Marcia Almeida de Melo

Defluorination of sodium fluoroacetate by bacteria from soil and plants in Brazil.

The aim of this work was to isolate and identify bacteria able to degrade sodium fluoroacetate from soil and plant samples collected in areas where the fluoroacetate-containing plants Mascagnia rigida and Palicourea aenofusca are found. The samples were cultivated in mineral medium added with 20 mmol L−1 sodium fluoroacetate. Seven isolates were identified by 16S rRNA gene sequencing as Paenibacillus sp. (ECPB01), Burkholderia sp. (ECPB02), Cupriavidus sp. (ECPB03), Staphylococcus sp. (ECPB04), Ancylobacter sp. (ECPB05), Ralstonia sp. (ECPB06), and Stenotrophomonas sp. (ECPB07). All seven isolates degraded sodium-fluoroacetate-containing in the medium, reaching defluorination rate of fluoride ion of 20 mmol L−1. Six of them are reported for the first time as able to degrade sodium fluoroacetate (SF). In the future, some of these microorganisms can be used to establish in the rumen an engineered bacterial population able to degrade sodium fluoroacetate and protect ruminants from the poisoning by this compound.

Isolation and Identification of Sodium Fluoroacetate Degrading Bacteria from Caprine Rumen in Brazil

The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100 μL in mineral medium added with 20 mmol L−1 sodium fluoroacetate (SF), incubated at 39°C in an orbital shaker. Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08) and Ancylobacter dichloromethanicus (ECPB09). These bacteria degraded sodium fluoroacetate, releasing 20 mmol L−1 of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L−1 of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.

An assessment of whole blood and fractions by nested PCR as a DNA source for diagnosing canine ehrlichiosis and anaplasmosis.

Ehrlichiosis and anaplasmosis are tick-borne diseases. Ehrlichia canis and Anaplasma platys infect mainly white cells and platelets, respectively. The main DNA source for PCR is peripheral blood, but the potential of blood cell fractions has not been extensively investigated. This study aims at assessment of whole blood (WB) and blood fractions potential in nested PCR (nPCR) to diagnose canine ehrlichiosis and anaplasmosis. The 16S rRNA gene was amplified in 71.4, 17.8, 31.57, and 30% of the WB, granulocyte (G), mononuclear cells (M), and buffy coat (BC) samples. Compared to the WB, the sensitivity of the PCR was 42.86% for the M, and BC fractions, 21.43% for the G, and 33.33% for the blood clot (C). There was fair agreement between the WB and M, BC and C, and slight with the G. Fair agreement occurred between the nPCR and morulae in the blood smear. One animal was coinfected with A. platys and E. canis. This study provided the first evidence of A. platys infection in dogs in Paraíba, Brazil, and demonstrated that WB is a better DNA source than blood fractions to detect Ehrlichia and Anaplasma by nPCR, probably because of the plasma bacterial concentration following host cell lysis.

Protothecosis by Prototheca wickerhamii in goats.

This article reports a new case of protothecosis by Prototheca wickerhamii in goats. The animal presented severe respiratory difficulty and nodules, sometimes ulcerated, in the nasal vestibule, mucocutaneous junction of the nostrils and skin of the face. Prototheca wickerhamii was isolated from the lesions. The animal had no clinical or haematologiccl evidence of immunodepression. The agent was highly resistant to antimicrobial drugs. The goat was treated unsuccessfully with fluconazole and euthanised 10 months after the diagnosis of the disease. Histological lesions were necrotising pyogranulomatous dermatitis, rhinitis and osteomyelitis with myriads of walled sporangia characteristic of P. wickerhamii. It is suggested that in goats, protothecosis is characterised by a chronic, slowly progressive infection, which affects immunologically competent goats, causing multifocal, ulcerative, pyogranulomatous and necrotising lesions of the mucosa of the nasal vestibule, mucocutaneous junctions of the nostrils and skin of the face.

Use of transgenic Aedes aegypti in Brazil: risk perception and assessment.

