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.

A comparison of rectal and oral cultivable microbiota in wild and captive black lion tamarins (Leontopithecus chrysopygus, Mikan 1823)

The black lion tamarin (Leontopithecus chrysopygus) is an endangered primate species, restricted to the Atlantic Forest fragments of Sao Paulo state, Brazil, with an estimated wild population of ~1600 individuals. Integrative studies between zoo (ex situ) and wild (in situ) animals are crucial to modern conservation programs. They can demonstrate a substantial impact with the One Health concept, an interdisciplinary research frontier regarding the relations between human, animal, and environmental health. Studies of wild populations of Leontopithecus spp. are scarce and should be encouraged to provide baseline information to develop preventive and curative medicine in zoos and other conservation programs. Studying these animals in the wild can offer important reference parameters for the species. Comparing bacterial communities between in situ and ex situ populations can help us understand both conditions and the dynamics of potentially pathogenic microorganisms. To increase our understanding of resident microorganisms among these groups, we collected oral and rectal samples from captive (zoo) and wild black lion tamarins. We employed a culture method for the identification of aerobic bacteria. Thirty-three specimens were sampled (24 zoo and 8 wild animals) and 18 bacterial genera were identified. We found primarily Gram-positive bacteria in wild animals, whereas in zoo animals, Gram-negative bacteria were dominant. Some of the bacterial species we identified are potentially pathogenic, whereas several others are being reported here for the first time in this host species. Our results reinforce the importance of integrative studies for the future management and conservation of this endangered primate species.

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.

Arthropod-borne pathogens circulating in free-roaming domestic cats in a zoo environment in Brazil

Recently, tick and flea-borne pathogens have been detected in wild carnivores maintained in captivity inBrazilian zoos. Since free-roaming cats are frequently found in Brazilian zoos, they could act as reservoirsfor arthropod-borne pathogens, which could be transmitted to endangered wild carnivores maintainedin captivity in these institutions. On the other hand, stray cats in zoos may play a role as sentinels topathogens that circulate among wild animals in captivity. The present work aimed to detect the presenceof Anaplasmataceae agents, hemoplasmas, Bartonella species, piroplasmas, and Hepatozoon sp. DNA inblood samples of 37 free-roaming cats in a Brazilian zoo. Three (8%) cats were positive for Anaplasma spp.closed related to Anaplasma phagocytophilum; 12 (32%) cats were positive for hemoplasmas [two (5%)for Mycoplasma haemofelis, five (13.5%) for Candidatus Mycoplasma haemominutum, and five (13.5%) forCandidatus Mycoplasma turicensis]; 11 (30%) were positive for Bartonella spp., six (16%) were positiveBabesia vogeli and one (3%) for Theileria sp. Coinfection with multiple arthropod-borne agentes wasobserved in sampled cats. None of sampled cats were positive for Ehrlichia spp., Cytauxzoon spp., orHepatozoon spp. in PCR. This is the first molecular detection of Babesia vogeli and Theileria sp. in domesticcats in Brazil. The control of the population of free-roaming cats in these conservation institutions ismuch needed aiming to prevent the potential transmission to endangered wild animals maintained incaptivity, such as wild neotropical wild felids, as well as to human beings visiting zoos

Plasmodium (Novyella) nucleophilum from an Egyptian Goose in São Paulo Zoo, Brazil: microscopic confirmation and molecular characterization

Plasmodium (Novyella) nucleophilum was identified using microscopy and PCR, in an Egyptian Goose (Alopochen aegyptiacus) that died in São Paulo Zoo, Brazil. This parasite is characterized by elongated gametocytes, small meronts with scant cytoplasm, less than eight merozoites and mainly for having all the stages appressed to the nuclei of infected erythrocytes. Additionally, Plasmodium (Haemamoeba) sp. was identified by microscopy in the same blood sample. The latter parasite lacks nucleophilic blood stages and is characterized by large roundish trophozoites, each with a large prominent centrally collated vacuole. This co-infection was not confirmed by PCR amplification of the mitochondrial cytochrome b (cytb) gene and sequencing; only one Plasmodium sp. cytb sequence was detected in the blood sample. Since parasitemia of P. nucleophilum (2.4%) was much higher than that of P. (Haemamoeba) sp. (0.2%), PCR may have favored the amplification of the cytb sequence of the former. Phylogenetic analysis is in agreement with this conclusion because the reported cytb sequence was positioned in the same branch of sequences of several Novyella species. This is the first assignment of the mitochondrial cytb gene sequence to P. nucleophilum. The P. (Haemamoeba) parasite is particularly similar to Plasmodium (Haemamoeba) tejerai, because its advanced trophozoites and young erythrocytic meronts possess a large vacuole with prominent pigment granules arranged around it, the characteristic features of development in this species. For definitive identification of P. (Haemamoeba) species, mature meronts and gametocytes are required; however, these were absent from the thin blood smear. Representative images of the blood stages of P. nucleophilum and P. (Haemamoeba) sp. are provided. Together with microscopy data, the P. nucleophilum cytb sequence will assist in molecular identification (barcoding) of this Plasmodium species in other birds.