Occurrence of Free-Living Amoebae in Non-Human Primate Gut.

The gut microbiome reflects health and predicts possible disease in hosts. A holistic view of this community is needed, focusing on identifying species and dissecting how species interact with their host and each other, regardless of whether their presence is beneficial, inconsequential, or detrimental. The distribution of gut-associated eukaryotes within and across non-human primates is likely driven by host behavior and ecology. To ascertain the existence of free-living amoebae (FLA) in the gut of wild and captive non-human primates, 101 stool samples were collected and submitted to culture-dependent microscopy examination and DNA sequencing. Free-living amoebae were detected in 45.4% (46/101) of fecal samples analyzed, and their morphological characteristics matched those of Acanthamoeba spp., Vermamoeba spp., heterolobosean amoeboflagellates and fan-shaped amoebae of the family Vannellidae. Sequence analysis of the PCR products revealed that the suspected amoebae are highly homologous (99% identity and 100% query coverage) with Acanthamoeba T4 genotype and Vermamoeba vermiformis amoebae. The results showed a great diversity of amoebae in the non-human primate's microbiome, which may pose a potential risk to the health of NHPs. To our knowledge, this is the first report of free-living amoebae in non-human primates that are naturally infected. However, it is unknown whether gut-borne amoebae exploit a viable ecological niche or are simply transient residents in the gut.

Staphylococcus nasal colonization in three species of non-human primates.

Bacterial nasal colonization is common in many mammals and Staphylococcus represents the main pathogen isolated. Staphylococcus nasal carriage in humans constitutes a risk factor for Staphylococcus infections pointing out the need for animal experimentation for nasal colonization studies, especially for vaccine development. A limitation in addressing this hypothesis has been a lack of appropriate animal model. Murine models do not mimic human nasal colonization studies. Non-human primates (NHP) remain the best classical models for nasal colonization studies. In this study, we analyzed nasal colonization between two species of Old World monkeys (cynomolgus and rhesus) and a New World monkey (squirrel monkey) from breeding colony at Fiocruz (Brazil). Sixty male and female NHP with the average age of 1-21 years old, comprising twenty animals of each species, were analyzed. Nine different Staphylococcus species (S. aureus, S. cohnii, S. saprophyticus, S. haemolyticus, S. xylosus, S. warneri, S. nepalensis, S. simiae, and S. kloosi) were identified by MALDI-TOF and 16S rRNA gene sequence analyses. Antibiotic resistance was not detected among the isolated bacterial population. S. aureus was the main isolate (19 strains), present in all species, predominant in cynomolgus monkeys (9/20) and squirrel monkeys (7/20). spa typing was used to examine the clonal structure and genetic profile of Staphylococcus aureus isolates. Eight (8) spa types were identified among the S. aureus strains. A major cluster was identified, corresponding to a new spa type t20455, and no spa types found in this study were seen before in Brazil.

Early puberty in short-haired Guinea pigs kept in laboratory animal facilities.

Lab animals, such as Guinea pigs (Cavia porcellus), are crucial for scientific development, as they play an important role in the development and quality control chain of vaccines and drugs distributed by the Brazilian public health system. Investigating their biological and physiological parameters is fundamental to raise and keep these animals, so the handling of the facilities that hold them can be updated whenever new information comes up, with the well-being of the animals and alignment with the 3 Rs in mind. In the search for understanding reproductive aspects of Guinea pigs, the present study had the main goal of studying puberty by means of estrous cycle analysis in short-haired Guinea pigs. Guinea pigs have a vaginal occlusive membrane that covers the vaginal orifice. Its rupture takes place gradually and naturally, moments before labor and during estrus. The present study followed 42 females as for the presentation of the vaginal occlusive membrane. Once the membranes ruptured spontaneously, a swab was collected to study vaginal cytology. Membrane rupture was observed in 39 females; six females showed membrane rupture with less than 21 days of age (17 to 21 days). Twenty-three females were characterized as being in estrus due to cytology showing a prevalence of anucleated superficial cells. One of these females was younger than 21 days old. The opening of the vaginal occlusive membrane took place most frequently in intervals between 17 and 18 days, and the membrane remained open between one and three consecutive days. It was possible to follow three cycles of membrane opening on six females. The present study showed the need to adapt handling guidelines for C. porcellus kept in research animal facilities. The early age of puberty imposes the need of separate the female daughters from their fathers at 16 days old.

Low Aedes aegypti Vector Competence for Zika Virus from Viremic Rhesus Macaques.

Despite worldwide efforts to understand the transmission dynamics of Zika virus (ZIKV), scanty evaluation has been made on the vector competence of Aedes aegypti fed directly on viremic human and non-human primates (NHPs). We blood-fed Ae. aegypti from two districts in Rio de Janeiro on six ZIKV infected pregnant rhesus macaques at several time points, half of which were treated with Sofosbuvir (SOF). Mosquitoes were analyzed for vector competence after 3, 7 and 14 days of incubation. Although viremia extended up to eight days post monkey inoculation, only mosquitoes fed on the day of the peak of viremia, recorded on day two, became infected. The influence of SOF treatment could not be assessed because the drug was administered just after mosquito feeding on day two. The global infection, dissemination and transmission rates were quite low (4.09%, 1.91% and 0.54%, respectively); no mosquito was infected when viremia was below 1.26 × 105 RNA copies/mL. In conclusion, Ae. aegypti vector competence for ZIKV from macaques is low, likely to be due to low viral load and the short duration of ZIKV viremia in primates suitable for infecting susceptible mosquitoes. If ZIKV infection in human and macaques behaves similarly, transmission of the Zika virus in nature is most strongly affected by vector density.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Fecal glucocorticoid metabolites and assay validation: Stress response evaluation in captive brown howler monkeys (Alouatta clamitans).