Abstract The OX513A strain of Aedes aegypti, which was developed by the British company Oxitec, expresses a self-limiting transgene that prevents larvae from developing to adulthood. In April 2014, the Brazilian National Technical Commission on Biosafety completed a risk assessment of OX513A and concluded that the strain did not present new biological risks to humans or the environment and could be released in Brazil. At that point, Brazil became the first country to approve the unconstrained release of a genetically modified mosquito. During the assessment, the commission produced a comprehensive list of – and systematically analysed – the perceived hazards. Such hazards included the potential survival to adulthood of immature stages carrying the transgene – should the transgene fail to be expressed or be turned off by exposure to sufficient environmental tetracycline. Other perceived hazards included the potential allergenicity and/or toxicity of the proteins expressed by the gene, the potential for gene flow or increased transmission of human pathogens and the occupation of vacant breeding sites by other vector species. The Zika epidemic both elevated the perceived importance of Ae. aegypti as a vector – among policy-makers and regulators as well as the general public – and increased concerns over the release of males of the OX513A strain. We have therefore reassessed the potential hazards. We found that release of the transgenic mosquitoes would still be both safe and of great potential value in the control of diseases spread by Ae. aegypti, such as chikungunya, dengue and Zika.

Survey of Ehrlichia canis, Babesia spp. and Hepatozoon spp. in dogs from a semiarid region of Brazil.

This study assessed the occurrence of Ehrlichia spp., Babesia spp. and Hepatozoon spp. infections in 100 tick-harboring dogs from a semiarid region of the State of Paraíba, Northeastern Brazil. Blood samples and ticks were collected from the animals, and a questionnaire was submitted to dog owners to obtain general data. Blood samples were used to perform hemogram, direct blood smear and immunological and molecular hemoparasite detection. The 1,151 ticks collected were identified as Rhipicephalus sanguineus; direct smears revealed E. canis-like morulae in the monocytes of 4% (4/100) of the non-vaccinated female dogs, and 34% and 25% of the dogs tested positive for Ehrlichia canis by indirect immunofluorescence assay (IFA) and polymerase chain reaction (PCR), respectively. Blood smear examination revealed Babesia-suggestive merozoites in the erythrocytes of 2% (2/100) of the animals. Babesia vogeli was detected by PCR in ten animals (10%) and was correlated with young age (p = 0.007) and thrombocytopenia (p = 0.01). None of the animals showed Hepatozoon spp. positivity. These results indicate that E. canis is the main tick-borne canine pathogen in the study area and provide the first report of B. vogeli infection in dogs from Paraiba State.

Caracterização clínica e laboratorial de cães naturalmente infectados com Trypanosoma cruzi no semiárido nordestino

Objetivou-se, com este estudo, evidenciar os sinais clinicos e laboratoriais desta enfermidade para auxiliar na caracterizacao da doenca de forma natural na area semi-arida da regiao nordeste. Foram avaliados 10 caes positivos para Trypanosoma cruzi, identificados mediante analises sorologicas de reacao de imunofluorescencia indireta (RIFI) e enzyme linked immunosorbent assay (ELISA); analise molecular pela Reacao em Cadeia Polimerase (PCR), microscopia direta e hemocultura. Os caes chagasicos foram submetidos a avaliacao fisica, verificacao da pressao arterial, exames eletrocardiograficos, radiograficos, hematologicos (eritrograma e leucograma) e bioquimicos (ureia, creatinina, ALT, AST, PT, albumina, globulina, CK, CK-MB e cTnI). O exame fisico e os valores das pressoes arteriais dos caes apresentaram dentro dos parâmetros de normalidade, enquanto que na eletrocardiografia observou-se FC normal com ritmo sinusal, com excecao de um cao, que apresentou taquicardia sinusal (168 bat/min). No ECG de oito caes houve aumento da duracao de P (47±6,5ms) sugestivo de aumento atrial, nao confirmado radiograficamente. Foi observado supradesnivelamento do segmento ST em um cao. Nos resultados hematologicos constatou-se trombocitopenia (187,4x103 ±137,2x103) e anemia (5,0x106 ±1,39x106/uL). Os valores medios da hemoglobina (11±2,7g/dL) e do hematocrito (34±10,5%) estavam abaixo dos limites de normalidade. A serie branca apresentou-se dentro dos limites de normalidade, com excecao da eosinofilia observada em tres caes. Individualmente, registrou-se em dois caes, leucocitose, linfocitose e neutrofilia. Na avaliacao bioquimica, registrou-se hiperproteinemia (7,2±0,9g/dL), hipoalbuminemia (2,2±0,4g/dL), hiperglobulinemia (5,1±1,0g/dL) e aumento da CK (196±171U/L). Nao houve alteracao nas enzimas ALT e AST. A isoenzima CK-MB e o cTnI alteraram somente em tres caes. Os caes infectados naturalmente no semiarido nordestino apresentam caracteristicas relacionaveis a forma cronica indeterminada, ou seja, caes assintomaticos. A identificacao dos caes infectados naturalmente sem caracteristicas patognomonicas da doenca de Chagas ressalta a importância desta enfermidade no processo diagnostico com as demais que manifestam perfis inespecificos associados ou nao as doencas cardiovasculares.

