Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

The objective of this research was to develop a primer for a polymerase chain reaction specific for Xylella fastidiosa strains that cause Pierce's Disease (PD) in grapes (Vitis vinifera). The DNA amplification of 23 different strains of X. fastidiosa, using a set of primers REP1-R (5'-IIIICGICGIATCCIGGC-3') and REP 2 (5'-ICGICTTATCIGGCCTAC-3') using the following program: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min and 72 ºC/1 min and 30 s) 72 ºC/5 min, produced a fragment of 630 bp that differentiated the strains that cause disease in grapes from the other strains. However, REP banding patterns could not be considered reliable for detection because the REP1-R and REP 2 primers correspond to repetitive sequences, which are found throughout the bacterial genome. The amplified product of 630 bp was eluted from the agarose gel, purified and sequenced. The nucleotide sequence information was used to identify and synthesize an specific oligonucleotide for X. fastidiosa strains that cause Pierce's Disease denominated Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3') which was used jointly with the REP-2 primer at the following conditions: 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. The DNAs isolated from strains of X. fastidiosa from other hosts [almond (Prumus amygdalus), citrus (Citrus spp.), coffee (Coffea arabica), elm (Ulmus americana), mulberry (Morus rubra), oak (Quercus rubra), periwinkle wilt (Catharantus roseus), plums (Prunus salicina) and ragweed (Ambrosia artemisiifolia)] and also from other Gram negative and positive bacteria were submitted to amplification with a pair of primers Xf-1/REP 2 to verify its specificity. A fragment, about 350 bp, was amplified only when the DNA from strains of X. fastidiosa isolated from grapes was employed.

Citrus exocortis viroid and Hop Stunt viroid Doubly infecting grapevines in Brazil

Viroids, non-protein-coding small (246-401 nt) circular single-stranded RNAs with autonomous replication, are currently classified into two families. Within the family Pospiviroidae, Citrus exocortis viroid (CEVd) belongs to the genus Pospiviroid while Hop stunt viroid (HSVd) is the single member of the genus Hostuviroid. These pathogens are distributed worldwide and infect a large number of hosts. In Brazil, isolates of CEVd and HSVd have been detected in both citrus and grapevine. To characterize and study the genetic variability of these viroids, total RNA from leaves of grapevine Vitis vinifera 'Cabernet Sauvignon' and V. labrusca 'Niagara Rosada' from Bento Gonçalves, RS, was used as a template for RT-PCR amplification with specific primers for the five viroids described infecting grapevines [HSVd, CEVd, Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid (AGVd)]. Leaf samples of Citrus medica infected with CEVd from São Paulo were also analyzed. The resulting products were separated by agarose gel electrophoresis and DNA fragments of the expected size were eluted, cloned and sequenced. The grapevine samples analyzed were doubly infected by CEVd and HSVd. A phylogenetic analysis showed that the Brazilian grapevine HSVd variants clustered with other grapevine HSVd variants, forming a specific group separated from citrus variants, whereas the Brazilian CEVd variants clustered with other citrus and grapevine variants.

Mining in Developing Countries: A Case Study of Sustainability Challenges

Mining has severe impacts on its surrounding. Particularly in the developing countries it has degraded the environment and signigicantly altered the socio-economical dynamics of the hosts. Especially relocation disrupts people from their homes, livelihoods, cultures and social activities. Mining industry has failed to develop the local host and streghten its governance structures; instead it has further degraded the development of mineral rich third world countries, which are among the world poorest ones. Cash flows derived from mining companies have not benefitted the crass-root level that however, bears most of the detrimental impacts. Especially if the governance structure of the host is weak, the sudden wealth is likely to accelerate disparities, corruption and even fuel wars. Environmental degradation, miscommunication, mistrust and disputes over land use have created conflicts between the communities and a mining company in Obuasi, Ghana; a case study of this thesis. The disputes are deeply rooted and further fuelled by unrealistic expectations and broken promises. The relations with artisanal and illegal miners have been especially troublesome. Illegal activities, mainly encroachment of the land and assets of the mine, such as vandalising tailings pipes have resulted in profits losses, environmental degradation and security hazards. All challenges mentioned above have to be addressed locally with site-specific solutions. It is vital to increase two-way communication, initiate collaboration and build capacity of the stakeholders such as local communities, NGOs and governance authorities. The locals must be engaged to create livelihood opportunities that are designed with and for them. Capacity can also be strengthened through education and skills training, such as women’s literacy programs. In order to diminish the overdependence of locals to the mine, the activities have to be self -sufficient and able to survive without external financial and managerial inputs. Additionally adequate and fair compensation practises and dispute resolution methods that are understood and accepted by all parties have to be agreed on as early as possible.

