Vector Competence of Aedes albopictus for Yellow Fever Virus: Risk of Reemergence of Urban Yellow Fever in Brazil.

The risk of the emergence and reemergence of zoonoses is high in regions that are under the strong influence of anthropogenic actions, as they contribute to the risk of vector disease transmission. Yellow fever (YF) is among the main pathogenic arboviral diseases in the world, and the Culicidae Aedes albopictus has been proposed as having the potential to transmit the yellow fever virus (YFV). This mosquito inhabits both urban and wild environments, and under experimental conditions, it has been shown to be susceptible to infection by YFV. In this study, the vector competence of the mosquito Ae. albopictus for the YFV was investigated. Female Ae. albopictus were exposed to non-human primates (NHP) of the genus Callithrix infected with YFV via a needle inoculation. Subsequently, on the 14th and 21st days post-infection, the legs, heads, thorax/abdomen and saliva of the arthropods were collected and analyzed by viral isolation and molecular analysis techniques to verify the infection, dissemination and transmission. The presence of YFV was detected in the saliva samples through viral isolation and in the head, thorax/abdomen and legs both by viral isolation and by molecular detection. The susceptibility of Ae. albopictus to YFV confers a potential risk of reemergence of urban YF in Brazil.

New records of Amblyomma ticks parasitizing neotropical primates in Brazil.

Amblyomma is an important tick genus for animal and human health, with some species being the vectors of zoonotic pathogens, such as Rickettsia rickettsii, in the Neotropical region. Knowing their hosts may help to understand the distribution of these agents and decrease the occurrence of clinical cases. Primates are intelligent and adaptable animals that can get close to humans in the search for food. So, they may be an important epidemiological link for the spread of these ticks. Beyond that, primates also suffer from these infections, serving as sentinels for different diseases. Thus, the present study aims to report the parasitism by Amblyomma spp. on six species of Neotropical primates from different locations in Brazil. The 337 collected ticks were morphologically identified using stereomicroscopes and taxonomic keys, and six distinct species of ticks were identified. We report here the first record of nymphs of the tick species Amblyomma cajennense sensu stricto on Alouatta belzebul, a male of Amblyomma fuscum on Alouatta guariba clamitans, nymphs of Amblyomma sculptum on Leontopithecus chrysopygus and Callithrix aurita, as well as nymphs of Amblyomma geayi on Saimiri collinsi. Of the 337 tick specimens collected, 256 (75,96%) were nymphs. The importance of primates in the life cycle of these species remains to be elucidated.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy.

BACKGROUND: Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE: To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS: Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS: Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS: Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp. ) I: gross anatomical and histopathological findings in organs at necropsy

BACKGROUND Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Collection, Evaluation, and Coagulum Dissolution of Semen from Goeldi's Monkey, Callimico goeldii.

Research on Neotropical primates' reproduction is necessary due to the lack of available information and the increasing threat to these species. Callimico goeldii is listed as Vulnerable on the IUCN Red List of Threatened Species. This study aimed to test rectal electrostimulation for semen collection and evaluate seminal characteristics. Therefore, semen from 6 captive Goeldi's monkeys was collected and, for the first time, seminal characteristics are described. Coagulum formation was noted in all ejaculates, and we obtained partial or complete liquefaction of the samples. Results were (means ± SD): volume = 26.9 ± 11.87 µL; pH = 7.61 ± 0.28; concentration = 143.18 ± 174.96 × 106 spermatozoa/mL; total sperm motility = 83.33 ± 5.16%; linear progressive motility = 46 ± 24.08%; plasma membrane integrity = 36.38 ± 16.11%; acrosome integrity using fast-green/bengal-rose staining = 63.41 ± 11.72%, and kit Spermac® = 69.36 ± 11.81%; abnormal sperm = 72.5 ± 17.7%, with 16.2 ± 7.7% major defects and 56.3 ± 10% minor defects; sperm with high mitochondrial activity class I = 16.45 ± 22.25%. Rectal electrostimulation was an efficient method for semen collection in this species. Investigations are required to improve semen collection and handling, including cryopreservation methods.

Antibody-enhanced dengue disease generates a marked CNS inflammatory response in the black-tufted marmoset Callithrix penicillata.

Severe dengue disease is often associated with long-term neurological impairments, but it is unclear what mechanisms are associated with neurological sequelae. Previously, we demonstrated antibody-enhanced dengue disease (ADE) dengue in an immunocompetent mouse model with a dengue virus 2 (DENV2) antibody injection followed by DENV3 virus infection. Here we migrated this ADE model to Callithrix penicillata. To mimic human multiple infections of endemic zones where abundant vectors and multiple serotypes co-exist, three animals received weekly subcutaneous injections of DENV3 (genotype III)-infected supernatant of C6/36 cell cultures, followed 24 h later by anti-DENV2 antibody for 12 weeks. There were six control animals, two of which received weekly anti-DENV2 antibodies, and four further animals received no injections. After multiple infections, brain, liver, and spleen samples were collected and tissue was immunolabeled for DENV3 antigens, ionized calcium binding adapter molecule 1, Ki-67, TNFα. There were marked morphological changes in the microglial population of ADE monkeys characterized by more highly ramified microglial processes, higher numbers of trees and larger surface areas. These changes were associated with intense TNFα-positive immunolabeling. It is unclear why ADE should generate such microglial activation given that IgG does not cross the blood-brain barrier, but this study reveals that in ADE dengue therapy targeting the CNS host response is likely to be important.

