Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Despite great efforts of pig holders, veterinarians, researchers and vaccine developers, the virus still causes major production losses. It is clear that efficient and correct monitoring and rational development of vaccines are crucial in the combat against this pathogen. PRRSV-specific monoclonal antibodies (mAbs) are essential tools for both diagnostic and research purposes. This study describes the production of PRRSV GP3-, GP5- and N-specific hybridomas and an extensive characterization of the mAbs. The N-specific mAbs generated in this study appear to be useful tools for diagnostics, as they were found to react with genetically very different PRRSV isolates and may serve to discriminate between European and American type PRRSV isolates. These mAbs also allowed detection of the PRRSV N protein in both formalin-fixed, paraffin-embedded tissue sections and frozen tissue sections of PRRSV-infected lungs, further illustrating their diagnostic value. Different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested shows virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the "major neutralizing epitope" suggested for American type PRRSV. The current findings argue against an important role of the identified antigenic regions in direct antibody-mediated neutralization of European type PRRSV in vivo. However, it is also clear that findings concerning a specific PRRSV epitope cannot always be generalized, as the antigenic determinants and their biological properties may differ radically between different virus isolates.     

Indicators of induced subacute ruminal acidosis (SARA) in Danish Holstein cows

Background

The prevalence of subacute ruminal acidosis (SARA) in dairy cows is high with large impact on economy and welfare. Its current field diagnosis is based on point ruminal pH measurements by oral probe or rumenocentesis. These techniques are invasive and inaccurate, and better markers for the diagnosis of SARA are needed. The goal of this study was to evaluate clinical signs of SARA and to investigate the use of blood, faecal and urinary parameters as indicators of SARA. Six lactating, rumen cannulated, Danish Holstein cows were used in a cross-over study with three periods. The first and second periods included two cows on control diet and two cows on nutritional SARA challenge. The third period only included two cows on SARA challenge. Control diet was a conventional total mixed ration [45.5% dry matter (DM), 17.8% crude protein, 43.8% neutral detergent fibre, and 22.5% acid detergent fibre (DM basis)]. SARA challenge was conducted by substituting control diet with grain pellets (50% wheat/barley) over 3 days to reach 40% grain in the diet. Ruminal pH was measured continuously. Blood samples were collected once daily at 7 h after feeding. Samples of faeces and urine were collected at feeding, and at 7 and 12 h after feeding. Blood samples were analysed for pCO2, pO2, pH, electrolytes, lactate, glucose, packed cell volume (PCV), and total plasma protein concentration. Milk composition, ruminal VFA, and pH of faeces and urine were measured.

Results

SARA was associated with decreased (P < 0.05) minimum ruminal, faecal and urinary pH. Daily times and areas of ruminal pH below 5.8, and 5.6 were increased to levels representative for SARA. Significant differences were detected in milk composition and ruminal VFAs. Blood calcium concentration was decreased (P < 0.05), and pCO₂ tended to be increased (P = 0.10). Significant differences were not detected in other parameters.

Conclusions

SARA challenge was associated with changes in faecal and urinary pH, blood calcium concentration and pCO₂. These may be helpful as indicators of SARA. However changes were small, and diurnal variations were present. None of these parameters are able to stand alone as indicators of SARA.     

A simple and accurate method for determining wheat grain fructan content and average degree of polymerization

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.     

Ophthalmomyiasis interna anterior in a dog: keratotomy and extraction of a Cuterebra sp. larva

A 1‐year‐old female spayed mixed‐breed dog was presented for evaluation of acute onset of lethargy and blepharospasm OD. Slit‐lamp biomicroscopy revealed a segmented larva embedded in the inferonasal iris. Moderate anterior uveitis was present. After pharmacologic dilation, mild vitreal hemorrhage and chorioretinal migration tracks were identified. The dog was treated empirically for uveitis for 5 days, after which a keratotomy and larval foreign body extraction was performed. The dog recovered uneventfully from intraocular surgery and remained visual and comfortable. Parasite evaluation confirmed the larva to be a Cuterebra sp. To the authors’ knowledge, this is the first report of canine ophthalmomyiasis interna anterior, and the first report of successful surgical removal from the anterior segment in a dog with preservation of vision.     

Protective role of antifreeze proteins on sterlet (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis>) sperm during cryopreservation

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.     

Sea cucumbers in the Seychelles: effects of marine protected areas on high‐value species

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Evaluation of potential feed sources,and technical and economic considerations of small‐scale commercial striped catfish (Pangasius hypothalamus) pond farming systems in the Mekong Delta of Vietnam

A technical and socio‐economic survey was conducted in Dong Thap, Can Tho and An Giang, provinces of Vietnam in 2009. The results showed that most of the small scale farmers of striped catfish (Pangasius hypothalamus) carry out fish pond culture, developed and operated by self‐learned experience and from neighbours knowledge. The mean pond depth varied from 3.5 to 4.0 m, ranging from 2 to 6 m. Most of the fish farms have extremely high stocking density, with an average of about 40 fish m^?2, in some cases up to 70 fish per m². The average yields and net income of small‐scales pond farming is about 21–27 kg per m² crop^?1 and US$ 0.184–0.329 kg^?1 crop^?1 respectively. These figures are not significantly different compared with large‐scale pond farming. About 75%, 86% and 80% of fish farmers in Dong Thap, Can Tho and An Giang province, respectively, received positive net returns. Farm‐made feed is still playing an important role because of higher net profit compared with commercial pellet feeds. Rice bran and trash fish are the main feed ingredients for farm‐made feed formulations. Striped catfish farmers are now actively searching for locally produced plant protein sources as alternatives to marine trash fish or fish meal.     

In Vitro Digestibility of Different Commercial Edible Algae Products

Digestibility is an important factor in determining the level of nutritive factors utilization. Determination of in vitro digestibility in a Daisy incubator and assessment of protein digestibility using Kjeldahl method of algal food products after pepsin, pancreatin, and combined hydrolysis of both enzymes was described. The influence of different hydrolysis times on algal digestibility was investigated in brown seaweeds, red seaweeds, and green alga. The highest digestibility values were generally determined in red seaweeds after 24 h of combined hydrolysis by pepsin and pancreatin. Direct dependence between digestion time and digestion efficiency was verified and statistically proven.     

Repression of the 14-3-3 gene affects the amino acid and mineral composition of potato tubers

Recently, transgenic potato plants were created showing underexpression of the 20R isoform of the 14-3-3 protein. The transgenic plants grown in tissue culture showed a significant increase in nitrate reductase activity and a decrease in nitrate level. The transgenic line with the lowest 14-3-3 quantity was field-trialed (1997-2000) and analyzed. The reduction in the 14-3-3 protein level consistently resulted in a starch content increase and in an increase in the ratio of soluble sugars to starch in the tubers, although the latter was only barely visible. The determination of amino acid composition in the tubers showed a significant increase in methionine, proline, and arginine content and a slight but consistent increase in hydrophobic amino acid and lysine content in the cells of the transgenic potato plants. We also observed an increase in the crude protein content, from 19 to 22.1% of the control value in consecutive years. It is proposed that all of these changes might have resulted from the downregulation of nitrate reductase and sucrose phosphate synthase activities by 14-3-3, although other potential mechanisms cannot be excluded (e.g., an increase in enzyme protein level). 14-3-3-repressed transgenic plants showed a significant increase in calcium content in their tubers. It is thus proposed that a function of the isolated 14-3-3 isoform is in the control of amino acid synthesis and calcium metabolism. However, the mechanism of this control is as yet unknown.