A Tropical Composting Operation Unit at São Paulo Zoo as a Soure of Bacterial Proteolytic Enzymes

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the Sao Paulo Zoo Park (SPZPF), Sao Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Genomic Surveillance of Yellow Fever Virus Epidemic Waves in São Paulo, Brazil, 2017 – 2018

São Paulo (SP), a densely populated state in southeast Brazil that contains one of the world´s largest urban regions, has experienced its largest yellow fever virus (YFV) outbreak in decades. Surveillance in non-human primates (NHP) is important in order to detect YFV early during an epidemic or epizootic, to quantify the magnitude of the outbreak in NHP, and to evaluate the risk of YFV spillover infection in human populations. To better understand the genetic diversity and spatial distribution of YFV during the current outbreak in southeast Brazil, we generated 46 new virus genomes from YFV positive cases identified in 18 different municipalities in SP, mostly sampled from non-human primates between April 2017 and February 2018. Our data show that most NHP cases in São Paulo state were likely caused by the introduction of a single YFV lineage from Minas Gerais to São Paulo. Phylogenetic and phylogeographic analyses of these data indicate that YFV spread southwards from Minas Gerais into São Paulo state at a typical rate of <1km per day. These results shed light in the sylvatic transmission of yellow fever in highly fragmented forested regions in São Paulo state and highlight the importance of continued operational research and surveillance of zoonotic pathogens in sentinel populations.

Molecular and morphological characterization of Hepatozoon spp. in Brazilian snakes

The genus Hepatozoon represents one of six genera in the hemogregarine group. Some studies in snakes indicated effects in the host, from slight influences on fitness to severe effects on growth rate, reproduction and offspring survival rates. Diagnosis and identification are usually through blood smear analyses; but not all infected animals show parasitemia. Based on this, the present study established an adapted molecular protocol to identify Hepatozoon spp. to be used as a complementary test for routine diagnoses at the Clinical Analysis Laboratory at S?o Paulo Zoological Park Foundation. The study was conducted with 113 individuals. Microscopical analysis and molecular techniques were used to identify the parasite. Microscopic analyses showed 13.3% of the samples to be positive. The first pair of primers, targeting 18S rRNA gene, amplified parasite DNA in 6.3% of the samples. The second pair of primers, targeting Apicoplast fragment, were used only on samples that were identified microscopically as being positive, detecting the presence of parasite DNA in 93.3% of these. Phylogenetic analysis of the resulting sequences found five clusters for the 18S gene and five clusters for the Apicoplast fragment. Studies involving Hepatozoon spp. are still scarce and limited, mainly in snakes and the impacts of this parasite on the vertebrate host, so diagnostic studies are essential for wildlife conservation, especially in ex situ work.

Uso de imagens térmicas infra-vermelho para localização e captura de mico-leão-preto (Leontopithecus chrysopygus Mikan, 1823) in situ

Devido à dificuldade de capturar os animais diante dos sítios de pernoite utilizados pelos micos-leões pretos (Leontopithecus chrysopygus) da Floresta Nacional Capão Bonito, SP, avaliamos o uso de equipamento para captação de imagens térmicas infra-vermelho como método complementar para localização dos grupos e posterior captura para coleta de material biológico. Neste sentido, nós discutimos a hipótese de que o calor corpóreo pode aquecer o tronco da arvore onde os animais estavam abrigados, facilitando sua localização. O uso da termografia favoreceu a localização e captura dos grupos quando associado a identificação e georreferenciamento de ocos de arvores para posterior análise do gradiente térmico.

Estudo do Comportamento Reprodutivo do Gato-palheiro, Leopardus colocola (Molina, 1782) (Carnívora: Felidae) em Ambiente Cativo

Muitas espécies são difíceis de serem estudadas em ambiente natural devido aos hábitos reservados que impedem estudos ecológicos mais aprofundados, como por exemplo, investigações sobre a ecologia e comportamento reprodutivo. Este estudo objetivou avaliar os comportamentos reprodutivos do gato-palheiro (Leopardus colocola) mantido em zoológico, de forma a contribuir com informações sobre a espécie. Como resultados foram registrados e avaliados 598 comportamentos divididos entre individuais, sociais e reprodutivos. O ambiente cativo propiciou a oportunidade de gerar informações comportamentais sobre a espécie com foco no manejo e nos programas de reprodução ex situ.