Polymorphic color vision in captive Uta Hick's cuxiús, or bearded sakis (Chiropotes utahickae).

The pitheciines (Chiropotes, Pithecia, and Cacajao) are frugivorous Neotropical primates that specialize on the predation of seeds from unripe fruits, usually cryptic against the foliage. However, little is known about the color vision distribution within this taxon, and even less about the abilities shared by these animals regarding discrimination of chromatic targets. The aim of this study was to evaluate the color vision perception of captive Uta Hick's cuxiús, or bearded sakis (Chiropotes utahickae) through a behavioral paradigm of color visual discrimination, as well as to estimate, by genetic studies, the number and kinds of medium to long wavelength cone photopigment (opsins) encoded by this species. Among 12 cuxiús (7 males and 5 females) studied only 1 female was diagnosed as a trichromat. Results from genotyping were in line with our behavioral data and showed that cuxiús carried one (dichromat) or two (trichromat) medium to long wavelength pigments alleles, demonstrating a color vision polymorphism in C. utahickae similar to the majority of Neotropical Primates.

Callithrix penicillata: a feasible experimental model for dengue virus infection.

Although the murine models have the feasibility to reproduce some signs of dengue Virus (DENV) infection, the use of isogenic hosts with polarized immune response patterns does not reproduce the particularities of human disease. Our goal was to investigate the kinetics of peripheral blood biomarkers in immunocompetent Callithrix penicillata non-human primates subcutaneously infected with DENV-3. The viral load of infected animals was determinated by quantitative real time PCR. Measurements of DENV-3/IgM were performed, and several parameters were assessed by hemogram: red blood cells count, hemoglobin, hematocrit, white blood cells count, neutrophils, monocytes, lymphocytes, and platelets count. The coagulogram was performed by prothrombin time (PT), and activated partial thromboplastin time (APTT) assays. The renal function was monitored by urea and creatinine, and the liver function by the aspartate (AST), and alanine (ALT) aminotransferases. Also, the level of the cytokines IL-6, TNF-α, IL-2, IFN-γ, IL-4 and IL-5 was quantified during the experimental study. Data analysis was performed considering relevant differences when baseline fold changes were found outside from 0.75 to 1.5 range. Our data demonstrated that infected animals presented relevant signs of dengue disease, including peaks of viremia at 5 days-post-infection (dpi), peaks of anti-DENV-3 IgM at 15 dpi and hemaglutination inhibition assay (HIA) from 15 to at 60 dpi. Despite early monocytosis, slight neutrophilia and lymphocytosis, animals developed persistent leucopenia starting at 4 dpi. Anemia episodes were steady at 3-4 dpi. Patent thrombocytopenia was observed from 1 to 15 dpi with sporadic decrease of APTT. A substantial increase of ALT and AST was observed with higher peak at 4 dpi. Moreover, early increases of TNF-alpha and IFN-gamma besides late increase of IFN-gamma were observed. The analysis of biomarkers network pointed out two relevant strong axes during early stages of dengue fever, a protective axes TNF-alpha/Lymphocytes/Platelets, and a pathological IL-2/IL-6/Viremia/Monocyte/PT bond. Later on, the biomarker network highlighted the interaction IFN-gamma/PLT/DENV-3(IgM;HAI)/PT, and the involvement of type-2 cytokines (IL-4; IL-5). Our findings demonstrated that C. penicillata is a feasible experimental model for dengue virus infection, which could be useful to pathogenesis studies, discovery of novel antiviral drugs as well as to evaluate vaccine candidates against DENV.

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.

Conservação do sêmen e liquefação do coágulo seminal de macaco-prego (Cebus apella) em água de coco em pó (ACP-118®), em diferentes temperaturas / Semen conservation and seminal clot liquefaction of capuchin monkey (Cebus apella) in powdered coconut water extender (PCW) at different temperatures

O objetivo do estudo foi avaliar a água de coco em pó (ACP) na conservação do sêmen e liquefação do coágulo seminal de Cebus apella. O sêmen de seis machos adultos foi coletado por eletroejaculação (EEJ), diluído em solução à base de ACP-118® e submetido à incubação em banho-maria a 33, 35 e 37°C, por 24 horas. Avaliou-se a integridade espermática por meio da coloração eosina-nigrosina a cada uma hora durante as seis horas iniciais e após 24 horas de incubação. Os volumes médios e as concentrações espermáticas das frações coagulada e líquida foram de 0,20±0,02 e 0,20±0,10mL; 1,1±0,3x10(8) e 1,3±0,9x10(7) espermatozoides mL-1, respectivamente. Somente em uma amostra da fração líquida foram verificados espermatozoides com motilidade (20 por cento) e vigor (4), perdurando por 40 minutos. A maior parte do coágulo liquefez em ACP-118® após 12 horas de incubação. O melhor tratamento observado foi sob 33°C, por manter até 47±12,8 por cento de espermatozoides vivos após 24 horas. Conclui-se que o diluente à base de ACP é eficiente na liquefação do coágulo seminal e na manutenção da integridade espermática até 24 horas após a EEJ, nas temperaturas de 33, 35 e 37°C.

