Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env.

Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.

Isolation from African Sykes' monkeys (Cercopithecus mitis) of a lentivirus related to human and simian immunodeficiency viruses.

Analysis of serum samples from 100 wild-caught or colony-born Sykes' monkeys (Cercopithecus mitis) in Kenya revealed that 59 animals had antibodies cross-reactive to human immunodeficiency virus type 2 (HIV-2) and to simian immunodeficiency viruses (SIVs). A lentivirus, designated SIVsyk, was isolated from five of six seropositive asymptomatic Sykes' monkeys, but in four cases isolation was possible only after depletion of CD8+ lymphocytes and cocultivation of the CD4(+)-enriched cell population with peripheral blood mononuclear cells from seronegative Sykes' monkeys. SIVsyk resembled other SIVs and HIVs morphologically, had an Mg2(+)-dependent reverse transcriptase enzyme, and replicated in and was cytopathic for CEMx174 and Sup-T1 cells. SIVsyk differred substantially from other SIVs, however, in that it failed to replicate in normal human, mangabey, and macaque peripheral blood mononuclear cells and serum from seropositive Sykes' monkeys immunoprecipitated env antigens from HIV-1 as well as from HIV-2, SIVsmm, and SIVagm. These data demonstrate a high prevalence of natural infection in Sykes' monkeys in Kenya with a lentivirus that appears to be unique with respect to its host range and antigenic cross-reactivity.

Hippocampal damage associated with prolonged and fatal stress in primates.

Sustained exposure to glucocorticoids (GCs), adrenal hormones secreted during stress, can cause neural degeneration in the rat. This is particularly so in the hippocampus, a principal neural target site for GCs, in which GCs can exacerbate the rate of neuron death during normal aging, as well as the severity of neuronal damage after various neurological insults. Thus, stress can be a potent modulator of hippocampal degeneration in the rat. The present report suggests a similar association in the primate. Eight vervet monkeys, housed in a primate center in Kenya, that had died spontaneously from 1984 to 1986, were found at necropsy to have multiple gastric ulcers; a retrospective, neuropathological study was then done of this opportunistic population. Compared with controls euthanized for other research purposes, ulcerated monkeys had marked hippocampal degeneration that was apparent both quantitatively and qualitatively, and both ultrastructurally and on the light-microscopic level. Minimal damage occurred outside the hippocampus. Damage was unlikely to have been due to an agonal or post-mortem artifact. Instead, ulcerated monkeys appear to have been subject to sustained social stress, perhaps in the form of social subordinance in captive breeding groups: most came from social groups, had significantly high incidences of bite wounds at necropsy, and had hyperplastic adrenal cortices, indicative of sustained GC release. Moreover, the specific hippocampal cell fields damaged in ulcerated animals matched those damaged by GCs in the rodent hippocampus. Thus, this represents the first evidence suggesting that sustained stress, via GC hypersecretion, might be neurodegenerative in the primate.

Reproduction in the vervet monkey (Cercopithecus aethiops): III. The menstrual cycle.

The menstrual cycles of 17 multiparous vervet monkeys were studied. Based on estradiol, progesterone, and LH profiles, ovulation is predicted to occur on day 13 of the 32.4-day menstrual cycle. Estradiol peaked on the day preceding the LH peak in 75% of cycles. Average luteal phase length (progesterone greater than 4 nmol/l) was 18 days, with progesterone rising above 4 nmol/l on the day of the LH peak. Vaginal cytology and perianal skin coloration exhibited too much within- and among-animal variability to be reliable indicators of menstrual cycle stages. Uterine biopsies of the proliferative phase were characterized by mild pseudostratification of the columnar epithelium and absence of glandular secretion; in contrast, those of the luteal phase had marked pseudostratification of the tall columnar epithelium with glandular secretions in the lumen. A few follicular-phase samples contained structures such as tortuous uterine glands with secretions. Such structures are more characteristic of the luteal phase. It is suggested that their presence can be explained by incomplete sloughing of the endometrium at menstruation, as this is known to be light or convert in this species.

Serological survey and virus isolation of simian T-cell leukemia/T-lymphotropic virus type I (STLV-I) in non-human primates in their native countries.

