Ultrastructural aspects of lingual papillae in squirrel monkey (Saimiri sciureus)

Saimiri sciureus is one of the smallest Cebidae native of Amazon region and also found at the biological reserve of northeast Atlantic forest. It is an omnivore animal, with diversified diet that directly influences the lingual mucosa, which includes certain types of papillae with different organization levels. The present study attempted to describe the morphological and ultrastructure aspects of the dorsal surface of the S. sciureus. Five tongues of de S. sciureus were analyzed from three males and two females who died from natural causes and were obtained from breeding colonies of CENP-Ananindeua-PA. Main macroscopic features were a general triangular shape with a craniocaudal elongation pointed apex. Tissue samples-apex, body, and root of tongue-were fixed in modified Karnovsky solution, following standard scanning protocol, mounted in stubs, coated by gold, and analyzed by Scanning Electron Macroscopy (SEM). Four types of papillae were described: filiform (along all tissue extension with 154 mu m of diameter), fungiform (along all tissue extension with 272 mu m of diameter), vallate [just three units in caudal (dorsal) portion with 830 mu m of diameter] and foliate (one pair at caudolateral surface with similar to 13 projections and 3000 mu m in length). Data analysis indicates that the distribution and ultra structural morphology of the S. sciureus lingual papillae are some similar to other primates. Microsc. Res. Tech. 74:484-487, 2011. (C) 2010 Wiley-Liss, Inc.

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica. (C) 2010 Elsevier Ltd. All rights reserved.

Ecological aspects of the free-living ticks (Acari: Ixodidae) on animal trails within Atlantic rainforest in south-eastern Brazil

In a recent ecological study of the ticks on animal trails within an area of Atlantic rainforest in south-eastern Brazil, Amblyomma aureolatum, A. brasiliense, A. incisum, A. ovale and Haemaphysalis juxtakochi were found questing on the vegetation. Most of the ticks recorded by a small, man-made dam on the forest border were A. dubitatum but a few A. brasiliense and A. cajennense, one A. incisum and one H. juxtakochi were also found. The seasonal activity of the ticks indicated that A. incisum and A. brasiliense had one generation/year. On the animal trails, most tick species and stages quested on the vegetation at a height of 30-40 cm above ground level. The questing larvae and adults of A. incisum tended to be found higher, however, with the greatest numbers recorded 40-50 cm (larvae) or 60-70 cm (adults) above ground level. Most of the adult ticks (81.1% -100%), nymphs (78.6%-100%) and larval clusters (100%) found on a forest trail remained questing at the same location over a 24-h period. Carbon-dioxide traps in the rainforest attracted, 50% of the ticks observed questing on the nearby vegetation and, curiously, the CO(2) traps set deep in the forest attracted far fewer ticks than similar traps set by the dam. The ecological relationships between the ticks, their hosts and the rainforest environment are discussed.

Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2)

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil. (C) 2007 Elsevier Ltd. All rights reserved.

OCCURRENCE AND MOLECULAR DIAGNOSIS OF CRYPTOSPORIDIUM SERPENTIS IN CAPTIVE SNAKES IN SAO PAULO, BRAZIL

The present study aimed to determine whether Cryptosporidium oocysts were present in stools from captive snakes at Fundacao Parque Zoo logic (Zoological Park Foundation) in Sno Paulo, Brazil. Two collections were performed; the first in July 2008 and the second in February 2009. Fecal samples were collected from 74 enclosures that housed 101 individuals of 23 snake species. The stool specimens collected from 16 out of the 74 enclosures (21.6%) contained Cryptosporidium spp. oocysts; all of them were confirmed as Cryptosporidium serpent is, using molecular techniques. Only in three (18.7%) out of the 16 enclosures with positive samples were there animals with clinical signs compatible with infection by C. serpentis, such as regurgitation and significant progressive weight loss. From the results, it was concluded that diagnostic examinations need to be performed periodically, even on clinically healthy animals, as a preventive measure.

Biological aspects of Amblyomma brasiliense (Acari : Ixodidae) under laboratory conditions

Although Amblyomma brasiliense Arag (a) over tildeo 1908 has been reported as one of the most aggressive ticks to humans in Brazil, information about the biology of this tick species is virtually inexistent. This work reports data on the life cycle of A. brasiliense fed on rabbits and pigs and maintained in an incubator at 20 degrees C, 90% RH and 12 h of light for off-host development. Tick yield of adult females fed on pigs and rabbits was 81.2% and 58.3%, respectively. Females fed on pigs had mean engorgement weight of 862.3 mg and egg mass of 208 mg, while females fed on rabbits had mean engorgement weight of 606.1 mg and egg mass of 160 mg; these values did not differ statistically between host species. Feeding period of female ticks fed on pigs (10 days) was significantly shorter than that on rabbits (17 days). Mean preoviposition period was slightly longer (35.9 days) for ticks fed on pigs than on rabbits (30 days). The minimum incubation period of eggs of ticks from both host species was similar and over 100 days. Egg production efficiency was low for females fed on both hosts (less than 30% and 20% for ticks from pigs and rabbits, respectively). More than 55% of larvae and 79% of nymphs fed on rabbits, set free inside the feeding chambers, engorged successfully. These ticks attained an engorgement weight of 1.3 and 18.2 mg, respectively, and fed for approximately 5 days. The minimum pre-molt period was 30 days for engorged larvae and over 44 days for nymphs. Molting success was low, less than 50% in the case of larvae and less than 20% for nymphs. Further studies are required to better determine the off-host requirements of this tick species.

