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.

Differential Yellow Fever Susceptibility in New World Nonhuman Primates, Comparison with Humans, and Implications for Surveillance.

A major outbreak of yellow fever (YF) occurred in Brazil during 2016-2018. Epizootics in New World nonhuman primates are sentinel events for YF virus circulation. However, genus-specific susceptibilities and suitability for YF surveillance remain poorly understood. We obtained and compared epidemiologic, histopathologic, immunohistochemical, and molecular results from 93 human and 1,752 primate cases submitted during the recent YF outbreak in Brazil (2017), with the support of the Brazilian National YF Surveillance Program. We detected heterogeneous YF-associated profiles among the various genera of primates we analyzed. Alouatta primates were the most reliable sentinel; Sapajus and Callicebus primates had higher viral loads but lower proportional mortality rates. Callithrix primates were the least sensitive, showing lower viral loads, lower proportional mortality rates, and no demonstrable YF virus antigen or extensive lesions in liver, despite detectable viral RNA. These differences in susceptibility, viral load, and mortality rates should be considered in strategic surveillance of epizootics and control measures for YF.

Yellow Fever as Cause of Death of Titi Monkeys (Callicebus Spp.).

From 2016 to 2018, an epidemic wave of yellow fever (YF) occurred in Brazil, affecting a large number of Platyrrhini monkeys. Titi monkeys (Callicebus spp.) were severely affected yet pathological characterizations are lacking. This study characterized epizootic YF in 43 titi monkeys (Callicebus spp.) with respect to the microscopic lesions in liver, kidney, spleen, heart, brain, and lung, as well as the distribution of immunolabeling for YF virus antigen, and the flaviviral load in the liver. Of 43 titi monkeys examined, 18 (42%) were positive for yellow fever virus (YFV) by immunohistochemistry or reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Affected livers had consistent marked panlobular necrotizing hepatitis, lipidosis, and mild inflammation, with intense immunolabeling for YFV mainly in centrilobular hepatocytes (zone 1; P = .05). In the spleen, consistent findings were variable lymphoid depletion (10/11), lymphoid necrosis (lymphocytolysis; 4/11), and immunolabeling for YFV in histiocytic cells (3/16). The main finding in the kidney was multifocal acute necrosis of tubular epithelium (5/7) that was occasionally associated with intracytoplasmic immunolabeling for YFV (6/15). These data indicate that titi monkeys are susceptible to YFV infection, developing severe hepatic lesions and high viral loads, comparable to humans and Alouatta spp. Thus, Callicebus spp. may be reliable sentinels for YF surveillance.

Histopathological kidney changes and myoglobinuria in neotropical non-human primates attacked by dogs, Brazil.

BACKGROUND: Non-human primates (NHPs) are susceptible to dogs' attacks, events that may cause muscle damage along with stress, and could be in some extent compatible with capture myopathy, a syndrome that results in myoglobinuria and renal damage. METHODS: We aimed to evaluate by histopathology pre-existing lesions and subsequent sequelae related to dogs' attacks, acute tubular necrosis (ATN) and myoglobinuria, as well as the usefulness of Pearls Stain and IHC to diagnose it. Histopathology was performed in available organs, and sections of kidney submitted to Prussian blue stain and myoglobin immunohistochemistry. RESULTS: During January 2014-June 2016, 16/145 (11%) of NHPs received by Adolfo Lutz Institute, Brazil were reported as attacked by dogs. A high frequency of young and debilitated animals was found. Myoglobinuria was observed in more than half animals (9/16; 56.2%), from which (5/9; 55.5%) presented ATN. CONCLUSIONS: Kidney lesions are plausible findings in NHPs attacked by dogs.

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.

Ultimobranchial body cyst in an adult horse: clinical, histopathologic, and immunohistochemical features.

A 9-y-old Mangalarga Marchador gelding was referred to a veterinary hospital because of a swelling on the upper right side of the neck. Ultrasound examination revealed a multilocular structure adjacent to the thyroid gland with low echogenic content suggestive of fluid. The mass was removed surgically. Histologically, the cystic cavities in the surgical sample were filled with abundant eosinophilic secreta and lined by cuboidal, segmentally ciliated, columnar epithelium with interspersed goblet cells. Segmental crowding of the multilayered lining of the cyst was noted. Immunohistochemistry suggested the presence of both C cells and follicular cells, given the positivity of the immunomarkers calcitonin and TTF-1, respectively. The histogenesis of ultimobranchial cysts is uncertain. Based on clinical, histopathologic, and immunohistochemical identification, the cystic structure in this case is compatible with an ultimobranchial body cyst.