Infection with a simian retrovirus (STLV-I) closely related to human T-lymphotropic virus type I (HTLV-I) was investigated in non-human primates living in their native countries in Africa and Asia. Serum antibodies cross-reacting with HTLV-I antigens were detected in 85 of 567 non-human primates of 30 species. Seropositive animals were found among African green monkeys, olive baboons, Sykes' monkeys, mandrills and patas monkeys in several countries in Africa, and cynomolgus monkeys, Celebes macaques and siamangs in Indonesia. The frequency of seropositivity was much higher in adult than in young African green monkeys, cynomolgus monkeys and Celebes macaques. STLV-Is were isolated by establishing II lines of virus-producing lymphoid cells in the presence of interleukin-2 from 5 species of seropositive non-human primates, i.e. the African green monkey, Sykes' monkey, Celebes macaque, cynomolgus monkey and siamang. All these cell lines had T-cell markers and Tac antigen, and the cell lines from the African green monkey and Sykes' monkeys were Leu2a+ while those from other species were Leu3a+. These cell lines expressed viral antigens reacting with human sera from adult T-cell leukemia (ATL) patients and monoclonal antibodies (MAbs) against p19 and p24 of HTLV-I core proteins, and produced virus particles having RNA-dependent DNA polymerase activity. Cellular DNAs from these cell lines contained provirus sequences homologous to HTLV-I, shown by Southern blot hybridization. The restriction patterns of these provirus genomes were different from those of HTLV-I and were also dissimilar in the different species.

Early implantation and embryonic development of the baboon: stages 5, 6 and 7.

Implantation stages of the olive baboon, Papio cynocephalus anubis, showing embryonic development equivalent to Carnegie stages 5, 6 and 7 of development, were collected by hysterotomy and examined histologically. The younger specimens (stage 5) consisted of a thick trophoblastic plate composed of cytotrophoblast and syncytiotrophoblast with multiple small clefts, and a bilaminar disk embryo with a small slit-like amniotic cavity. An epithelial plaque response was present in the uterine epithelium immediately peripheral to the implantation site, within an area of pronounced uterine edema. The bilaminar embryonic disk consisted of columnar epiblast cells underlying the amniotic cavity, and thickened visceral endodermal cells that form part of the yolk sac. The slightly further developed placenta (stage 6) consisted predominantly of cytotrophoblast including primary villi and syncytiotrophoblast lining large spaces containing maternal blood. Secondary placental villi were present in the oldest group (stage 7), and there was modest decidualization of the uterine stroma. An epithelial plaque response persisted, but varied in extent. The sequence of events in early development in the baboon is similar to that in the rhesus monkey insofar as blood space formation and endometrial responses are concerned. However, the plaque response is not so great as in the rhesus; there is no secondary placenta, and the decidual response is slightly more extensive.

Bovine tuberculosis in a wild baboon population: epidemiological aspects.

A follow-up of an outbreak of Mycobacterium bovis in a population of feral baboons in Kenya was undertaken after one year by necropsy of euthanized, clinically ill animals, and tuberculin testing of others. It was concluded that the source of the infection was animals feeding on village slaughterhouse offal of M. bovis infected cows. Secondary (direct) baboon-baboon transfer of the disease appeared to be minimal or nonexistent.

Pregnancy outcome in free-ranging vervet monkeys (Cercopithecus aethiops).

A preliminary assessment of individual female differences in conception rate and fetal wastage has been determined for a population of wild vervet monkeys (Cercopithecus aethiops). One of three troops of vervet monkeys, the subjects of a long-term behavioral study, was trapped and blood was obtained for electrophoretic analysis. Pregnant females exhibited a distinctive serum aminopeptidase phenotype allowing a conclusive determination of pregnancy. Of the seven females diagnosed as pregnant, three later gave birth. Of the females that aborted, two were nulliparous and one was very old. Studies of captive animals have indicated that age and rank may affect a female's ability to carry a fetus to term. These factors, rather than the trapping procedure, may have been responsible for most of the fetal loss in the trapped troop. A comparison of all three troops for a 3-year period indicated that there were fluctuations in yearly birth success of individual females, as well as a relatively high miscarriage rate. The results of this study indicate the advantages of obtaining joint behavioral and biological data.

STLV-I antibodies in feral populations of East African vervet monkeys (Cercopithecus aethiops).

Serum samples from feral populations of African green monkeys (Cercopithecus aethiops) were screened for antibodies to the simian T-lymphotropic virus, type I (STLV-I). Blood samples had been collected from 336 monkeys in 4 regions of central and southern Kenya in 1978 and 1979, from 114 monkeys in central Ethiopia in 1973, and from 85 monkeys from the Kampala region of Uganda in 1966. A total of 178/535 monkeys (33%) were seropositive (STLV-I+). Only 4/114 monkeys (4%) from Ethiopia were seropositive compared to 25/85 Ugandan monkeys (29%) and 149/336 Kenyan monkeys (44%). Epidemiological analysis of the Kenyan monkeys showed that 37% of the males and 54% of the females were STLV-I+, and that there was a progressive increase in the proportion of STLV-I+ monkeys of both sexes with age, rising from an average of 16% in infants (less than 9 months) to an average of 69% in adults (greater than 42 months). The proportion of STLV-I+ monkeys was higher among females in each age category. Seropositivity for antibodies to STLV-I had no apparent effect on the health of monkeys, and no association with the occurrence of Hepatocystis parasitemia was seen in this species. The analysis of data from infants of STLV-I+ mothers showed that seroconversion had occurred in 1 of 3 cases, suggesting that vertical transmission of the STLV-I virus is not an inevitable consequence for infants of seropositive mothers.