Powdery mildew of ornamental species caused by Oidiopsis haplophylli in Brazil

Oidiopsis haplophylli (syn. Oidiopsis sicula) was identified as the causal agent of powdery mildew diseases occurring on five ornamental species in Brazil. This disease was observed in plastic house-grown lisianthus (Eustoma grandiflorum: Gentianaceae), in nasturtium (Tropaeolum majus: Tropaeolaceae) cultivated under open field conditions and in greenhouse-grown calla lily (Zantedeschia aethiopica: Araceae), impatiens (Impatiens balsamina: Balsaminaceae) and balloon plant (Asclepias physocarpa: Asclepiadaceae). Typical disease symptoms consisted of chlorotic areas on the upper leaf surface corresponding to a fungal colony in the abaxial surface. With the disease progression, these chlorotic areas eventually turned to necrotic (brown) lesions. Fungi morphology on all hosts was similar to that described for the imperfect stage of Leveillula taurica (O. haplophylli). The Koch's postulates were fulfilled by inoculating symptom-free plants via leaf-to-leaf contact with fungal colonies. Additional inoculations using an isolate of O. haplophylli from sweet pepper (Capsicum annuum) demonstrated that it is pathogenic to all five species belonging to distinct botanical families, indicating lack of host specialization. This is the first formal report of a powdery mildew disease on lisianthus, calla lilly, impatiens and nasturtium in Brazil. It is, to our knowledge, the first report of O. haplophyllii infecting A. physocarpa, extending the host range of this atypical powdery mildew-inducing fungus. This disease might become important on these ornamental crops especially in protected cultivation and also under field conditions in hot and dry areas of Brazil.

Phylogenetic analysis of Tomato mosaic virus from Hemerocallis sp. and Impatiens hawkeri

The culture and commercialization of ornamental plants have considerably increased in the last years. To supply the commercial demand, several Hemerocallis and Impatiens varieties have been bred for appreciated qualities such as flowers with a diversity of shapes and colors. With the aim of characterizing the tobamovirus isolated from Hemerocallis sp. (tobamo-H) and Impatiens hawkeri (tobamo-I) from the USA and São Paulo, respectively, as well as to establish phylogenetic relationships between them and other Tobamovirus species, the viruses were submitted to RNA extraction, RT-PCR amplification, coat-protein gene sequencing and phylogenetic analyses. Comparison of tobamovirus homologous sequences yielded values superior to 98.5% of identity with Tomato mosaic virus (ToMV) isolates at the nucleotide level. In relation to tobamo-H, 100% of identity with ToMV from tomatoes from Australia and Peru was found. Based on maximum likelihood (ML) analysis it was suggested that tobamo-H and tobamo-I share a common ancestor with ToMV, Tobacco mosaic virus, Odontoglossum ringspot virus and Pepper mild mottle virus. The tree topology reconstructed under ML methodology shows a monophyletic group, supported by 100% of bootstrap, consisting of various ToMV isolates from different hosts, including some ornamentals, from different geographical locations. The results indicate that Hemerocallis sp. and I. hawkeri are infected by ToMV. This is the first report of the occurrence of this virus in ornamental species in Brazil.

Development and characterisation of microsatellite markers for the fungus Lasiodiplodia theobromae

Lasiodiplodia theobromae is an important fungal pathogen of higher plants from tropical and sub-tropical regions. The fungus infects divergent hosts in a wide range of environmental conditions, suggesting that it is highly variable. The aim of this study was to develop new polymorphic microsatellite markers from a Brazilian isolate of L. theobromae that can be used in population studies of this and related fungi. The nine microsatellite markers developed included six that revealed allelic polymorphisms among nine isolates of the disease collected from infected plants in Brazil. Preliminary evaluation of the markers suggested substantial genetic variability among Brazilian L. theobromae populations. These markers have potential utility for evolutionary and epidemiologic studies of this fungus.

Characterization of Xanthomonas axonopodis pv. phaseoli isolates

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Defects in Persistent Luminescence Materials