Phenotypic and molecular characterization of recent and archived Erysipelothrix spp. isolated from Brazilian swine

One hundred fifty-one Erysipelothrix spp. isolates from Brazilian swine were characterized by serotyping, determination of antimicrobial susceptibility, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE). Among all isolates, 139 were classified in 18 different serotypes and serotype 2b was the most frequent. The susceptibility profiles of the isolates were very similar among each other, which did not permit subtyping Erysipelothrix spp. isolates by the antimicrobial susceptibility testing. Despite the fact that AFLP and PFGE provided the same discriminatory index (0.98), PFGE was more discriminatory than AFLP, given the types of groups it generates. Regardless the technique employed (AFLP or PFGE), no discrimination between recent and historical isolates was established, neither a fixed epidemiologic pattern for their grouping was observed. Nevertheless, AFLP could be an interesting alternative for discriminating the Erysipelothrix species, while PFGE could be an indication for discerning this bacterium according to the serotypes. (C) 2011 Elsevier Inc. All rights reserved.

Rabies virus in insectivorous bats: Implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology

Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil. (C) 2010 Elsevier Inc. All rights reserved.

Molecular Epidemiology of Avian Infectious Bronchitis in Brazil from 2007 to 2008 in Breeders, Broilers, and Layers

Multiple lineages of Brazilian strains from 2007 to 2008 of avian infectious bronchitis virus (IBV) were detected in flocks of breeders, broilers, and layers. Organs samples from 20 IBV-positive flocks with variable clinical signs were submitted to the partial amplification of S gene (nucleotides 726-1071) of IBV. Fifteen of the 20 sequenced strains segregated in a unique Brazilian cluster subdivided in three subclusters (Brazil 01, 02, and 03). Whereas three strains could be classified as Massachusetts (Mass) genotype, the remaining two strains, originating from flocks with reproductive and respiratory disorders, grouped within the 4/91-793B genotype, a genotype that has not been detected before in Brazil. The potential relevance of the findings to the poultry industry is discussed because the low level of identity of the sequenced part of the S gene from 17 of 20 detected field strains and the vaccines of the Massachusetts serotype used suggest that the level of cross-protection by the Massachusetts vaccines might be low.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.

Hepatic Hemosiderosis in Red-Spectacled Amazons (Amazona pretrei) and Correlation with Nutritional Aspects

Alimentary habits of free-living Psittaciformes vary significantly among different species. Amazona pretrei is under risk of extinction and has very specific free-living dietary habits, which are based on Parana pine seeds. Hemosiderosis is a pathologic process characterized by intracellular accumulation of iron without other evident lesions. It is associated with increased prevalence of infections, neoplasms, and hepatopathies. The purpose of this study was to quantify hepatic hemosiderin deposits in captive A. pretrei and verify their association with nutritional parameters. Liver samples were processed for histopathology and stained with Prussian blue. The sections were analyzed by computerized morphometry to quantify the hepatic hemosiderin deposits. The hepatic hemosiderosis rates showed positive correlation with age and time in captivity. These results suggest that the menus and commercial rations for Psittacidae must be carefully revised.

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Physical, epidemiological, and molecular evaluation of infection by Cryptosporidium galli in Passeriformes

Due to the scarcity of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the periodicity of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, and its clinical signs, mortality, and molecular characterization. Four hundred eighty fecal samples were collected from 40 birds, including 372 samples from 31 adult birds and 108 samples from nine young birds (up to 12 months old), housed in five aviaries, monthly from September 2007 to September 2008, with the exception of April. The birds originated from aviaries in which the following species were raised: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Cyanocompsa brissonii), and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate at 4A degrees C until processing. The oocysts were purified by centrifugal flotation in Sheather`s solution, followed by genomic DNA extraction and molecular characterization of oocysts using the nested polymerase chain reaction for amplification of fragments of the 18S subunit of rRNA gene. Intermittent shedding of oocysts was observed by positive amplification for Cryptosporidium spp. in 91 (24.5%) samples of adult birds and 14 (13%) of young birds. The sequencing of the amplified fragments enabled the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in only one aviary and was associated with concomitant infection with Escherichia coli and Isospora sp.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4 degrees C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicurs) and a peach-faced lovebird (Agapornis roseicolis). (C) 2009 Elsevier B.V. All rights reserved.