Assessment of different sperm functional tests in golden-headed lion tamarins (Leontopithecus chrysomelas)

The golden‐headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil’s Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm‐egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage‐induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle‐specific tests. Our findings showed that sperm‐binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap‐freezing. Eosin‐nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome‐intact spermatozoa was found using Fast Green‐Rose Bengal (FG‐RB), Coomassie Blue (CB), and FITC‐PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV‐irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3′‐diaminobenzidine (DAB) and JC‐1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG‐RB, CB, TB, and DAB protocols.

Semen cryopreservation in golden-headed lion tamarin, Leontopithecus chrysomelas

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden‐headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden‐headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, suscept- ibility to oxidative stress, and sperm–egg‐binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short‐term measure for the conservation of golden‐headed lion tamarins.

Draft genome sequence of a multidrug-resistant CTX-M-65-producing Escherichia coli ST156 colonizing a giant anteater (Myrmecophaga tridactyla) in a Zoo

This study aimed to report the draft genome sequence of a multidrug-resistant (MDR) Escherichia coli colonizing a giant anteater (Myrmecophaga tridactyla) in a Brazilian Zoo.
The genome was sequenced using the Illumina MiSeq Platform and de novo genome assembly was performed using SPAdes v. 3.9. The draft genome sequence was annotated using NCBI Prokaryotic Genome Annotation Pipeline. Antibiotic resistance genes, virulence genes, sequence type, serotype and plasmid incompatibility groups were identified using tools from the Center for Genomic Epidemiology.

The genome presented 4970 coding sequences and a GC content of 50.2%. Several antimicrobial resistance genes associated with resistance to ß-lactams (blaTEM-1A and blaCTX-M-65), aminoglycosides [aph(6)-ld, aph(3″)-lb, aph(4)-la, aac(3)-lVa, aadA1 and aadA2], tetracyclines (tetB), sulphonamides (sul2 and sul3), trimethoprim (dfrA8 and dfrA12) and phenicols (floR and cmlA1) were identified. Moreover, mutations in quinolone resistance-determining regions (QRDR) were found. This E. coli isolate also presented virulence genes and belonged to serotype ONT:H25 and ST156 (CC156).

This is the first report of a draft genome sequence of a CTX-M-65-producing E. coli ST156 obtained from a zoo animal, which can be used by genomic surveillance platforms, in order to track transmission dynamics of extended-spectrum ß-lactamase (ESBL)-producing E. coli at the human-animal interface.

Giardia spp., ten years of parasitological data in the biggest zoo of Latin America

Giardia spp. (Diplomonadida: Hexamitidae) is an important and widely studied protozoan parasite with worldwide distribution. Nowadays have six described species, and the most important probably is Giardia duodenalis due to the zoonotical potential that some assemblages have. Many studies analysing samples from wild animals have detected Giardia in captive environment, including the zoonotic type. The aim of this study was to determine the prevalence of Giardia sp. in wild captive animals at Sao Paulo Zoo, using conventional parasitological techniques (direct smear, passive flotation with saturated sodium chloride solution and simple gravity sedimentation), from 2006 to 2016. In total, 7066 coprological exams were performed during this period with samples from mammals, birds, reptiles and amphibians. The prevalence of Giardia infections was of 1.5% (103/7066). Mammals had the higher prevalence of infections with 2% (77/3872), followed by birds with 1.1% (25/2186) and reptiles with only one positive sample (1/894). All samples from amphibians were negative. Representatives of thirteen families presented positive results for this parasite: Dromaidae, Phasianidae, Ramphastidae, Cervidae, Giraffidae, Canidae, Felidae, Herpestidae, Myrmecophagidae, Callithrichidae, Cebidae, Hylobatidae and Dipsadidae. This study presents the first report of Giardia sp. in Pavo muticus and Brachyteles arachnoides. Infections were prevalent in Cebidae and Ramphastidae species. The findings of this study highlight the importance of identifying which Giardia assemblage are involved in the infections and if they may have a zoonotic potential.