BACKGROUND: The advent of non-invasive methods provides a powerful alternative to stress studies as the use of stressful handling techniques is no longer needed. However, many factors influence hormone metabolism such as sex, diet, and metabolic rate. Thus, validation should be species- and matrix-specific. METHODS: To assess stress response in brown howler monkeys Alouatta clamitans, we adopted an ACTH challenge test and parallelism to provide physiological and laboratorial validation. Radioimmunoassay was used to measure fecal levels of corticosterone. RESULTS AND CONCLUSIONS: All challenged animals presented a peak in fecal glucocorticoids levels the day after the treatment, while control animals did not. There were no significant sex differences, but females with infants had higher levels of corticosterone. Corticosterone levels showed parallelism to the standard curve of the diagnostics kit. Collectively, the data suggest that the method was validated and is useful for monitoring stress, thereby helping in conservation programs both in captivity and in the wild. Transit time information may be coupled with travel distance in seed dispersal studies.

Detection of mycobacterial infection in non-human primates using the Xpert MTB/RIF molecular assay.

Tuberculosis is a major public health concern, and diagnostic strategies applied to animal populations are scarce. As part of ongoing efforts to control tuberculosis dissemination at our animal facility, two non-human primates (NHP, Saimiri sciureus) presenting cutaneous lesions were examined for mycobacterial infection. Both animals tested positive for acid-fast bacilli and Mycobacterium tuberculosis using a molecular assay (IS6110 PCR). Animals were euthanized and several samples were tested for M. tuberculosis using the Xpert MTB/RIF assay. Many samples were positive for M. tuberculosis and rifampicin resistance, and some produced mycobacterial growth. Oral swabs from cage mates were then tested with Xpert MTB/RIF, and the majority tested positive for M. tuberculosis and rifampicin resistance, and produced growth in culture. To our knowledge, this is the first report of multidrug-resistant mycobacterial infection in NHP. Additionally, our data shows that the Xpert MTB/RIF assay can be useful as a screening tool for tuberculosis infection in NHP.

Evaluation of the fecal steroid concentrations in Alouatta belzebul (Primates, Atelidae) in the National Forest of Tapirape-Aquiri in Pará, Brazil.

BACKGROUND: The studies on fecal steroid metabolites published with free-living primates are limited mainly by the difficulty in obtaining samples. METHODS: A radioimmunoassay was used to measure the fecal steroid concentrations in Alouatta belzebul in the National Forest of Tapirape-Aquiri in Brazil. RESULTS AND CONCLUSIONS: Androgens were significantly higher for the adult males from the Area of Influence (AI-I group) when compared to those from the Control Area (CA group) (P < 0.05). Progestin and estrogen concentrations were higher in the females from the CA group than in those from the AI-I for both the adult females and females with offspring; however, P < 0.05 was only observed in the concentrations of fecal progestins from the adult females. The physiological differences between the AI-I and CA groups suggest that the cause was a sum of factors, such as an exposure to sound waves, feeding habits, daily activity patterns, and the habituation of the animals.

Renal evaluation of Aotus azarai infulatus by ultrasonography and serum chemistry profile.

This study aimed to characterize anatomical and biochemical properties of owl monkey kidneys in order to provide normal reference values. Sixty-nine Aotus azarai infulatus (45 males and 24 females) were divided into four different age groups (AG1: 3 months-1 year; AG2: 2-3 years; AG3: 4-6 years; and AG4: over 7 years old). The monkeys were evaluated with a serum chemistry profile, focusing on serum creatinine (SCr) and blood urea nitrogen (BUN) and with ultrasound. Mean body mass differed among the age groups. This significance was attributed to AG1 body mass being significantly lower than in AG2 and that in both AG2 and AG3 being significantly lower than in the two older age groups (AG3 and AG4). SCr and BUN concentrations differed significantly between the sexes and SCr level correlated positively with age. In contrast, renal measurements did not differ between males and females. Left and right renal volumes did not differ significantly within age groups, or among AG2, AG3, and AG4. Renal volumes in AG1, however, while not differing from those in AG2, did differ significantly from those in AG3 and AG4. In conclusion, this study provides ultrasonographic reference values for the morphology the kidneys in A. a. infulatus. Evidence is also provided that SCr and BUN levels in owl monkeys are influenced by the sex and age of the individual, factors that should be considered when interpreting test results.

Variation in clitoral length in rhesus macaques (Macaca mulatta).

Clitoromegaly in the neonatal period is an important morphologic sign that can be useful for sexual determination in aberrant cases. In rhesus monkeys, differentiation of the external genitalia occurs early during gestation (at 55 to 60 d) and is complete by approximately 80 d. Most of the critical steps in genital differentiation in primates occur prenatally. We sought to determine clitoral size in normal rhesus monkeys (Macaca mulatta) and possible effects of age and inheritance. Clitoral length was highly variable and had no relationship to fertility. Statistical evaluation revealed no association in the distribution of daughters with and without clitoris between mothers with and without clitoris. However, even when mated with several female monkeys, some male macaques produced primarily daughters without clitoris.