Persistent luminescence materials can store energy from solar radiation or artificial lighting and release it over a period of several hours without a continuous excitation source. These materials are widely used to improve human safety in emergency and traffic signalization. They can also be utilized in novel applications including solar cells, medical diagnostics, radiation detectors and structural damage sensors. The development of these materials is currently based on methods based on trial and error. The tailoring of new materials is also hindered by the lack of knowledge on the role of their intrinsic and extrinsic lattice defects in the appropriate mechanisms. The goal of this work was to clarify the persistent luminescence mechanisms by combining ab initio density functional theory (DFT) calculations with selected experimental methods. The DFT approach enables a full control of both the nature of the defects and their locations in the host lattice. The materials studied in the present work, the distrontium magnesium disilicate (Sr₂MgSi₂O₇) and strontium aluminate (SrAl₂O₄) are among the most efficient persistent luminescence hosts when doped with divalent europium Eu²⁺ and co-doped with trivalent rare earth ions R³⁺ (R: Y, La-Nd, Sm, Gd-Lu). The polycrystalline materials were prepared with the solid state method and their structural and phase purity was confirmed by X-ray powder diffraction. Their local crystal structure was studied by high-resolution transmission electron microscopy. The crystal and electronic structure of the nondoped as well as Eu²⁺, R^2+/3+ and other defect containing materials were studied using DFT calculations. The experimental trap depths were obtained using thermoluminescence (TL) spectroscopy. The emission and excitation of Sr₂MgSi₂O₇:Eu²+,Dy³⁺ were also studied. Significant modifications in the local crystal structure due to the Eu²⁺ ion and lattice defects were found by the experimental and DFT methods. The charge compensation effects induced by the R³⁺ co-doping further increased the number of defects and distortions in the host lattice. As for the electronic structure of Sr₂MgSi₂O₇ and SrAl₂O₄, the experimental band gap energy of the host materials was well reproduced by the calculations. The DFT calculated Eu²⁺ and R^2+/3+ 4fn as well as 4f^n-15d¹ ground states in the Sr₂MgSi₂O₇ band structure provide an independent verification for an empirical model which is constructed using rather sparse experimental data for the R³⁺ and especially the R²⁺ ions. The intrinsic and defect induced electron traps were found to act together as energy storage sites contributing to the materials’ efficient persistent luminescence. The calculated trap energy range agreed with the trap structure of Sr₂MgSi₂O₇ obtained using TL measurements. More experimental studies should be carried out for SrAl₂O₄ to compare with the DFT calculations. The calculated and experimental results show that the electron traps created by both the rare earth ions and vacancies are modified due to the defect aggregation and charge compensation effects. The relationships between this modification and the energy storage properties of the solid state materials are discussed.

O estado da arte da cirurgia do baço, no início do século XXI

In the last fifty years evolution of scientific knowledge on the spleen provoked an entirely new approach to splenic surgery. It was shown that virulence may emerge as a significant consequence of environmental and evolutionary changes of some microbial communities, and devastating pathogenetic results of these changes become visible in human hosts without the splenic function. In other words: the spleen plays a pivotal role in the dynamic balance between biodiversity, microorganisms and immunogenecity in human hosts. Therefore, to preserve the "splenic immunologic repertoire" became an increasing commitment among surgeons. Understanding the integration of these multiple information on spleen, seems central to understand the new splenic surgery. Partial splenectomies (Réglées) - based on anatomical, experimental and clinical studies, developed at the University of Minas Gerais since the fifties - were successfully applied initially to treat the traumatic injuries of the spleen; in a following step, partial splenectomy were used to control hematological diseases. "Réglées" techniques on the spleen have conquered "ethical support, consilience status and clinical governance" to give birth to surgical therapeutic decisions on the spleen, in order to spare the structural integrity of the immune system. Splenic réglées procedures became a seminal achievement of splenic surgical practice. Initial results of "Partial splenectomies" - with conventional surgical armamentaria and techniques - were confirmed and improved by the introduction of techniques based on laparoscopic and endovascular approaches. And current usage of surgical splenic saving procedures propitiated the emergence of an appropriate lexicon for medical communication and became an "end point" of a "long-standing surgical inhibition" over the spleen.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Occurrence of parasitism by Dioctophyma renale in ring-tailed coatis (Nasua nasua) of the Tiete Ecological Park, São Paulo, Brazil

Dioctophymosis is a worldwide renal parasitosis caused by the Dioctophyma renale nematode, which results in progressive destruction of renal tissue. Aquatics annelids are considered the main intermediate hosts and the literature refers as permanent hosts of dogs, wild mammals and even humans. During procedures for population control of coatis (Nasua nasua) in the Ecological Park of Tietê (PET), was noticed the presence of parasitosis by D. renale. From 68 animals, males and females, young and adults, submitted to exploratory laparotomy, 51 were positive for the presence of worms, 9 were found only in the right kidney. In 10 cases, in addition to right kidney parasitism, worms were also observed in the abdominal cavity. In 24 cases D. renale was found only in the abdominal cavity and in 8 animals the right kidney was reduced to a small rigid structure. The study showed that the preferred site for parasitism of the worm, considered erratic, was the abdominal cavity in 66.66% of the cases.

