Marcos Bryan Heinemann

Prevalence and Molecular Epidemiology of Noroviruses in Hospitalized Children With Acute Gastroenteritis in Rio de Janeiro, Brazil, 2004

Background: The role of noroviruses (NoV) as a cause of gastroenteritis outbreaks is well documented; however, the importance of NoV infections in hospitalized children is not well established. The aim of this study was to determine the prevalence and the genetic diversity of NoV in hospitalized children. Methods: Three-hundred eighteen fecal samples were collected from January to December 2004, from children with acute gastroenteritis in 3 public hospitals in Rio de Janeiro, Brazil. The prevalence and genetic diversity of NoV was carried out by using genome amplification and sequencing of polymerase and capsid genes. Results: NoV infections were detected in 65 (20%) of the samples, of which 11 (4%) were mixed infections with rotavirus. Infants up to 1-year-old were the most affected and a peak of virus detection was observed in autumn and spring seasons. Dehydration and diarrhea were the inclusion criterion; coughing (51%), vomiting (33%), and fever (22%) were the main clinical manifestations. Phylogenetic analysis showed that Genogroup II and GII/4 were prevalent. Two potential recombinant strains based in the different clustering pattern were observed. Conclusions: This study demonstrated the importance of NoV infections causing severe acute gastroenteritis in hospitalized children in Rio de Janeiro, Brazil. Molecular epidemiology surveillance determining the circulation pattern of different genotypes and recombinant strains is helpful for designing prevention strategies of NoV transmission in children. Studies concerning the prevalence and the molecular epidemiology of gastroenteric viruses in hospitalized children are particularly important to evaluate the impact of the rotavirus vaccine in Brazil.

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses.

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.

Prevalence of Toxoplasma gondii and Neospora caninum infections in sheep from Federal District, central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%<CI 0.95<49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%<CI 0.95<10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.

High seroprevalence of caseous lymphadenitis in Brazilian goat herds revealed by Corynebacterium pseudotuberculosis secreted proteins-based ELISA

We conducted a seroepidemiological survey to determine the prevalence of caseous lymphadenitis (CLA) in goat herds in Minas Gerais state, Brazil. Serum samples were collected from goats (n=676) from 108 rural properties in 2001, covering most of the sub-regions of this ca. 586,500 square kilometer state. Antibodies against Corynebacterium pseudotuberculosis secreted proteins were detected by an indirect enzyme-linked immunosorbent assay (ELISA). Most of the animals (78.9%) tested positive for CLA; 98% of flocks presented at least one seropositive animal. Goats managed under an extensive production system had a significantly higher seroprevalence of CLA than those in intensive and semi-intensive operations. The age distribution of the animals in the flocks affected the prevalence of this disease; however, goat breed did not. We found seropositivity against C. pseudotuberculosis to be highly prevalent in these Brazilian goat herds; consequently, appropriate management practices for the control of CLA should be implemented.

Estudo epidemiológico da leptospirose bovina e humana na Amazônia oriental brasileira

The seroprevalence study for leptospirosis in bovines and humans was realized in family holder farms along the Transamazon Highway. The prevalence of bovine leptospirosis was 97% [90.9 - 99.5%] of farms with at least one positive animal according to microscopic agglutination test for the leptospirosis diagnostic. In 61.2% of the tested herds, the serovar hardjo was the most common, followed by the serovar bratislava (9%) and the serovar shermani (4.5%). The serologic prevalence of leptospirosis in humans was 32.8% [23.4 - 43.5%] in family groups with at least one positive individual according to microscopic agglutination test for the leptospirosis diagnostic. In 9% of family groups, the serovar bratislava was the most common, while serovar hardjo and grippotyphosa accounted for 6% and 4.5%, respectively. The impact of these results is discussed in relation to animal production and public health. Suggestions have been proposed in order to improve the situation in the region.

Ticks (Acari: Ixodidae) Associated with Rural Dogs in Uruara ´, Eastern Amazon, Brazil

Abstract A survey of 124 dogs for ticks was conducted from August to November 1998 in 68 small farms of Uruará municipality in Eastern Amazon-Brazil. We collected 55 ticks from 18 dogs (14.5%). Amblyomma oblongoguttatum Koch, Amblyomma ovale Koch, and Amblyomma cajennense (F.) were found on nine, six, and one dog, respectively. Fourteen small farms (20.5%) had at least one dog with ticks.

Genetic diversity of Brazilian strains of porcine circovirus type 2 (PCV-2) revealed by analysis of the cap gene (ORF-2)

SummaryPorcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, 19 of 870 samples from pigs from different Brazilian states were found to be positive for PCV-2 by polymerase chain reaction (PCR). A fragment of 700 nt of the cap gene (ORF-2) from the 19 PCV-2-positive samples were sequenced using three pairs of primers (Fa/Ra, Fb/Rb and Fc/Rc). Maximum parsimony genealogy with a heuristic algorithm using the 19 field strain studied here, 21 sequences from GenBank and PCV-1 as an out-group showed the existence of two major clusters (A and B) and the Brazilian strains segregating in both of them. PCV-2 was found in pigs with various clinical signs. No association between clusters of PCV-2 and different states or clinical signs were observed, demonstrating that the exact role of PCV-2 in porcine circovirus diseases (PCVD) in Brazil still needs to be clarified. These results contribute to the molecular characterization of PCV-2, which serve as a basis for the epimiology of PCV-2 infection.

Detection of contaminants in cell cultures, sera and trypsin

The aim of this study was standardization and application of polymerase chain reaction (PCR) for the detection of contaminants in cell cultures, sera and trypsin. Five PCR protocols were standardized to assess the presence of genetic material from mycoplasma, porcine circovirus 1 (PCV1), bovine leukemia virus (BLV) or bovine viral diarrhea virus (BVDV) in cell culture samples. PCR reactions for the genes GAPDH and beta-actin were used to evaluate the efficiency of nucleic acid extraction. The PCR protocols were applied to 88 cell culture samples from eight laboratories. The tests were also used to assess potential contamination in 10 trypsin samples and 13 fetal calf serum samples from different lots from five of the laboratories. The results showed the occurrence of the following as DNA cell culture contaminants: 34.1% for mycoplasma, 35.2% for PCV1, 23.9% for BVDV RNA and 2.3% for BLV. In fetal calf sera and trypsin samples BVDV RNA and PCV1 DNA was detected. The results demonstrated that cell culture, sera and trypsin used by different laboratories show a high rate of contaminants. The results highlight the need for monitoring cell cultures and controlling for biological contaminants in laboratories and cell banks working with these materials.

Molecular epidemiology of Brazilian pseudorabies viral isolates

Pseudorabies is a disease caused by pseudorabies virus (PRV) and is responsible for considerable economic losses in the swine industry. The objective of this work was to use molecular epidemiology as a tool to facilitate the study of PRV outbreaks in Brazil. The standard PRV strain Shope, the vaccine strain Bartha and isolates from the south and the southeast regions of Brazil, were amplified for gE and gC partial genes by PCR. Results indicated that Brazilian PRV isolates are grouped in two clusters, A and B, except for one isolate that grouped with Bartha and Shope. Most Brazilian PRV isolates belonged to cluster B and diverged from virus isolated from other countries.