Monitoring of Leptospira in captive turtles by DNA analysis / [Monitoramento de Leptospira em quelônios mantidos em cativeiro pela análise de DNA]

Existem poucos estudos sobre doenças infecciosas em animais silvestres. O objetivo deste estudo foi pesquisar DNA de Leptospira spp. em sangue de tartarugas mantidas em cativeiro, pertencentes ao Bosque Rodrigues Alves (Jardim Zoobotânico da Amazônia). O DNA foi isolado das amostras de sangue coletadas de 148 tartarugas pertencentes a seis espécies diferentes. A reação em cadeia da polimerase (PCR) foi realizada utilizando-se iniciadores específicos para DNA de Leptospira spp. Nenhuma das amostras apresentou resultado positivo para Leptospira spp.(AU)

Isolation and characterization of the aerobic bacterial microbiota of the esophagus and its probable association with obstructive caseous lesions in green turtles (Chelonia mydas) / Isolamento e caracterização da microbiota bacteriana aeróbia do esôfago e sua provável associação com lesões caseosas obstrutivas em tartarugas-verdes (Chelonia mydas)

Caseous lesions in the esophagus of green turtles (Chelonia mydas) from the coast of Brazil have been described as obstructive lesions and can lead to the death of these animals. However, their etiology remains unclear. The aim of this study was to isolate and characterize the aerobic bacterial microbiota of the esophagus of green turtles (C. mydas) from the Brazilian coast and to verify its possible participation in the etiology of caseous lesions. For this, 42 animals were used, 33 alive and healthy and 9 naturally dead that had esophageal lesions confirmed by necropsy, from Anchieta and Piúma beaches, Espírito Santo. Microbiological tests and morphological evaluation of the esophagus were performed. We isolated 14 different bacterial agents from healthy animal samples, with the prevalence of Pseudomonas aeruginosa being (36.36%), Staphylococcus aureus (33.33%), Aeromonas hydrophila (27.27%), and Vibrio alginolyticus (24.24%). In dead animals, only three distinct agents were isolated: S. aureus (50.00%), A. hydrophila (25.00%), and V. alginolyticus (25.00%). Morphological evaluation revealed a predominance of the lesions at the gastroesophageal junction, with multifocal-to-coalescent distribution, discrete intensity, and absence of obstruction. Ulcerations and caseous exudates, inflammatory infiltrates, parasitic eggs, and giant foreign body cells were also observed as well as bacterial lumps and glandular alterations, such as necrosis, adenitis, and fragments of adult parasites. There was a positive correlation between bacterial lumps and microbiological culture and a negative correlation between bacterial lumps and microbiological culture with parasites. Thus, it was noted that the esophageal aerobic microbiota of C. mydas was predominantly composed of Gram-negative bacteria such as P. aeruginosa, A. hydrophila, and V. alginolyticus, in addition to several enterobacteria and Gram-positive bacteria, such as S. aureus. These agents are opportunists and may be involved in the etiology of caseous esophagitis in association with other pathogens as co-factors working in association or, even in a secondary way.(AU)