The aim of this study was to evaluate the powdered coconut water (PCW) in the semen conservation and seminal clot liquefaction. The semen of six adult male Cebus apella was collected by electroejaculation (EEJ), diluted in ACP-118® extender and stayed in water bath at 33, 35 and 37°C for 24 hours. The sperm integrity was evaluated by eosin-nigrosine staining every one hour during the six initial hours and after 24 hours of incubation. The average volumes and sperm concentrations of clotted and liquid fractions were 0.20±0.02 and 0.20±0.10mL, 1.1±0.3x108 and 1.3±0.9x107 sperm mL-1, respectively. Immediately after collection, only in a sample of liquid fraction was observed 20 percent motility and vigor 4, which stopped after 40 minutes. Most of the clot was liquefied in ACP-118® after 12 hours of incubation. The best observed treatment was 33°C, because it kept 47±12.8 percent of sperm integrity after 24 hours. It was concluded that the PCW extender is effective in the liquefaction of seminal clot and maintenance of sperm viability 24 hours after the EEJ at 33, 35 and 37°C.

Uterine evaluation and gestation diagnosis in owl monkey (Aotus azarai infulatus) using the B mode ultrasound.

BACKGROUND: Gynecological and obstetrical ultrasonography has become an indispensable tool in the routine management, health evaluation and research on captive non-human primates. METHODS: Ultrasound was used to evaluate the uterus and estimate the gestation of owl monkeys. Twelve couples were selected, where five were primiparous and seven multiparous females from the National Primate Center reproductive colony, Ananindeua-PA, Brazil. The procedures were carried out using the GE Logiq 100 MP, equipped with a 7.5 MHz linear probe. RESULTS: The females showed a simple uterus, of elongated shape, regular outline and homogeneous echogenic texture. In the uterine measurements craniocaudal diameter, dorsoventral diameter and uterine volume (UV), significant differences were identified (P < 0.05) between ultrasound examinations of primiparous and multiparous females. The UV showed a positive correlation with the number of births. The gestational sac and the embryonic echo were visible between 28 and 38 days after mating. Between 48 and 68 days after mating, embryonic death was identified in all the gestations. CONCLUSIONS: The chemical (use of tranquilizers) and husbandry factors (capture stress) may be related to the prenatal death. The establishing methods of conditioning the female to the ultrasonographic exam may offer a solution to this problem.

Pelvimetria em macacos-da-noite (Aotus azaraiinfulatus – KUHL, 1820 / Pelvimetry on owl monkeys (Aotus azarai infulatus – KUHL, 1820)

Analisou-se os diâmetros pelvicos de 72 Aotus azarai infulatus, adultos, 42 machos e 30 fêmeas não prenhes, correlacionando-os com o dimorfismo sexual, a biometria corpórea e a origem dos animais por meio de radiografias em projeção ventro-dorsal, digitalizadas. As médias verificadas foram: para o comprimento do corpo 30,94 cm; comprimento da cauda 35,63 cm; perímetro do tórax 18,97 cm; perímetro da pelve 17,11 cm e o peso 0,96 Kg, em média. As médias dos diâmetros pélvicos foram de 2,64 cm para o diâmetro diagonal direito; 2,66 cm para o diâmetro diagonal esquerdo; 1,97 cm para o diâmetro biilíaco médio; 1,41 cm para o diâmetro biilíaco superior;1,58 cm para o diâmetro biilíaco inferior; 2,48 cm para o diâmetro sacro-púbico e 3,85 cm(elevado a 2) para a área de entrada da pelve. Concluiu-se que, tanto nos machos quanto nas fêmeas, a pelve de Aotus azarai infulatus pode ser classificada como dolicopélvica, tendo-se verificado dimorfismo sexual pélvico nos adultos.

Related data to the diameters of the pelvis from 72 Aotus azarai infulatus, owl monkeys, 42 adult males and 30 adult non-pregnant females, were obtained in vivo by ventrodorsal projection radiographic exams, correlated with sex, the measures of the body and their origin.The mean values of the body length (30.94 cm), tail length (35.63cm), thoracic perimeter (18.97 cm), hip perimeter (17.11 cm) and the weight (0.96 Kg) were verified. The radiographic images were digitalized and measured, and the mean values were 1.41 cm for the superior biiliac diameter; 1.58 cm for the inferior biiliac diameter; 1.97cm for the medium biiliac diameter; 2.64 cm for the right diagonal diameter; 2.66 cm for the left diagonal diameter; 2.48 cm for thesacrum-pubic diameter; 3.85 cm2 for the inlet pelvic area. In conclusion, the pelvis from males and females Aotus azarai infulatus can be classified as dolicopelvic and there is pelvic sexual dimorphism in adult owl monkeys (Aotus azarai infulatus).