Isosporoid Coccidia (Apicomplexa: Eimeriidae) parasites of tanagers (Passeriformes: Thraupidae) from the Marambaia Island, Brazil

In recent years some coccidian parasites of birds were recorded in Marambaia Island, which is a protected environment with a great biodiversity of birds, mainly tanagers. In this current study Isospora tiesangui, I. sepetibensis, I. ramphoceli, I. navarroi, I. cadimi and I. marambaiensis were identified according to their respective thraupid hosts of the Marambaia Island. These species were characterized with histograms, linear regression and analysis of variance (ANOVA). The main feature of identification was the morphology of the sporocyst, mainly Stieda and substieda bodies, since the morphometry did not provide sufficient differentiation. Besides, Dacnis cayana and Thraupis palmarum were reported as new hosts to I. sepetibensis and I. navarroi respectively.

Anti-Rickettsia spp. antibodies in free-ranging and captive capybaras from southern Brazil

Capybaras (Hydrochaeris hydrochaeris) are among the main hosts of Amblyomma spp. ticks, which is able to transmit Rickettsia species to human beings and animals. Since they are often infested with potential vector ticks, capybaras may be used as sentinels for rickettsiosis, such as the Brazilian Spotted Fever. The aim of the present study was to determine the prevalence of antibodies against Rickettsia spp. using the indirect immunofluorescence assay (IFA) in 21 free-ranging and 10 captive animals from the Zoological Park of the 'Bela Vista Biological Sanctuary' (BVBS), Itaipu Binational, Foz do Iguaçu, Southern Brazil. Antigens of six rickettsial species already identified in Brazil (Rickettsia rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis) were used for IFA. Ticks from each capybara were collected for posterior taxonomic identification. A total of 19 (61.3%) samples reacted to at least one of tested species. Seropositivity was found in 14 (45.2%), 12 (38.7%), 5 (16.1%), 4 (12.9%), 3 (9.7%) and 3 (9.7%) animals for R. rickettsii, R. bellii, R. parkeri, R. amblyommii, R. felis and R. rhipicephali, respectively. Two captive capybaras presented suggestive titers of R. rickettsii infection and one sample showed homologous reaction to R. parkeri. Only one free-ranging capybara presented evidence R. bellii infection. Ticks collected on capybaras were identified as Amblyomma dubitatum e Amblyomma sp. Results evidenced the rickettsial circulation in the area, suggesting a potential role of capybaras on bacterial life cycle.

Sheep abortion associated with Neospora caninum in Mato Grosso do Sul, Brazil

Canids are the main hosts of Neospora caninum, but cattle, (sheep, goats and horses may serve as intermediary hosts. N. caninum infection of pregnant intermediary hosts may provoke abortion and neonatal infections. This study is the first to report lamb abortion associated with N. caninum in Mato Grosso do Sul. Epidemiological data were obtained from interviews with sheep producers. For microscopic examination, fragments of different organs removed from 4 sheep fetuses, aborted and necropsied, were fixed in 10% formaldehyde, embedded in paraffin and subjected to the hematoxylin-eosin staining protocol and immunohistochemistry (IHC) to test for N. caninum and Toxoplasma gondii. The abortion outbreak studied was reported from a herd of 268 Santa Inês sheep (including 186 pregnant ewes), with 10 abortion cases in the last third of gestation. Four fetuses were examined, 3 from a same ewe. At necropsy, one fetus exhibited crackling in the lung and all its organs were reddish. Histological findings detected mononuclear cell infiltrates among myocardium fibers and around blood vessels, in addition to circular structures with basophilic points resembling protozoans. IHC tests revealed strongly positive staining for N. caninum and weakly positive for T. gondii, characterizing N. caninum infection.

Infection by Toxoplasma gondii in Neotropical non-human primates

Toxoplasma gondii (Nicolle et Manceaux, 1909) is an obligatory intracellular protozoan parasite of warm animals, including human and non-human primates. Domestic and wild felids are considered definitive hosts. Several authors have already identified lesions in New World primates caused by T. gondii. Nevertheless, little is known about serological studies on those animals. With this reason, New World non-human primates of the genera Cebus and Callithrix that were apprehended by governmental authorities and sent to the Wildlife Screening Center (Cetas)/IBAMA, at the municipality of Seropédica, state of Rio Janeiro, were bled and sera were submitted to the indirect hemagglutination test for detection of anti-T. gondii antibodies. From 21 sera of Cebus primates, 76.19% (16/21) had anti-T. gondii antibodies. Titles varied from 16 to 2048. In samples from 21 Callithrix, only 4.5% (1/22) had anti-T. gondii antibodies. Only one animal had a title of 32. During all the time those animals were clinical evaluated until sample was collected; none of them had any clinical sign or sequel related to infection by T. gondii. The fact that the origin of these primates is unknown and that there is no information about their feeding habits before captivity makes it difficult to determine the source of T. gondii infection.