A ocorrência de lesão caseosa no esôfago de tartarugas-verdes (Chelonia mydas) da costa do Brasil tem sido descrita como de caráter obstrutivo e pode causar a morte dos animais. No entanto, sua etiologia permanece pouco esclarecida. Objetivou-se isolar e caracterizar a microbiota aeróbica esofágica das tartarugas-verdes (C. mydas) da costa brasileira e verificar sua possível participação na etiologia das lesões caseosas. Foram utilizados 42 animais, 33 vivos e hígidos e nove mortos naturalmente que apresentavam lesão esofágica confirmada pela necropsia, provenientes de Anchieta e Piúma, Espírito Santo, nos quais foram feitos testes microbiológicos e avaliação morfológica do esôfago. Foram isolados 14 agentes bacterianos diferentes nas amostras de animais saudáveis, com prevalência de Pseudomonas aeruginosa (36,36%), Staphylococcus aureus (33,33%), Aeromonas hydrophila (27,27%) e Vibrio alginolyticus (24,24%). Nos animais mortos, foram isolados apenas três agentes distintos: S. aureus (50,00%), A. hydrophila (25,00%) e V. alginolyticus (25,00%). A avaliação morfológica revelou predominância da lesão em junção gastroesofágica, com distribuição multifocal a coalescente, intensidade discreta e ausência de obstrução. Observou-se ainda ulceração e exsudato caseoso, infiltrado inflamatório, ovos de parasitos e células gigantes do tipo corpo estranho, além de grumos bacterianos e de alterações glandulares, como necrose, adenite e fragmentos de parasitos adultos. Houve correlação positiva dos grumos bacterianos com cultivo microbiológico e negativa dos grumos bacterianos e cultivo microbiológico com parasitos. Assim, nota-se que a microbiota esofágica aeróbica de C. mydas é constituída predominantemente por bactérias Gram-negativas como P. aeruginosa, A. hydrophila e V. alginolyticus, além de diversas enterobatérias e por Gram-positivas, como S. aureus. Esses agentes são oportunistas e podem estar envolvidos na etiologia da esofagite caseosa em associação a outros patógenos como co-fatores agindo em associação, ou mesmo, por via de infecção secundária.(AU)

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains / Frequência das repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs) em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium

Abstract The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

Resumo A fidelidade dos genomas ​​é defendida por mecanismos conhecidos como sistemas de repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs). Três tipos de sistemas CRISPR II (CRISPR1-cas, CRISPR2 e CRISPR3-cas) têm sido identificados em cepas de enterococos isolados de amostras clínicas e ambientais. O objetivo deste estudo foi observar a distribuição dos CRISPR1-cas, CRISPR2 e CRISPR3-cas em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium isoladas de amostras alimentícias e fecais, incluindo animais marinhos selvagens. A presenca dos CRISPRs foi determinada por PCR em 120 cepas de enterococos, sendo 67 E. faecalis e 53 E. faecium. É o primeiro relato da presença do sistema CRISPRs nas estirpes E. faecalis e E. faecium isoladas de amostras fecais de animais marinhos selvagens. Os resultados mostraram que em cepas não-clínicas, os CRISPRs foram mais frequentemente detectados em E. faecalis do que em E. faecium. E as frequências de CRISPR1-cas e CRISPR2 foram maiores (60%) em cepas de E. faecalis isoladas de fezes de animais, quando comparadas à amostras de alimentos. Ambas as cepas apresentaram baixas freqüências de CRISPR3-cas (8,95% e 1,88%). Em conclusão, as diferenças nos habitats das espécies de enterococos podem estar relacionadas com os resultados observados na distribuição dos sistemas CRISPRs.

Isolamento e determinação de sensibilidade e resistência a antimicrobianos de cepas bacterianas presentes na cloaca de Trachemys scripta elegans (Wied, 1839) criadas em cativeiro em Petrolina, PE / Isolation and determination of antimicrobials sensitivity and resistance of bacterial strains present in the cloaca Trachemys scripta elegans (Wied, 1839) raised in captivity in Petrolina/PE, Brazil

