Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 - 2018.

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.

Myeloid and histiocytic sarcomas in subantarctic fur seals Arctocephalus tropicalis, Brazil.

Histopathological and immunohistochemical methods were used to diagnose round cell tumors in 2 subantarctic fur seals Arctocephalus tropicalis with marked anemia. Although wild-born, both individuals were placed under human care while juveniles in a Brazilian aquarium. Both pinnipeds were PCR tested for herpesvirus, and 1 was infected with otariid gammaherpesvirus 5 (OtHV-5), previously described in a subantarctic fur seal stranded in Brazil. Although some gammaherpesviruses can cause sarcomas and other neoplasms, it was not possible to definitively associate OtHV-5 with the neoplasm. To our knowledge, these are the first neoplasm records in subantarctic fur seals.

Benign Peripheral Nerve Sheath Tumor in a Wild Toco Toucan ( Ramphastos toco ).

Peripheral nerve sheath tumors are a heterogeneous group of neoplasms that comprise neurofibromas, schwannomas, neurilemmomas, and perineuromas. In animals, peripheral nerve sheath neoplasms are most commonly diagnosed in dogs and cattle, followed by horses, goats, and cats, but their occurrence is uncommon in birds. An adult, free-living, male toco (common) toucan ( Ramphastos toco ) was admitted to the zoo animal clinic with weight loss, dehydration, and presence of a soft nodule adhered to the medial portion of the left pectoral muscle. Clinical, cytologic, and computed tomography scan results were indicative of a neoplasm. The toucan died during surgical resection of the mass. Necropsy, histopathologic, and immunohistochemical findings confirmed the diagnosis of benign peripheral nerve sheath tumor. To our knowledge, benign peripheral nerve sheath tumor has not previously been reported in a toucan or any other species in the order Piciformes.

Cytokine profile and parasite load in lymph nodes of dogs naturally infected with Leishmania infantum from distinct epidemiological scenarios in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.

Feline Sporothrix spp. detection using cell blocks from brushings and fine-needle aspirates: Performance and comparisons with culture and histopathology.

BACKGROUND: Sporotrichosis is an emerging zoonotic mycosis that presents as a cutaneous lymphatic or disseminated disease, caused by fungi from the Sporothrix schenkii (S schenkii) clinical clade. Its importance is growing, primarily due to an outbreak that occurred in Brazil, affecting mainly cats and people. OBJECTIVES: In Brazil, an S schenkii diagnosis is often made using cultures, which allows genus identification and sufficient growth to perform molecular biology testing. Despite its advantages, fungal cultures are slow to develop and can delay public health measures, highlighting the importance of developing additional diagnostics techniques. METHODS: Cell block cytology (CBLC) is an older method that regained importance after liquid-based cytology (LBC) was introduced, and it has been previously and successfully applied to veterinary diagnostics. We aimed to standardize and compare CBLC from cervical brush exfoliation of open wounds and fine-needle aspirates with culture and immunohistochemistry of skin biopsies for sporotrichosis in cats, as a novel method. RESULTS: For this purpose, we selected 40 cats with skin lesions suspected of having sporotrichosis in Guarulhos city, São Paulo state, Brazil. We achieved 97.5% and 95% positivity using CBLC and culture, respectively, and 100% of feline skin biopsies were positive for Sporothrix spp on histopathology/immunohistochemistry. CONCLUSIONS: Cell block cytology is an efficient and rapid tool to diagnose sporotrichosis in cats, particularly during epidemics.

Comparative Immunopathology of Cetacean morbillivirus Infection in Free-Ranging Dolphins From Western Mediterranean, Northeast-Central, and Southwestern Atlantic.

Cetacean morbillivirus (CeMV; Paramyxoviridae) causes epizootic and interepizootic fatalities in odontocetes and mysticetes worldwide. Studies suggest there is different species-specific susceptibility to CeMV infection, with striped dolphins (Stenella coeruleoalba), bottlenose dolphins (Tursiops truncatus), and Guiana dolphins (Sotalia guianensis) ranking among the most susceptible cetacean hosts. The pathogenesis of CeMV infection is not fully resolved. Since no previous studies have evaluated the organ-specific immunopathogenetic features of CeMV infection in tissues from infected dolphins, this study was aimed at characterizing and comparing immunophenotypic profiles of local immune responses in lymphoid organs (lymph nodes, spleen), lung and CNS in CeMV-molecularly (RT-PCR)-positive cetaceans from Western Mediterranean, Northeast-Central, and Southwestern Atlantic. Immunohistochemical (IHC) analyses targeted molecules of immunologic interest: caspase 3, CD3, CD20, CD57, CD68, FoxP3, MHCII, Iba1, IFNγ, IgG, IL4, IL10, lysozyme, TGFß, and PAX5. We detected consistent CeMV-associated inflammatory response patterns. Within CNS, inflammation was dominated by CD3+ (T cells), and CD20+ and PAX5+ (B cells) lymphocytes, accompanied by fewer Iba1+, CD68+, and lysozyme+ histiocytes, mainly in striped dolphins and bottlenose dolphins. Multicentric lymphoid depletion was characterized by reduced numbers of T cells and B cells, more pronounced in Guiana dolphins. Striped dolphins and bottlenose dolphins often had hyperplastic (regenerative) phenomena involving the aforementioned cell populations, particularly chronically infected animals. In the lung, there was mild to moderate increase in T cells, B cells, and histiocytes. Additionally, there was a generalized increased expression of caspase 3 in lymphoid, lung, and CNS tissues. Apoptosis, therefore, is believed to play a major role in generalized lymphoid depletion and likely overt immunosuppression during CeMV infection. No differences were detected regarding cytokine immunoreactivity in lymph nodes, spleen, and lung from infected and non-infected dolphins by semiquantitative analysis; however, there was striking immunoreactivity for IFNγ in the CNS of infected dolphins. These novel results set the basis for tissue-specific immunophenotypic responses during CeMV infection in three highly susceptible delphinid species. They also suggest a complex interplay between viral and host's immune factors, thereby contributing to gain valuable insights into similarities, and differences of CeMV infection's immunopathogenesis in relation to body tissues, CeMV strains, and cetacean hosts.

