Virulence-associated gene profiling of Streptococcus suis isolates by PCR

Definition of virulent Streptococcus suis strains is controversial. One successful approach for identification of virulent European strains is differentiation of capsular serotypes (or the corresponding cps types) and subsequent detection of virulence-associated factors, namely the extracellular factor (EF, epf), the muramidase-released protein (MRP, mrp) and the hemolysin suilysin (SLY, sly). In this work we present a novel multiplex PCR (MP-PCR) and an mrp variant PCR for identification and characterization of virulent S. suis strains. These new methods were used to identify association of disease with particular profiles of virulence-associated genes. The MP-PCR allowed identification of S. suis through detection of the housekeeping gene gdh, differentiation of four cps types (1, 2, 7 and 9), and detection of epf, mrp, sly and arcA (arginine deiminase from S. suis). Furthermore, this study describes the first PCR assay for differentiation of at least six mrp variants. Expression of the corresponding size variants of MRP was shown for four of the six mrp variants, but was undetectable for the two larger mrp variants in the particular strains investigated. The results of this study suggest that cps7 strains are associated with pneumonia and that variation of mrp is very pronounced among these strains. Gene profiles of invasive, pneumonia and carrier S. suis isolates by combination of PCR assays allowed differentiation of 24 different genotypes among cps1, 2, 7 and 9 strains. Forty-five percent of the invasive S. suis diseases investigated in this study were caused by only two of these genotypes, namely cps2/mrp+/epf+/sly+ and cps9/mrp(*)/epf-/sly+. Thus, this study demonstrates for the first time a uniform profile of the particular virulence-associated genes for the vast majority of the investigated invasive cps9 strains.     

Quantification,Morphology and Ultrastructure of Preantral Follicles of Buffalo (Bubalus bubalis) Foetuses

The objective of this study was to determine the number, morphology and ultrastructure of preantral ovarian follicles of buffalo (Bubalus bubalis) foetuses at different ages. Quantification revealed number of primordial, primary and secondary follicles of 48 857 ± 17 506, 26 000 ± 20 452, 18 428 ± 10 875 and 18 375 ± 19 690, 225 ± 349, 326 ± 288 at 12–34 cm and 35–60 cm crown rump length (CRL), respectively. Follicular diameter values were 28.9 (±3.4), 34.7 (±5.9) and 59.4 (±12.6) μm; oocyte diameters were 21.7 (±2.8), 24.3 (±3.4) and 33.0 (±7.7) μm, and the numbers of follicular cells in the follicle equatorial section were 7.1 (±1.4), 12.0 (±2.4) and 13.8 (±2.4) for primordial, primary and secondary follicles, respectively. The primordial follicle consisted of an oocyte surrounded by a layer of flattened follicular cells with a normally eccentric oocyte nucleus. Dispersed Golgi complex, smooth endoplasmic reticulum, rounded mitochondria and several lipid vesicles were observed in the cytoplasm and cell junctions between the follicle cell membranes and the oocyte. This work describes the number, morphometry and ultrastructure of preantral follicles of buffalo foetuses, concluding that folliculogenesis is established between 8 and 34 cm CRL and that follicle number varies individually and according to age and that further studies are needed in this species.     

Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes

The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6DeltaLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5DeltaLORF11 plaques were evident at 12-13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5DeltaLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.     

Platelet-Rich Plasma in the Treatment of Induced Tendinopathy in Horses: Histologic Evaluation

This study was carried out to evaluate the effect of platelet-rich plasma (PRP) on the treatment of tendinopathy induced in the superficial digital flexor tendon (TFDS) of horses, by using histologic evaluation. Six healthy crossbred geldings aged 8 to 15 years (12 ± 3) were used. The TFDS tendinopathy was provoked in both forelimbs, by intratendinous administration of 2.5 mg collagenase (2.5 mg/mL), and this procedure was considered as the beginning of the experimental phase. At 12 days after induction of the tendinopathy, the animals were subjected to the following treatments: (1) in the lesion caused in the right superficial digital flexor tendon (PRP-treated group), 2.5 mL PRP activated with calcium chloride at 0.0125 mol/L at concentrations from 320,000 to 500,000 platelets/μL, were injected; (2) in the tendinopathy of the left SDFT (control group), 2.5 mL 0.9% saline solution was administrated. Thirty-six days after the treatments, a biopsy of the injured area was performed for histologic evaluation. In both groups, the histologic analysis showed an increase in the fibroblastic density, as well as the presence of neovascularization, lymphocytes, and plasmocytes infiltrate and tissue organization at variable intensity. In the PRP-treated group, the SDFT was more organized, with the collagen fibers and fibroblasts being better arranged on the tendon matrix. The numbers of the fibroblasts and blood vessels did not differ between the groups. Histologic evaluation 36 days after tendinopathy showed that injuries under a single PRP treatment present a more uniform and organized tissue repair when compared with the control group.     

