally inoculated with the simian immunodeficiency virus (SIV). METHODS: An archival methodology was used. Colony records at four Regional Primate Research Centers were screened, and data pertaining to demographics, contents of the inoculum, medical history before and after inoculation, and housing relocations and social companions were coded. The final sample size totaled 298 individuals. RESULTS: Following statistical control of covariates, housing relocations and social separations in the 90-day period before SIV inoculation and in the 30-day period after inoculation were associated with decreased survival. There was evidence that housing disruptions occurring earlier after inoculation were associated with shorter survival. Finally, a subset of animals was found to have been socially housed after SIV inoculation; such experience had negative consequences for survival. CONCLUSIONS: The results indicate that psychosocial experiences that likely produce a stressful state are associated with shorter survival in SIV-infected monkeys.

Social stress results in altered glucocorticoid regulation and shorter survival in simian acquired immune deficiency syndrome.

From early in the AIDS epidemic, psychosocial stressors have been proposed as contributors to the variation in disease course. To test this hypothesis, rhesus macaques were assigned to stable or unstable social conditions and were inoculated with the simian immunodeficiency virus. Animals in the unstable condition displayed more agonism and less affiliation, shorter survival, and lower basal concentrations of plasma cortisol compared with stable animals. Early after inoculation, but before the emergence of group differences in cortisol levels, animals receiving social threats had higher concentrations of simian immunodeficiency virus RNA in plasma, and those engaging in affiliation had lower concentrations. The results indicate that social factors can have a significant impact on the course of immunodeficiency disease. Socially induced changes in pituitary-adrenal hormones may be one mechanism mediating this relationship.

Identification of a human population infected with simian foamy viruses.

Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.

Individual differences in peripheral blood immunological and hormonal measures in adult male rhesus macaques (Macaca mulatta): evidence for temporal and situational consistency.

A growing body of research has indicated that consistent individual differences exist in physiological systems with which the immune system interacts. Few data have been reported that demonstrate stable individual differences in immunological measures, however. In the present study, enumerative measures of immune system activity were examined in 36 adult male rhesus macaques over a 13 month period under baseline conditions as well as under conditions of pharmacological and physical challenge. Blood samples were assayed for plasma concentrations of ACTH and cortisol, as well as neutrophil, total lymphocyte, CD4+ and CD8+ lymphocyte numbers, and the CD4/CD8 ratio. Analyses revealed that individual differences in the CD4/CD8 ratio and, to a lesser extent, plasma ACTH and cortisol concentrations, and neutrophil and CD4+ lymphocyte numbers were consistent across situations and times, despite changes in mean values during the various blood sampling sessions. The results suggest that the CD4/CD8 ratio might be considered trait-like and a useful immunological measure of biobehavioral organization.

Screening for simian type-D retrovirus infection in macaques, using nested polymerase chain reaction.

A nested polymerase chain reaction (PCR) assay was developed to detect proviral DNA in peripheral blood mononuclear cells (PBMC) of macaques infected with simian type-D retrovirus (SRV/D). Primers were designed to amplify gag gene sequences of SRV/D serotype 1, 2, and 3 viral genomes and were used in a single assay for simultaneous detection of infection with SRV/D-1, SRV/D-2, or SRV/D-3. Results of plasmid dilution studies indicate sensitivity of nested PCR in the range of 1 to 10 genomic copies. The PBMC samples from 395 macaques of unknown SRV/D status, obtained from several primate facilities, were tested in parallel by Western blot (immunoblot) analysis, virus isolation, and nested PCR. Infection was detected in 60 (15.2%) animals by nested PCR, in 40 (10.1%) animals by virus isolation, and in 28 (7.1%) animals by immunoblot. All 40 culture-positive samples were positive by nested PCR. In addition, 11 of 23 immunoblot-positive/virus isolation-negative samples, 2 of 20 immunoblot-indeterminate/virus isolation-negative samples, and 7 of 312 immunoblot-negative/virus isolation-negative samples were identified as positive by nested PCR. Nested PCR is a sensitive and specific assay for simultaneous screening for infection with serotypes 1, 2, and 3 of simian type D retrovirus, and is a powerful tool for rapid screening and surveillance in macaque colonies.

Experimental measles. I. Pathogenesis in the normal and the immunized host.

An animal model to study measles pathogenesis and the correlates of protective immunity was established using rhesus monkeys. A measles isolate, obtained during an epidemic of measles in the primate colony at the University of California, Davis, was passaged through rhesus monkeys and amplified in rhesus mononuclear cells to create a pathogenic virus stock. Sequence analysis of the nucleoprotein and hemagglutinin genes of this isolate revealed strong homology with the Chicago 89 strain of measles virus. Conjunctival/intranasal inoculation of juvenile rhesus monkeys with this virus resulted in skin rash, pneumonia, and systemic infection with dissemination to other mucosal sites and to the lymphoid tissues. Inflammation and necrosis occurred in the lungs and lymphoid tissues and many cell types were infected with measles virus on Day 7 postinoculation (p.i.). The most commonly infected cell type was the B lymphocyte in lymphoid follicles. Measles antigen was found in follicular dendritic cells on Day 14 p.i. In contrast to naive monkeys infected with measles virus, animals vaccinated with the attenuated Moraten strain did not develop clinical or pathologic signs of measles after challenge. However, moderate to marked hyperplasia occurred in the lymph nodes and spleen of a vaccinated animal on Day 7 after pathogenic virus challenge, suggesting that an effective measles vaccine limits but does not prevent infection with wild-type measles virus.