Disseminated paracoccidioidomycosis in a captive western black-handed tamarin (Saguinus niger).

Paracoccidioidomycosis (PCM) is an endemic fungal disease that occurs in Latin America and primarily affects humans. The disease has been rarely documented in non-human primates. This report details a disseminated and fatal case of PCM caused by Paracoccidioides brasiliensis in a western black-handed tamarin (Saguinus niger) under human care. Histopathological examination revealed extensive pyogranulomatous inflammation in the lungs, spleen, liver, lymph nodes, kidneys, epididymis, right testicle, heart, adrenal gland and intestines, associated with characteristic yeast forms consistent with Paracoccidioides spp and confirmed by immunohistochemistry. Molecular analysis indicated a high nucleotide similarity with P. brasiliensis sequences for both the 18S rRNA and gp43 genes. This naturally occurring infection highlights the susceptibility of these animals to PCM and their role in ecoepidemiology warrants further investigation.

A rare case of primary hepatic poorly differentiated leiomyosarcoma in a goat.

A 12-year-old Saanen goat presented with a history of hyporexia and sudden recumbency. Euthanasia was indicated due to suspicion of hepatic neoplasia associated with senility. Necropsy revealed generalized oedema and increased liver size and weight (33 × 38 × 17 cm and 10.6 kg, respectively), with a firm, multilobular mass. Fusiform to polygonal neoplastic cells, with marked pleomorphism, anisocytosis and anisokaryosis, were seen on histopathological examination of the hepatic mass. The neoplastic cells were immunohistochemically positive for alpha-smooth muscle actin and vimentin but immunonegative for pancytokeratin. The Ki-67 index was 18.8%. A poorly differentiated leiomyosarcoma was diagnosed on the basis of the gross, histopathological and immunohistochemical findings and should be included in the differential diagnosis of liver disease in goats.

RBD and Spike DNA-Based Immunization in Rabbits Elicited IgG Avidity Maturation and High Neutralizing Antibody Responses against SARS-CoV-2.

Neutralizing antibodies (nAbs) are a critical part of coronavirus disease 2019 (COVID-19) research as they are used to gain insight into the immune response to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections. Among the technologies available for generating nAbs, DNA-based immunization methods are an alternative to conventional protocols. In this pilot study, we investigated whether DNA-based immunization by needle injection in rabbits was a viable approach to produce a functional antibody response. We demonstrated that three doses of DNA plasmid carrying the gene encoding the full-length spike protein (S) or the receptor binding domain (RBD) of SARS-CoV-2 induced a time-dependent increase in IgG antibody avidity maturation. Moreover, the IgG antibodies displayed high cross neutralization by live SARS-CoV-2 and pseudoviruses neutralization assays. Thus, we established a simple, low cost and feasible DNA-based immunization protocol in rabbits that elicited high IgG avidity maturation and nAbs production against SARS-CoV-2, highlighting the importance of DNA-based platforms for developing new immunization strategies against SARS-CoV-2 and future emerging epidemics.

CLINICO-PATHOLOGIC FINDINGS AND PATHOGEN SCREENING IN FUR SEALS (ARCTOCEPHALUS AUSTRALIS AND ARCTOCEPHALUS TROPICALIS) STRANDED IN SOUTHEASTERN BRAZIL, 2018.

Among pinnipeds, four Otariidae species (eared seals) have been reported as occasional or frequent vagrants along the coast of Brazil, mainly in the southern region. These animals usually arrive debilitated during winter and are directed to rehabilitation. Nevertheless, available information on sanitary aspects of stranded pinnipeds in Brazil is limited. Increased fur seal strandings (n=23) were recorded during the 2018 winter season in southeast Brazil (Iguape, Ilha Comprida, and Ilha do Cardoso, Sao Paulo State) compared to 2017 (n=2). Of these 23 fur seals, two were found dead and were in a good postmortem condition, and four died during rehabilitation and were subsequently necropsied. The remaining fur seals were not analyzed due to advanced decomposition (9/23) or successful rehabilitation (8/23). Herein, we report the antemortem hematology (n=4) and postmortem pathologic, parasitologic, and molecular analysis results as well as the most likely cause of stranding and/ or death (CSD) in five free-ranging juvenile South American fur seals (Arctocephalus australis) and one free-ranging juvenile subantarctic fur seal (Arctocephalus tropicalis). All animals were males, and all but one had poor body condition. Pathologic examinations revealed a variety of lesions, predominantly hemodynamic disturbances, endoparasitism, and inflammatory disease processes of suspected infectious nature. Molecular analyses detected gammaherpesvirus infections in two South American seals and one subantarctic fur seal, Sarcocystis sp. in one subantarctic fur seal, and Neospora spp. in two South American fur seals. All seals were PCR-negative for morbillivirus, flavivirus, and Toxoplasma gondii. The most likely CSDs were: starvation (2), aspiration pneumonia (1), asphyxia (1), predator attack (1), and presumed systemic infectious disease (1). These findings expand the geographic range of various pathogens of pinnipeds and may be of value to first responders, clinicians, and diagnosticians.