Enclosure design and reproductive success of baboons used for reproductive research in Kenya.

Three 125mz enclosures were designed to enable collection of urine from female baboons (Papio sp.) during early pregnancy, while maintaining them in close continuous social contact to permanent breeding groups. Attached to each enclosure were individual cages to which females were confined for urine collections. Over the 18-month period following group formation, 60 of 67 females became pregnant and then 51 reconceived following surgical termination of pregnancy. A total of 181 pregnancies were recorded. Over 75% of reconceptions occurred within the first two cycles following surgery. The enclosures have proved to be extremely versatile, allowing sampling of individual animals maintained in group conditions with the ability to interact socially.

Partial protection of baboons against Schistosoma mansoni using radiation-attenuated cryopreserved schistosomula.

Three groups of five baboons were vaccinated in Kenya using three doses of 10,000 viable cryopreserved schistosomula attenuated with either 10, 20 or 60 krad 60Co-irradiation prepared in England. Animals were vaccinated at four-week intervals, challenged after a further six weeks with 2,000 cercariae and perfused at 10 weeks after challenge. High antibody titres to schistosomula mediating in vitro cytoadherence with P 388D1 macrophage-like cells were demonstrated in all vaccinated animals but not in controls. Significant titres to soluble egg antigen (SEA) were also demonstrated by ELISA in the 10 and 20 krad vaccinated groups following the first vaccination. The subsequent vaccinations and the challenge boosted this response considerably. Mean anti-SEA titres were only elevated above background in the 60-krad group six weeks after the third vaccination and in the challenge controls six weeks after challenge. Peripheral eosinophil counts were slightly reduced and neutrophil counts slightly elevated before challenge while eosinophil and erythrocyte counts were elevated and neutrophil counts depressed after challenge. PCV values were erratic in all groups. Eggs appeared in the faeces from six weeks after challenge and excretion rates were higher in all three vaccinated groups than in the challenge controls by necropsy 10 weeks after challenge. Body-weights were depressed in all groups after challenge but subsequently rose in the 10 and 20 krad groups. The 60 krad and challenge control groups lost 12.4% and 7.9% of body-weight respectively after challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

The suitability of East African primates as animal models of visceral leishmaniasis.

The susceptibility of four East African primate species to experimental infection with Leishmania donovani was investigated. Vervet monkeys (Cercopithecus aethiops), Sykes monkeys (C. mitis) and baboons (Papio cynocephalus) all supported low grade infections for periods ranging between four and eight months and subsequently showed evidence of self-cure. Greater bushbabies (Galago crassicaudatus) remained completely refractory throughout the course of the experiment. The significance of hepatic histiocytic nodules in the infected primates, similar to those observed in asymptomatic human visceral leishmaniasis, and the susceptibility of Old World primates to experimental infection are discussed.

Reproduction in the vervet monkey (Cercopithecus aethiops): II. Annual menstrual patterns and seasonality.

Menstrual patterns and progesterone levels were monitored for 5 years from a cohort of 28 female vervet monkeys that were individually caged indoors. Three distinct cycle types (short, normal, and prolonged) were defined according to cycle length. Mean length of the normal cycle (32.5 days) and menses duration (4.8 days) are in agreement with previous reports. Prolonged cycles (> 50 days) contributed 20% of the total, with a decreased incidence during the natural peak breeding period. Weekly progesterone measurements indicated that many prolonged cycles were associated with an extended luteal phase, while others were probably due to lack of ovulation. From these data it would appear that the vervet monkey, although not strongly seasonal, does favor a particular time of year for breeding in a colony housed indoors.

The detection of ovulation and early pregnancy in the baboon by direct measurement of conjugated steroids in urine.

The pattern of excretion of urinary steroid metabolites in the olive baboon (Papio anubis) was examined during the menstrual cycle and in conception cycles in which embryos were surgically removed at intervals between day 11 and day 21 (day 0 = day of preovulatory estrogen peak). Conjugated estrone and pregnanediol-3α-glucuronide were measured in overnight urine samples by direct, nonextraction assays, and the levels were indexed by creatinine. Results showed that measurement of urinary conjugated estrone reflected preovulatory estrogen output and that pregnanediol-3α-glucuronide was an abundant urinary metabolite of progesterone. There was a defined postovulatory increase in the excretion of conjugated estrone during conception cycles in eight of ten animals. The timing of the increase ranged between day 13 and day 19 and was related to the appearance of elevated levels of urinary gonadotrophin. In four animals, increased estrogen excretion was first detected after the day of embryo removal, but this was most likely a response to chorionic gonadotrophin secreted before surgery. The findings demonstrate that measurement of conjugated estrone offers a rapid and practical approach for monitoring ovulation and implantation in the baboon by a single assay technique.