Cytomegalovirus and simian immunodeficiency virus coinfection: longitudinal study of antibody responses and disease progression.

Antibody titers to rhesus cytomegalovirus (RhCMV) were prospectively analyzed over a period of 68 weeks in a longitudinal serosurvey of 17 RhCMV-seropositive rhesus macaques (Macaca mulatta) experimentally coinfected with simian immunodeficiency virus (SIV). These were compared with anti-RhCMV titers in 18 animals that were also naturally infected with RhCMV but not infected with SIV. Fluctuations in anti-RhCMV antibody titers were observed within 5 weeks of SIV inoculation, and two distinct patterns of RhCMV antibody response were observed in SIV-infected animals. Animals showing a progressive decline in anti-RhCMV immunoglobulin G (IgG) exhibited the most rapid disease progression, coincident with low anti-SIV and anti-tetanus toxoid IgG responses, high levels of p27 antigen in the plasma, and short survival. Animals exhibiting a more stable CMV-specific response after SIV inoculation had the least rapid disease course. Anti-RhCMV antibody titers in SIV-uninfected animals remained relatively stable during the period of study. Evidence that preinoculation immunologic measures predicted postinoculation outcome was equivocal.

Simultaneous detection of simian retrovirus type D serotypes 1, 2, and 3 by polymerase chain reaction.

Asymptomatic infection of macaques with macaques with simian retroviruses type D (SRV/D), the etiologic agents of one form of retrovirus-induced simian immunodeficiency disease, can confound experiments with the simian immunodeficiency virus (SIV), which also induces immunodeficiency disease in macaques. The SIV/macaque model is the preferred nonhuman primate model for AIDS-related research. Serological screening for SRV/D alone is insufficient because not all infected animals seroconvert, and virus isolation by cocultivation may require 4 to 6 weeks. We have established a DNA polymerase chain reaction (PCR) assay. One set of nested primers allows detection of SRV/D serotypes 1, 2, and 3 and distinguishes SRV-2 from the other two serotypes. The PCR assay is sensitive; a single proviral copy of SRV/D could be detected in 150,000 to 210,000 macaque peripheral blood mononuclear cells (PBMCs). When applied to a panel of virus isolation-positive macaque samples, the PCR assay was positive in 100% of the tests. No false-positive results were seen when known specific-pathogen-free (SPF) macaques were examined. We propose that macaques be screened with a combination of SRV/D serology and this DNA PCR assay prior to enrollment in experiments with SIV.

Accidental injuries associated with nonhuman primate exposure at two regional primate research centers (USA): 1988-1993.

Although occupationally acquired zoonoses of nonhuman primates have been well documented, the epidemiology of work-related injuries associated with occupational exposure to nonhuman primates has not been studied. To investigate such injuries, we retrospectively reviewed injury records at one regional primate research center and distributed a self-administered, anonymous questionnaire to at-risk personnel at two centers. Records of bite, animal-inflicted scratch, needlestick, cut, and mucous membrane exposure injuries were reviewed at one center for the 5-year period 1988 to 1993 to determine incidence and frequency of injuries and to identify possible risk factors. A total of 261 injuries were reported during this period, with an annual incidence for all injuries combined ranging from 43.5 to 65.5 injuries per 100,000 person workdays (pwd) at risk. For specific injuries the highest incidence was observed for animal-inflicted scratches and bites, with a rate of 82 and 81 per 100,000 pwd respectively. The job category Veterinary Resident was found to have the highest incidence for needlestick injuries (547 per 100,000 pwd), scratches (239 per 100,000 pwd), and cuts (171 per 100,000 pwd). The highest rates for bites were observed in the job categories Animal Health Technician and Animal Technician, with 171 and 150 per 100,000 pwd respectively; the category Staff Veterinarian had the highest rate of mucous membrane exposures (71 per 100,000 pwd). The frequency of all injuries was greatest in personnel employed < or = 2 years. Questionnaire responses indicated that having > 20 h per week of contact with nonhuman primates or contact with more than 50 nonhuman primates per week was associated with a significantly increased risk of bites, animal-inflicted scratches, needlesticks, and mucous membrane exposures. In addition, data analysis indicated that under-reporting of work-related injuries was high; 59% of scratches, 50% of mucous membrane exposures, 45% of cuts, 37% of bites, and 20% of needlestick injuries went unreported. Results of this study identify job categories with a high incidence of specific injuries, for which additional targeted training and prevention programs may be beneficial, as well as providing quantitative baseline data for evaluating the effectiveness of any new safety programs or practices.

Evidence of natural bluetongue virus infection among African carnivores.

Bluetongue is an International Office of Epizootics List A disease described as the century's most economically devastating affliction of sheep. Bluetongue (BLU) viruses were thought to infect only ruminants, shrews, and some rodents, but recently, inadvertent administration of BLU virus-contaminated vaccine resulted in mortality and abortion among domestic dogs. We present evidence of natural BLU virus infection among African carnivores that dramatically widens the spectrum of susceptible hosts. We hypothesize that such infection occurred after ingestion of meat and organs from BLU virus-infected prey species. The effect of BLU virus on endangered carnivores such as the cheetah and African wild dog requires urgent investigation. Also, the role of carnivores in the epizootiology of this disease needs elucidation.