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.     

Spatialization of Brazilian pig production: relationship between productive,physical, environmental,and socio-economic variables

Brazilian pig production spans over a large territory encompassing regions of different climatic and socio-economic realities. Production, physical, socio-economic, and environmental data were used to characterize pig production in the country. Multivariate analysis evaluated indices including number productivity, production levels, and income from pigs, together with the average area of pig farm and socio-economic variables such as municipal human development index, technical guidance received from agricultural cooperatives and industrial companies, number of family farms, and offtake; and finally, environmental variables: latitude, longitude, annual temperature range, solar radiation index, as well as temperature and humidity index. The Southern region has the largest herd, number of pigs sold/sow, and offtake rate (p < 0.05), followed by the Midwest and Southeast. No significant correlations were seen between production rates and productivity with the socio-economic and environmental variables in the regions of Brazil. Production indexes, productivity, and offtake rate discriminated Northeast and Midwest and Northeast and Southeast regions. The Northern region, with a large area, has few and far-between farms that rear pigs for subsistence. The Northeast region has large herds, but low productivity. Number of slaughtered pigs has been variable over the past three decades, with few states responsible for maintaining high production in Brazil. However, the activity can be effective in any region of the country with technology and technical assistance adapted to regional characteristics.     

Serum testosterone,sperm quality,cytological, physicochemical and biochemical characteristics of the prostatic fraction of dogs with prostatomegaly

Prostatomegaly is a common finding in older non‐neutered dogs. This study compared the serum testosterone, sperm quality and characteristics of the prostatic fraction between healthy dogs and dogs with prostatomegaly. Blood samples of ten dogs (five dogs from each group) were taken for serum testosterone measurement. Sperm motility, vigour, concentration, viability, membrane functionality and morphology were analysed in sperm‐rich fraction. Osmolality, pH, cell types, and albumin, haemoglobin, acid phosphatase, alkaline phosphatase, glucose, triglycerides, cholesterol, calcium, phosphorus, magnesium and chloride were analysed in prostatic fraction. Dogs with prostatomegaly have the lowest sperm motility, vigour, concentration and functional membrane. Dogs with prostatomegaly have the highest glucose, triglycerides and cholesterol. Glucose was the only constituent positively correlated with serum testosterone and prostate volume. It can be concluded that dogs with prostatomegaly have poorer sperm quality, and glucose, triglycerides and cholesterol in prostatic fraction can be used as prostatomegaly biomarkers.     

Supplementation of suckling beef calves with different levels of crude protein on tropical pasture

The effects of supplementation with different levels of crude protein on performance, intake and nutrient digestibility and efficiency of microbial protein synthesis in suckling beef calves on pasture were assessed. Fifty-five calves, with an average age of 100 days and an initial average body weight of 110?±?7.5 kg and their respective dams, were used. The experimental design was completely randomised with five treatments and 11 replications. The experimental treatments for calves were as follows: control = calves received only mineral mixture; supplementation levels = calves received supplement containing 8, 19, 30 or 41 % of crude protein (CP, at a rate of 0.5 % of body weight (BW)). The cows received only mineral mixture ad libitum. Supplemented calves had higher (P?<?0.1) average daily gain (ADG). Protein levels showed a quadratic effect (P?<?0.1) on average daily gain (ADG) of calves. There was no difference in total dry matter (DM) intake (P?>?0.1). However, intake of dry matter forage (DMF) presented cubic profiles (P?<?0.1), with CP levels in the supplements. Supplementation increased (P?<?0.1) the digestibility of nutrients, except for the digestibility of neutral detergent fibre. Supplementation increased (P?<?0.1) the production of microbial nitrogen and N losses in urine. It can be concluded that multiple supplementations optimise the performance of beef calves on creep feeding. The intake of supplements with CP levels between 8 and 30 % partially replaces of the pasture ingested by calves and increases the digestibility of the diet.