Carlos Sacristán

Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain

The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified: one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method.

Lacaziosis-like disease in Tursiops truncatus from Brazil: a histopathological and immunohistochemical approach

Cetacean lacaziosis-like disease or lobomycosis-like disease (LLD) is a chronic skin condition caused by a non-cultivable yeast of the order Onygenales, which also includes Lacazia loboi, as well as Paracoccidioides brasiliensis and P. lutzii, respectively responsible for lacaziosis and paracoccidioidomycosis in humans. Complete identification and phylogenetic classification of the LLD etiological agent still needs to be elucidated, but preliminary phylogenetic analyses have shown a closer relationship of the LLD agent to Paracoccidioides spp. than to L. loboi. Cases of LLD in South American cetaceans based on photographic identification have been reported; however, to date, only 3 histologically confirmed cases of LLD have been described. We evaluated multiple tissue samples from 4 Tursiops truncatus stranded in the states of Santa Catarina (n = 3) and Rio Grande do Sul (n = 1), southern Brazil. Macroscopically, all animals presented lesions consistent with LLD. Hematoxylin-eosin, periodic acid-Schiff, Grocotts methenamine silver, and Mayers mucicarmin stains were used for histological evaluation. Microscopically, numerous refractile yeasts (4-9 µm in diameter) were observed in skin samples (4/4), and for the first time in dolphins, also in a skeletal muscle abscess (1/4). Immunohistochemistry using anti-P. brasiliensis glycoprotein gp43 as a primary antibody, which is known to cross-react with L. loboi and the LLD agent, was performed and results were positive in all 4 cases. We describe 3 new cases of LLD in cetaceans based on histopathology and immunohistochemistry. This is the first report of LLD in the muscle of cetaceans.

A retrospective pathology study of two Neotropical deer species (1995-2015), Brazil: Marsh deer (Blastocerus dichotomus) and brown brocket deer (Mazama gouazoubira)

This retrospective study describes the biological and epidemiological aspects, gross and microscopical findings, and most likely causes of death (CD) in two species of Neotropical deer in Brazil. The animals were collected between 1995 and 2015 and represented 75 marsh deer (MD) and 136 brown brocket deer (BBD). Summarized, pneumonia was diagnosed microscopically in 48 MD and 52 BBD; 76 deer suffered trauma, involving dog attack (14 BBD) and vehicle-collision (14 BBD). Pulmonary edema (50 MD; 55 BBD) and congestion (57 MD; 78 BBD) were the most common findings for both species. Additionally, we diagnosed ruminal and myocardial mycosis in MD and BBD, respectively; ovarian dysgerminoma and pancreatic trematodiasis in BBD; and lesions suggestive of malignant catarrhal fever and orbiviral hemorrhagic disease in both species. The main CD in MD was: respiratory (41/75), alimentary, nutritional, trauma and euthanasia (3/75 each). Correspondingly, in BBD were: trauma (34/131), respiratory (30/131) and euthanasia (9/131). Respiratory disease was often defined by pulmonary edema and pneumonia. We provide evidence that respiratory disease, mainly pneumonia, is a critical pathological process in these Neotropical deer species. Although no etiological agents were identified, there is evidence of bacterial and viral involvement. Our results show trauma, mainly anthropogenic, as a common ailment in BBD. We propose to prioritize respiratory disease in future research focused on South American deer health aspects. We believe anthropogenic trauma may be a primary threat for populations of BBD.

Identification of Novel Gammaherpesviruses in a South American Fur Seal (Arctocephalus australis) with Ulcerative Skin Lesions

Abstract:  There are few studies on pathogens affecting free-ranging pinnipeds from South America. We employed molecular techniques to identify a gammaherpesvirus infection by two putative novel herpesvirus species: Otariid herpesvirus 5 (OtHV-5), possibly associated with ulcerative cutaneous lesions, and Otariid herpesvirus 6 (OtHV-6) in a wild South American fur seal (Arctocephalus australis) that stranded alive in Santa Catarina state, southern Brazil. Here we provide new information regarding pinniped herpesviruses, important for the design of future disease surveillance studies.

Pulmonary and systemic fungal infections in an Atlantic spotted dolphin and a Bryde’s whale, Brazil

We report the gross and microscopic findings and molecular identification of 2 cases of hyphate fungal infection in cetaceans from Brazil. The first case involved an adult male Atlantic spotted dolphin Stenella frontalis with localized pulmonary disease characterized by pyogranulomatous and necrotizing bronchopneumonia with intralesional hyphae. The second case involved an adult male Brydes whale Balaenoptera edeni with orchitis, periorchitis, mesenteric lymphadenitis and pyogranulomatous bronchopneumonia with intralesional hyphae. PCR analysis from the dolphins lung yielded Aspergillus fumigatus, and the fungus from the whales mesenteric lymph node showed the greatest identity to Nanniziopsis obscura and Stagonosporopsis cucurbitacearum These cases represent the first reports of pulmonary aspergillosis by A. fumigatus in an Atlantic spotted dolphin and systemic mycosis by a possibly novel Onygenales in marine mammals.

Molecular identification and microscopic characterization of poxvirus in a Guiana dolphin and a common bottlenose dolphin, Brazil

The poxviruses identified in cetaceans are associated with characteristic tattoo or ring skin lesions. However, little is known regarding the prevalence and progression of these lesions and the molecular characterization of cetacean poxviruses in the Southern Hemisphere. This manuscript describes the progression of poxvirus-like skin lesions in 5 free-ranging Guiana dolphins Sotalia guianensis. Additionally, 151 skin samples from 113 free-ranging cetaceans from Brazil, including 4 animals with tattoo skin lesions, were selected for poxvirus testing. Poxviral DNA polymerase gene PCR amplification was used to detect the virus in β-actin-positive samples (145/151). DNA topoisomerase I gene PCR was then used in Cetaceanpoxvirus (CePV)-positive cases (n = 2), which were further evaluated by histopathology and electron microscopy. Based on photo-identification, adult Guiana dolphins presented regressing or healed poxvirus-like lesions (2/2), while juveniles presented persistent (2/3) or healed and progressive lesions (1/3). CePV DNA was amplified in a common bottlenose dolphin Tursiops truncatus and in a Guiana dolphin. Intracytoplasmic inclusion bodies and viral particles consistent with poxvirus were identified by histology and electron microscopy, respectively. CePV-specific amino acid motifs were identified through phylogenetic analysis. Our findings corroborate previous studies that suggest the placement of poxviruses from cetaceans within the novel CePV genus. This is the first molecular identification of poxvirus in South American odontocetes.