Genetic diversity and population structure of Brazilian native bovine breeds

Conservation and improvement strategies should be based on the association between genetic and phenotypic characteristics. The objective of this work was to characterize five native Brazilian cattle breeds (Caracu, Crioulo Lageano, Curraleiro, National Polled and Pantaneiro) and two commercial breeds (Holstein and Nellore) using RAPD technique to estimate genetic distances and variability between and within breeds. Genetic relationships were investigated using 22 primers which generated 122 polymorphic bands. Analysis of molecular variance indicated that most of the genetic variation lay among individuals within populations. The genetic variabilities between pairs of breeds were statistically significant. The smallest genetic divergence was between Crioulo Lageano and Curraleiro.The National Polled, although historically considered to be of Bos taurus aquitanicus origin,similar to theCaracu, was grouped together with the other breeds of Bos taurus ibericus origin. Generally, the individual breeds formed distinct clusters except the National Polled. The RAPD technique was capable to distinguish genetically between the breeds studied; the Caracu, Crioulo Lageano, Curraleiro and Pantaneiro may be considered distinct genetic entities thereby proving the uniqueness of the populations; the National Polled has not been able to re-establish itself after its decline in the 1950s, thereby losing its genetic identity.

Secretory IgA, a major immunoglobulin in most bovine external secretions.

Bovine secretory IgA (SIgA), recently identified in colostrum, was shown to be homologous to human SIgA by immunologic cross-reaction. A quantitative study indicated that bovine SIgA, a minor component of colostrum, is a major immunoglobulin in most other external secretions including saliva, spermatic fluid, lacrimal, nasal and gastrointestinal secretions. SIgA was isolated from saliva. The free form of secretory component was found to be abundant in milk. A normal lactating cow produces about 1.2 g of this protein per day. Two forms of IgA were identified in serum: a normal serum IgA with no secretory antigenic determinant, and a small amount of SIgA. In vitro synthesis of SIgA by the salivary gland was studied by tissue cultures with incorporation of labeled amino acids.

Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy.

Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples. The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in negatively stained or conventionally embedded samples are preparation artifacts.

Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246) of the bovine CAST gene

The objective of this work was to genotype the single nucleotide polymorphism (SNP) A2959G (AF159246) of bovine CAST gene by PCR-RFLP technique, and to report its use for the first time. For this, 147 Bos indicus and Bos taurus x Bos indicus animals were genotyped. The accuracy of the method was confirmed through the direct sequencing of PCR products of nine individuals. The lowest frequency of the meat tenderness favorable allele (A) in Bos indicus was confirmed. The use of PCR-RFLP for the genotyping of the bovine CAST gene SNP was shown to be robust and inexpensive, which will greatly facilitate its analysis by laboratories with basic structure.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

cis- and trans-acting elements involved in reactivation of vaccinia virus early transcription.

We have previously shown that transcription from the vaccinia virus 7.5K early promoter is reactivated late in infection (J. Garcés, K. Masternak, B. Kunz, and R. Wittek, J. Virol. 67:5394-5401, 1993). To identify the sequence elements mediating reactivation, we constructed recombinant viruses harboring deletions, substitutions, or insertions in the 7.5K promoter or its flanking regions. The analysis of these viruses showed that sequences both upstream as well as downstream of the transcription initiation site contribute to reactivation of the 7.5K promoter. We tested whether reactivation could be explained by a high affinity of vaccinia virus early transcription factor to reactivated promoters. Bandshift experiments using purified protein showed that promoters which bind the factor with high affinity in general also have high early transcriptional activity. However, no correlation was found between affinity of the factor and reactivation. Interestingly, overexpression of recombinant early transcription factor in vaccinia virus-infected cells resulted in a shutdown of late transcription and in reactivation of promoters, which are normally not reactivated.

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

A bovine model of a respiratory Parachlamydia acanthamoebae infection.

The aim of this study was to evaluate the pathogenicity of Parachlamydia (P.) acanthamoebae as a potential agent of lower respiratory tract disease in a bovine model of induced lung infection. Intrabronchial inoculation with P. acanthamoebae was performed in healthy calves aged 2-3 months using two challenge doses: 10(8) and 10(10) bacteria per animal. Controls received 10(8) heat-inactivated bacteria. Challenge with 10(8) viable Parachlamydia resulted in a mild degree of general indisposition, whereas 10(10) bacteria induced a more severe respiratory illness becoming apparent 1-2 days post inoculation (dpi), affecting 9/9 (100%) animals and lasting for 6 days. The extent of macroscopic pulmonary lesions was as high as 6.6 (6.0)% [median (range)] of lung tissue at 2-4 dpi and correlated with parachlamydial genomic copy numbers detected by PCR, and with bacterial load estimated by immunohistochemistry in lung tissue. Clinical outcome, acute phase reactants, pathological findings and bacterial load exhibited an initial dose-dependent effect on severity. Animals fully recovered from clinical signs of respiratory disease within 5 days. The bovine lung was shown to be moderately susceptible to P. acanthamoebae, exhibiting a transient pneumonic inflammation after intrabronchial challenge. Further studies are warranted to determine the precise pathophysiologic pathways of host-pathogen interaction.

Evidence for Parachlamydia in bovine abortion.

Bovine abortion of unknown infectious aetiology still remains a major economic problem. In this study, we focused on a new possible abortigenic agent called Parachlamydia acanthamoebae. Retrospective samples (n=235) taken from late-term abortions in cattle were investigated by real-time diagnostic PCR for Chlamydiaceae and Parachlamydia spp., respectively. Histological sections of cases positive by real-time PCR for any Chlamydia-related agent were further examined by immunohistochemistry using specific antibodies. Chlamydophila abortus was detected only in three cases (1.3%) by real-time PCR and ArrayTube Microarray playing a less important role in bovine abortion compared to the situation in small ruminants in Switzerland. By real-time PCR as many as 43 of 235 (18.3%) cases turned out to be positive for Parachlamydia. The presence of Parachlamydia within placental lesions was confirmed in 35 cases (81.4%) by immunohistochemistry. The main histopathological feature in parachlamydial abortion was purulent to necrotizing placentitis (25/43). Parachlamydia should be considered as a new abortigenic agent in Swiss cattle. Since Parachlamydia may be involved in lower respiratory tract infections in humans, bovine abortion material should be handled with care given the possible zoonotic risk.

Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion.

Chlamydophila abortus and Waddlia chondrophila cause abortion in ruminants. We investigated the role of Parachlamydia acanthamoebae in bovine abortion. Results of immunohistochemical analyses were positive in 30 (70%) of 43 placentas from which Chlamydia-like DNA was amplified, which supports the role of Parachlamydia spp. in bovine abortion.