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.

Yellow fever surveillance challenge: Investigation of a marmoset non-autochthonous case.

Yellow fever (YF) surveillance in Brazil is focused mainly on the detection of epizootic events regarding New World non-human primates (NWNHP). We present a challenging case of a Callitrichidae (Callithrix spp) kept as a domiciliated pet that lived in the urban area of São Paulo municipality and was positive to YF virus by RT-qPCR and immunohistochemistry. After investigation, it was the first occurrence of non-autochthonous YF case of NWNHP described, with probable place of infection in the North shore of São Paulo state. This case illustrates the importance of coordinated laboratorial and field actions, and risks posed by transit of wildlife.

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.

Yellow fever surveillance challenge: Investigation of a marmoset non-autochthonous case

Yellow fever (YF) surveillance in Brazil is focused mainly on the detection of epizootic events regarding New World non-human primates (NWNHP). We present a challenging case of a Callitrichidae (Callithrix spp) kept as a domiciliated pet that lived in the urban area of São Paulo municipality and was positive to YF virus by RT-qPCR and immunohistochemistry. After investigation, it was the first occurrence of non-autochthonous YF case of NWNHP described, with probable place of infection in the North shore of São Paulo state. This case illustrates the importance of coordinated laboratorial and field actions, and risks posed by transit of wildlife.

Intestinal mucinous adenocarcinoma with metastases to epididymis, testis and tunica albuginea in a dog

A case of intestinal mucinous adenocarcinoma with metastasis to gonadal tissue is reported. A 13-year-old, male, poodle dog presented with intestinal and peritoneal masses, as well as infiltrative masses in testicular tunics. Samples were biopsied and submitted for histopathological analysis. Microscopically, intestinal lesion consisted of an adenocarcinoma (mucinous type), with infiltration of muscular layers and mesenteric adipose tissue. In gonadal tissue, there was neoplastic infiltration of epididymis and tunica albuginea (with a predominantly tubular pattern), and testicular parenchyma (with a predominantly signet-ring cell pattern). Immunohistochemistry was positive for CDX2 and pancytokeratin, and negative for vimentin, supporting the diagnosis of intestinal mucinous adenocarcinoma with metastases to epididymis, testis and tunica albuginea.

Steroidogenic factor 1 overexpression and gene amplification are more frequent in adrenocortical tumors from children than from adults.

BACKGROUND: Steroidogenic factor 1 (SF-1) is a key determinant of endocrine development and function of adrenal cortex. SF-1 overexpression and gene amplification were previously demonstrated in a small group of pediatric adrenocortical tumors. OBJECTIVE: Our objective was to determine the frequency of SF-1 protein expression and gene amplification in a large cohort of pediatric and adult adrenocortical tumors. PATIENTS: SF-1 protein expression was assessed in a cohort of 103 adrenocortical tumors from 36 children and 67 adults, whereas gene amplification was studied in 38 adrenocortical tumors (17 from children). METHODS: Tissue microarray, multiplex ligation-dependent probe amplification, and quantitative real-time PCR were used. RESULTS: A strong nuclear SF-1 expression was detected by tissue microarray in 56% (20 of 36) and 19% (13 of 67) of the pediatric and adult adrenocortical tumors, respectively (P = 0.0004). Increased SF-1 copy number was identified in 47% (eight of 17) and 10% (two of 21) of the pediatric and adult adrenocortical tumors, respectively (P = 0.02). All adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five (29%) pediatric adrenocortical tumors without SF-1 amplification. The frequency of SF-1 overexpression and gene amplification was similar in adrenocortical adenomas and carcinomas. CONCLUSION: We demonstrated a higher frequency of SF-1 overexpression and gene amplification in pediatric than in adult adrenocortical tumors, suggesting an important role of SF-1 in pediatric adrenocortical tumorigenesis.