Risk factors associated with seropositivity for Leishmania spp. and Trypanosoma cruzi in dogs in the state of Paraiba, Brazil

The aim of this survey was to determine the seropositivity and risk factors for Leishmania spp. and Trypanosoma cruzi in dogs in the State of Paraíba, Northeastern Brazil. A total of 1,043 dogs were tested, and the serological diagnoses of Chagas disease (CD) and canine visceral leishmaniasis (CVL) was performed by the indirect fluorescent antibody test (IFAT). Animals that tested seropositive for both diseases (by IFAT) were further subjected to ELISA. Of the 1,043 dogs 81 (7.8%; 95% CI = 6.1-9.4%) tested seropositive for Leishmania spp., while 83 were seropositive for T. cruzi (7.9%; 95% CI = 6.3-9.6%). Simultaneous serological reactions were detected in 49 animals (4.6%; 95% CI= 3.6-6.2%). Semi-domiciled housing (OR = 2.044), free housing (OR = 4.151), and soil (OR = 3.425) and soil/cement (OR = 3.065) environmental conditions were identified as risk factors for CVL seropositivity. The risk factors identified for CD seropositivity were semi-domiciled (OR = 2.353) or free housing (OR = 3.454), and contact with bovine (OR = 2.015). This study revealed the presence of dogs in the Paraíba State seropositive for CVL and CD, suggesting the need for revisiting and intensification of disease control measures through constant monitoring of the canine population.

Efficiency of inactive vaccines against contagious agalactia in Brazil

This paper aims to evaluate the efficiency of three inactive vaccines against contagious agalactia prepared with samples of Mycoplasma agalactiae isolated in Brazil and different adjuvants. Vaccine 1 adsorbed with aluminum hydroxide was administered in 23 goats (Gc1) and 13 sheep (Gov1); vaccine 2 containing Montanide IMS-2215-VG was administered in 22 goats (Gc2) and 12 sheep (Gov2) and vaccine 3, containing Montanide Gel-01 was administered in 22 goats (Gc3) and 12 sheep (Gov3). All animals were negative for Ma at indirect ELISA and received two doses of 2mL each, subcutaneously, within a 21 day interval. Five animals from each species were used as control. Seventy-five days after the booster, four animals from each vaccinated group and two from the control group were challenged with 5mL of Ma culture containing 107cfu/mL, orally and through immersion of the females udder in lactation. The serological response was analyzed during vaccination days (0 and 21) and at 51, 81, 111, 141 and 171 days after vaccination. The collection and analysis of the challenged animals were conducted at the day of the challenge (D0) and 7, 14, 21, 28, 35, 42, 49 and 56 days after the challenge. The three vaccines induced the production of antibodies, having no significant statistical difference (p<0.05). Animals from groups Gc1, Gc2 and Gov2 developed higher levels of antibodies, with significant statistical difference compared to the other vaccinated group and control group (p<0.05). After the challenge, the animals from the control presented an increase in regional lymph nodes and conjunctivitis, mastitis and arthritis. In four vaccinated animals, discrete conjunctivitis and congestion of the episcleral veins was observed. It is concluded that vaccines 1 and 2 induced levels of protective antibodies in goats and sheep, sufficient for clinical protection of the animals submitted to the experimental infection, indicating its use on the prevention of contagious agalactia.

Seroprevalence and risk factors associated with canine visceral leishmaniasis in the State of Paraíba, Brazil

INTRODUCTION The aim of this study was to determine the seroprevalence and risk factors associated with canine visceral leishmaniasis in Paraíba State. METHODS Blood samples were collected from 411 dogs in four municipalities of Paraíba State. The seroprevalence was assessed by using ELISA. RESULTS The seroprevalence ranged from 7.2% to 20%. The risk factors that were associated with the disease were the presence of ticks and contact with other animals. CONCLUSIONS The seroprevalence of canine visceral leishmaniasis is high in Paraíba, indicating the need for health authorities to resume visceral leishmaniasis control activities. The ruralization of suburban areas in the region tends to homogenize the risk factors between suburban and rural areas.