Molecular, serological, pathological, immunohistochemical and microbiological investigation of Brucella spp. in marine mammals of Brazil reveals new cetacean hosts.

Brucella-exposure and infection is increasingly recognized in marine mammals worldwide. To better understand the epidemiology and health impacts of Brucella spp. in marine mammals of Brazil, molecular (conventional PCR and/or real-time PCR), serological (Rose Bengal Test [RBT], Competitive [c]ELISA, Serum Agglutination Test [SAT]), pathological, immunohistochemical (IHC) and/or microbiological investigations were conducted in samples of 129 stranded or by-caught marine mammals (orders Cetartiodactyla [n = 124], Carnivora [n = 4] and Sirenia [n = 1]). Previous serological tests performed on available sera of 27 of the 129 animals (26 cetaceans and one manatee), indicated 10 seropositive cetaceans. Conventional PCR and/or real-time PCR performed in cases with available organs (n = 119) and/or blood or swabs (n = 10) revealed 4/129 (3.1%) Brucella-infected cetaceans (one of them with positive serology; the remaining three with no available sera). Pathological, IHC and/or microbiological analyses conducted in PCR/real-time PCR and/or seropositive cases (n = 13) revealed Brucella-type lesions, including meningitis/meningoencephalitis, pneumonia, necrotizing hepatitis, pericarditis and osteoarthritis in some of those animals, and positive IHC was found in all of them (excepting two live-stranded animals without available organs). Brucella spp. culture attempts were unsuccessful. Our results demonstrated exposure, asymptomatic, acute and chronic Brucella sp. infection in several cetacean species in the Brazilian coast, highlighting the role of this pathogen in stranding and/or death, particularly in Clymene dolphin (Stenella clymene) and short-finned pilot whale (Globicephala macrorhynchus) off Ceará State. Novel hosts susceptible to Brucella included the franciscana (Pontoporia blainvillei), the Guiana dolphin (Sotalia guianensis) and the spinner dolphin (Stenella longirostris). Additionally, three coinfection cases involving Brucella spp. and cetacean morbillivirus, Edwarsiella tarda and Proteus mirabilis were detected. To the best of our knowledge, this is the first long-term and large-scale survey of Brucella spp. in marine mammals of South America, widening the spectrum of susceptible hosts and geographical distribution range of this agent with zoonotic potential.

Experimental Zika virus infection induces spinal cord injury and encephalitis in newborn Swiss mice.

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America, with neurological symptons including meningoencephalitis and Guillain-Barré syndrome in adults, besides an apparent increased incidence of microcephaly in infants born to infected mothers. It is becoming a necessity to have a trustworthy animal model to better understand ZIKV infection. In this study we used newborn white Swiss mice as a model to investigate the ZIKV strain recently isolated in Brazil. ZIKV was inoculated via intracerebral and subcutaneous routes and analysed through gross histopathology and immunohistochemistry. Here we demonstrated first that the intracerebral group (ICG) displayed severe cerebral lesions, with neuronal death, presence of apoptotic bodies, white matter degeneration and neutrophil perivascular cuffing. In the subcutaneous group (SCG), we observed moderate cerebral lesions, morphologically similar to that found in ICG and additional myelopathy, with architectural loss, marked by neuronal death and apoptotic bodies. Interestingly, we found an intense astrogliosis in brain of both groups, with increased immunoexpression of GFAP (glial fibrillary acidic protein) and presence of hypertrophic astrocytes. The spinal cord of subcutaneous group (SCG) exhibited reduction of astrocytes, but those positive for GFAP were hypertrophic and presented prolonged cellular processes. Finally significant lesions in the central nervous system (CNS) were present in newborn mice inoculated by both routes, but SCG method led to an important neurological manifestations (including myelopathy), during a longer period of time and appears for us to be a better model for ZIKV infection.