Reproduction in the vervet monkey (Cercopithecus aethiops): I. Testicular volume, testosterone, and seasonality.

To test whether the male could contribute to the birth peak seen in both wild and captive vervets, testicular volume and peripheral testosterone concentration were measured monthly in nine adults throughout a 14-month period. Volume was an average of 15% greater during the months of June to September, the period of natural breeding activity in the wild, than at other times of the year. Testosterone concentration rose throughout the time period but did not correlate with testicular volume. Quarterly biopsies of the right testis in animals either used for or withheld from breeding revealed the presence of spermatozoa and the existence of spermatogenesis throughout the year. There was no correlation of testicular volume with breeding status or biopsy. If these results from individually caged animals are representative of group-caged and wild animals, then such small seasonal changes are unlikely to affect year-round breeding. Therefore, a physiological contribution by the male to natural birth peaks seen in the wild and captivity is not readily apparent.

Captive propagation of vervet monkeys (Cercopithecus aethiops) in harems.

A small breeding colony of captive vervet monkeys (Cercopithecus aethiops), consisting of ten single male harem groups, was established in Kenya. Vervets were extremely prone to stress after capture and manipulative procedures led to high mortalities. Six weeks were required for partial habituation, after which routine handling was not too problematic. However, complete adjustment to captivity took a minimum of one year. Establishment of stable breeding groups from adult animals initially proved difficult because of fighting among females. Once harem groups stabilized, reproductive rate was high with just under 90% pregnancies and 85% livebirths annually. Births exhibited a seasonal pattern similar to that reported from wild populations in Kenya.

Tuberculosis in wild olive baboons, Papio cynocephalus anubis (Lesson), in Kenya.

Several wild olive baboons from a single troop in the Masai Mara Game Reserve, Kenya were observed to be lethargic and emaciated. Five were trapped and tuberculin tested by intradermal inoculation of 0.1 cc (100 IU) mammalian old tuberculin in the upper eyelid. Two of the five showed positive reaction at 72 hr and were examined at necropsy. Gross lesions in both animals consisted of multiple nodules with caseation in the lung, spleen and tracheobronchial lymph nodes. There were multiple granulomas throughout the lung, spleen and the lymph nodes. Tissues were cultured on Lowenstein-Jensen media with and without pyruvic acid. Isolates were typed as Mycobacterium bovis.

Effects of ketamine anaesthesia, stress and repeated bleeding on the haematology of vervet monkeys.

Haematology values are presented for the vervet monkey (Cercopithecus aethiops), and the relative effects of high dose ketamine anaesthesia, stress of capture and repeated bleedings assessed. Anaesthesia resulted in decreased WBC and RBC values, attributed to depression of cardiovascular function. These effects were the reverse of those of alarm and strenuous exercise (leukocytosis and polycythaemia) during capture. Stress resulted in relatively high white and low red blood cell counts. Opposing effects of stress and anaesthesia led to comparable haematological values for trained, non-anaesthetized vervets and stressed, anaesthetized vervets. Effects of repeated bleedings were opposite in anaesthetized and non-anaesthetized animals. These effects, however, along with those of ketamine anaesthesia and stress, were relatively insignificant compared with the wide variation in haematological values found among individuals. The biological importance of these effects thus appeared to be slight. The concept of 'normal values' is discussed.

Experimental Schistosoma mansoni infection in vervet monkeys (Cercopithecus aethiops) in Kenya: I. Susceptibility to a primary infection.

Groups of five 3-kg Kenyan monkeys, Cercopithecus aethiops, were exposed individually to 150,600 or 1500 Schistosoma mansoni cercariae per monkey. Three monkeys died soon after the infections became patent and the survivors were autopsied 4 months after exposure. Mortality and most haematological, parasitological and pathological sequelae of infection were dose-related, but not the white cell response or changes in the levels of serum proteins or fibrinogen. No gross liver fibrosis was seen. Comparison of this study with earlier ones on related cercopithecine monkeys suggests that the vervet closely resembles the baboon in its response to S. mansoni infections. Difficulties in managing and maintaining vervets can be overcome by using colony-bred or properly adapted feral animals. Thus, the vervet provides a cheaper, more readily available primate model for experimental S. mansoni studies. A prolonged infection, sufficiently heavy to permit reliable parasitological monitoring without undue mortality, should be provided by 150 S. mansoni cercariae per kg body-weight, using the Kenyan strains of vervet and parasite.