Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.     

Bilateral thyroarytenoid cartilage lateralization and vocal fold excision with mucosoplasty for treatment of idiopathic laryngeal paralysis: 67 dogs (1998-2005)

Objective— To evaluate combined bilateral thyroarytenoid cartilage lateralization, vocal fold excision, and mucosoplasty technique (BTAL) through ventral median laryngotomy for treatment of laryngeal paralysis in dogs.
Design— Retrospective study.
Animals— Dogs (n=67) with laryngeal paralysis.
Methods— Medical records were reviewed for dogs with idiopathic laryngeal paralysis that had BTAL between January 1998 and March 2005. Retrieved data included signalment, history, physical and laryngoscopic examination findings, clinicopathologic tests, and results of recheck examination findings.
Results— BTAL was performed by a single surgeon. Short-term (<6 months) follow-up information was available for 67 dogs and long-term (>12 months) for 40 dogs. Major postoperative complications were surgical failures (13; 7 short term, 6 long term) and aspiration pneumonia (1). Mean recurrence of clinical signs was at 19 weeks (range, 2–30 weeks). Minor complications occurred in 22 (33%) dogs including occasional coughing or gagging, stridorous breathing during exercise, panting, noisy or heavy breathing, and aspiration pneumonia (3 dogs) that did not require hospitalization. All owners reported an improved quality of life and had no regrets with surgical outcome.
Conclusions— BTAL is seemingly an effective procedure for treatment of laryngeal paralysis.
Clinical Relevance— BTAL is associated with a low incidence of aspiration pneumonia; however, there is substantial risk of recurrence of clinical signs associated with narrowing of the glottis. Consequently, unilateral arytenoid lateralization currently represents the accepted approach to the treatment of laryngeal paralysis.     

The effects of osmolality on sperm quality in <Emphasis Type="Italic">Jenynsia multidentata</Emphasis> (Cyprinodontiformes: Anablepidae)

Sperm quality tests on fish are classically used for evaluating cryopreservation procedures, and they are also promising to assess aquatic toxicity and biomarkers of xenobiotic effects on reproduction. Osmotic shock from the storage medium is one of the main factors affecting sperm quality during evaluation. Thus, the objective of this study was to evaluate the effects of different osmolalities (240–460 mOsm/kg) for at least 4 days on the sperm quality parameters of the viviparous fish Jenynsia multidentata. The level of significance was (P < 0.05). The plasma osmolality of J. multidentata is 326 ± 3.9 mOsm/kg. The motility of fresh semen was higher in osmolalities of 280 and 300 mOsm/kg but did not differ between osmolalities from 240 to 320 mOsm/kg. Above 380 mOsm/kg, the motility observed was 0 %. Over the time period studied motility increased with increasing osmolality, and the most constant and long-lasting rates were between 300 and 320 mOsm/kg. On the 4th day of evaluation, higher membrane integrity rates were observed between 280 and 360 mOsm/kg, higher mitochondrial membrane potential was observed between 300 and 460 mOsm/kg, and higher DNA integrity rates were observed between 260 and 380 mOsm/kg. Moreover, osmolalities ≥460 and ≤240 resulted in the lowest motility and DNA integrity levels. Over 4 days, the plasma membrane integrity was significantly lower at ≤260 and ≥400 mOsm/kg, and the mitochondrial membrane potential was significantly lower only in osmolalities ≤240 mOsm/kg. Therefore, we conclude that for sperm quality preservation in J. multidentata, an osmolality of 300–320 mOsm/kg of the most suitable diluent is necessary. Furthermore, we conclude that the storage of sperm in a hyposmotic (<260 mOsm/kg) or hyperosmotic (>400 mOsm/kg) solution affects not only motility but also other sperm quality parameters.     

Influence of Growing Medium and Seed Size on Germination and Seedling Growth of Pinus gerardiana Wall.

Pinus gerardiana Wall. is an important nut-producing pine having restricted distribution in the world. It has been observed that natural regeneration in the species is extremely poor or entirely lacking. The species has erratic and infrequent seed years, dormancy-related problems, and slow growth of seedling, which reduces its regeneration process in natural habitats. Therefore, we investigated the effect of growing medium and seed size on germination and seedling growth of the Pinus gerardiana. The seeds were categorized into two sizes, viz, small (<2.35 cm) and large (>2.35 cm) and five growing medium treatments were used, viz, Soil:Sand:FYM; Soil:Sand:Moss:FYM; Soil:Moss:Vermicompost; Soil:Sand:Vermicompost; and Soil:Sand:Moss:Vermicompost, for assessing their impact on germination and seedling growth. It was observed that, among different growing medium treatments, higher germination and seedling growth parameters were recorded, when Soil:Sand:Moss:Vermicompost was used for the study. Between different seed sizes, higher germination and seedling growth were recorded when seed size was large.     

Discovery of lunasin peptide in triticale (X Triticosecale Wittmack)

Lunasin is a novel, cancer-preventive, anti-inflammatory and cholesterol-reducing peptide that was originally isolated from soy and later from barley, wheat and rye. We report the first discovery of lunasin in triticale (X Triticosecale Wittmack). Moreover, we report first data of lunasin content in winter rye and wheat genotypes grown in Northern Europe. These data are novel as previously published data on finding of lunasin in cereals were obtained in genotypes grown in Korea. Lunasin content was uncovered using a previously published procedure for isolation from cereals and identified by LC-MS/MS assay. We found that triticale was the most lunasin-rich cereal, with the tested genotypes displaying the following trend in lunasin content: genotype 0002-26 > Dinaro > DSGU 10/94 > 0213-22 > 0317-14 > 0006-31. The greatest lunasin content was 6.46 mg/g in the grain of triticale genotype 0002-26. In comparison, the highest lunasin content in rye variety Dankovske Diament was 1.5 mg/g, and the highest lunasin content in the winter wheat variety Fredis was 0.23 mg/g. We conclude that triticale can play a significant role as functional food, with great potential for the use of triticale products in human and animal diets.