Este estudo isolou e determinou o perfil de sensibilidade e de resistência a antimicrobianos de cepas bacterianas isoladas da cloaca de Trachemys scripta elegans (T. s. elegans) criadas em cativeiro. Após 120 dias de adaptação, amostras de swab cloacal obtidas de 20 animais adultos foram cultivadas e, após a identificação dos patógenos através de testes bioquímicos, submetidas ao teste de suscetibilidade a nove antimicrobianos. Enterobacter aerogenes (85%); Shigella spp. (10%) e Edwadsiella spp. (5%) foram isolados e identificados. Os isolados de E. aerogenes foram sensíveis à gentamicina (86%), enrofloxacina (79%), estreptomicina (50%), sulfazotrim (36%) e ampicilina (29%) e resistentes a penicilina (100%), eritromicina (93%), cefalexina (86%) ampicilina (71%) e sulfazotrim (64%). Isolados de Shigella spp. apresentaram sensibilidade à gentamicina (100%), enrofloxacina (50%), doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (50%) e sulfazotrim (50%) e resistência a doxicilina (50%), estreptomicina (50%), ampicilina (50%), penicilina (100%), cefalexina (50%) e sulfazotrim (50%), enquanto que os de Edwardsiella spp. foram sensíveis apenas à gentamicina (100%) e altamente resistentes (100%) aos demais antimicrobianos. Os resultados sugerem a participação de T. s. elegans na cadeia epidemiológica, como reservatório de patógenos importantes, como E. aerogenes, Shigella spp. e Edwardisiella spp., tornando importante a adoção de medidas preventivas pelo risco zoonótico que apresentam e corretas de tratamento e de controle em cativeiros e domicílios, assim como de estudos que enfoquem as características de sensibilidade e de resistência antimicrobiana dos isolados cloacais, pois a multirresistência a drogas pode ser transmitida aos humanos e comprometer o tratamento de indivíduos com doenças graves.(AU)

This study isolated and determined the profile of susceptibility and antimicrobials resistance of bacterial strains isolated from the cloaca Trachemys scripta elegans (T. s. elegans) raised in captivity. After 120 days of adaptation, cloacal swab samples obtained from 20 adults animals were grown and, after the pathogens identification through biochemical tests, submitted to the test of susceptibility to nine antimicrobials. Enterobacter aerogenes (85%); Shigella spp. (10%) and Edwadsiella spp. (5%) were isolated and identified. Isolates from E. aerogenes were sensitive to gentamicin (86%), enrofloxacin (79%), streptomycin (50%), sulfazotrim (36%) and ampicillin (29%) and resistant to penicillin (100%), erythromycin (93%), cephalexin (86%), ampicillin (71%) and sulfazotrim (64%). Isolates from Shigella spp. showed sensitivity to gentamicin (100%), enrofloxacin (50%), doxycycline (50%), streptomycin (50%), ampicillin (50%), penicillin (50%) and sulfazotrim (50%) and resistance to doxycycline (50 %), streptomycin (50%), ampicillin (50%), penicillin (100%), cephalexin (50%) and sulfazotrim (50%), while the Edwardsiella spp. were sensitive only to gentamicin (100%) and were highly resistant (100%) to other antibiotics. The results suggest the participation of T. s. elegans in the epidemiological chain, as reservoir of important pathogens, such as E. aerogenes, Shigella spp. and Edwardisiella spp., making it important to adopt preventive measures for zoonotic risk that present and correct treatment and control in captivity and households, as well as studies that address the sensitivity characteristics and antimicrobial resistance of isolates from cloaca, as it multidrug resistance to drugs can be transmitted to humans and compromise the treatment of patients with serious diseases.(AU)

Gastroenteritis por Salmonella spp. en tres lactantes asociada a contacto con tortugas acuáticas / Salmonella spp. gastroenteritis associated to pet turtles in three infants

Non-typhoidal salmonellosis is an important cause of acute diarrhea in children. Generally they are acquired by consumption of contaminated food, but contact with reptiles is an increasing cause of this zoonoses. We describe three pediatric cases of gastroenteritis by Salmonella (Montevideo, Newport and Pomona), related to having turtles as pets. In two cases, the bacteria were isolated from the patient’s stools and the turtle’s droppings. The same genetic subtype by PFGE was obtained in both isolates. All the children recovered. Information and public health measures should be undertaken to control this zoonoses which affects young children.

Las infecciones por Salmonella no tíficas, son una importante causa de diarrea aguda en niños. Generalmente adquiridas por alimentos contaminados, el contacto con animales, especialmente reptiles, también es responsable de esta zoonosis. Describimos tres casos de lactantes con gastroenteritis por Salmonella (S. Montevideo, S. Newport y S. Pomona), relacionados a contacto con tortugas mascotas. En dos de los casos se aisló Salmonella en las deposiciones de los niños y de las tortugas. En todos hubo proximidad entre los niños y los acuarios y/o tortugas, siendo probable la contaminación en la preparación de alimentos. El estudio de clonalidad por electroforesis de campo pulsado demostró el mismo subtipo genético en el aislado correspondiente al paciente y en el procedente de la mascota, lo cual confirmó la fuente zoonótica de los casos. Esta zoonosis debe ser abordada por las autoridades de salud y las sociedades científicas, ya que representa un importante riesgo para la salud, especialmente de niños pequeños.

Flora bacteriana cloacal y nasal de Lepidochelys olivacea (Testudines: Cheloniidae) en el pacífico norte de Costa Rica / Cloacal and nasal bacterial flora of Lepidochelys olivacea (Testudines: Cheloniidae) from the North Pacific coast of Costa Rica

The aerobic cloacal and nasal bacterial flora of 45 apparently healthy female olive ridley sea turtles (Lepidochelys olivacea )was studied at Nancite nesting beach,in Santa Rosa National Park (Costa Rican North Pacific)during July and August 2002.Bacterial samples were obtained by inserting sterile swabs directly into the cloaca and the nasal cavities of the turtles.Ninety-nine aerobic bacterial isolates, including 10 Gram-negative and 5 Gram-positive bacteria, were recovered.The most common bacteria cultured were Aeromonas spp. (13/45) and Citrobacter freundi (6/45)from cloacal samples and Bacillus spp.(32/45), Staphylococcus aureus (6/45)and Corynebacterium spp.(5/45)from nasal ducts.The results of the present study showed that the aerobic bacterial flora of nesting female olive ridleys was composed of several potential human and animal microbe pathogens.

Con el objetivo de determinar la flora normal aerobia, cloacal y nasal de la tortuga lora (Lepidochelys olivacea ), entre los meses de julio y agosto del 2002,se colectaron muestras bacteriológicas de 45 quelonios aparentemente sanos,durante el desove en Playa Nancite,Parque Nacional Santa Rosa, Costa Rica, a través del uso de hisopos estériles que se introdujeron en la cloaca y en uno de los conductos nasales. De las muestras recolectadas se obtuvieron e identificaron un total de 99 aislamientos, incluyendo 10 grupos de Gram-negativos y 5 de Gram-positivos. De cada tortuga se obtuvo un promedio de 0.7 bacterias de la cloaca y 1.4 de las cavidades nasales. Las bacterias más frecuente halladas fueron Aeromonas spp.(13/45) y Citrobacter freundi (6/45) en la cloaca, y Bacillus spp. (32/45),Staphylococcus aureus (6/45)y Corynebacterium spp.(5/45)en las cavidades nasales. En este investigación, la flora microbiana de las tortugas lora resultó constituida por microorganismos potencialmente patógenos para el ser humano y las tortugas.

An outbreak of salmonellosis linked to a marine turtle.

In September 1998, an outbreak of gastroenteritis occurred in a coastal Aboriginal community in the Northern Territory over a seven day period. An investigation was conducted by the Center for Disease Control, Territory Health Services. Thirty-six cases were detected and 17% (n=6) were hospitalized. Salmonella chester was isolated from eight of nine stool specimens. Sixty-two percent of cases interviewed (n=28) reported consumption of a green turtle (Chelonia mydas) within a median of 24 hours prior to onset of illness. Of the remainder, all but two were contacts of other cases. Salmonella chester was isolated from a section of partially cooked turtle meat. There are no previous published reports of salmonellosis associated with consumption of sea turtles despite them being a popular food source in coastal communities in the Pacific.

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.