Effects of carbohydrate source and polyethylene glycol on maturation and germination of somatic embryos in walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of somatic embryo maturation, germination, and conversion to plantlets is a major problem in many species including J. regia L. Germination efficiency of somatic embryos is very low in walnut. In this study, effects of two carbohydrate sources, sucrose and maltose (each at 3% and 6%), and two kinds of PEG (4000 and 6000) (each at four levels of 1.5%, 3%, 5% and 7.5%) on maturation and germination of walnut embryos were tested. The number of somatic embryos increased conspicuously on medium containing PEG. Furthermore, higher levels of PEG-4000 (7.5%) could remarkably enhance the morphogenesis of somatic embryos and the number of embryos produced. PEG-4000 stimulated somatic embryo maturation of walnut. This stimulatory effect was dependent on the carbohydrate source used. Sucrose in combination with PEG-4000 produced 50% of cotyledonary and normal somatic embryos. Different concentrations of PEG were effective on the number of embryos with a shoot meristem. PEG-4000 7.5% and sucrose 3.0% produced the highest rate (50.0%) of normal shooting embryos. However, PEG (4000, 6000) and maltose caused an unfavorable effect and increased the frequency of abnormal shaped somatic embryos.     

Effect of a Probiotic on Prevention of Diarrhea and Clostridium difficile and Clostridium perfringens Shedding in Foals

Background

Up to 60% of foals develop diarrhea within 6 months after birth. Preventive measures are limited but potentially probiotics could be used.

Objective

To evaluate the effect of a newly designed probiotic on the incidence of foal diarrhea in a randomized field trial.

Animals

Seventy‐two healthy neonatal foals.

Methods

Randomized, placebo‐controlled field trial. Foals were administered a placebo or probiotic for 3 weeks and monitored for an additional week. A total of 3 fecal samples were taken from each foal at biweekly intervals. Statistical modeling was applied for comparison of incidence and duration of diarrhea and fecal shedding of Clostridium perfringens and Clostridium difficile between treatment and age groups.

Results

The overall incidence of diarrhea was 41 of 72 (59%) and did not differ (P = 0.37) between treatment groups. Foals treated with probiotics were more likely to develop diarrhea requiring veterinary intervention (P = 0.007). Age had a significant effect on incidence of diarrhea (P < 0.001); foals 8–15 days old having the highest probability of developing diarrhea. Duration of diarrhea and soft feces were not significantly different between groups. The prevalence of C. perfringens shedding was 55% with no difference between treatment groups (P = 0.23). The prevalence of C. difficile shedding was 11%.

Conclusion and Clinical Importance

There was no benefit of administering a 3‐week course of probiotics, but potential adverse effects were noted. Whether the probiotics lacked a clinical effect, or the choice of strains or dose was inadequate, is unknown. Clostridial shedding was not influenced by probiotics despite in vitro activity of probiotics.     

Serum Profiles of Luteinizing Hormone,Oestradiol and Cholesterol and Ovarian Functions in Layer Poultry Birds (Gallus domesticus) Fed Diets Containing Furazolidone

This study was carried out to assess the serum profiles of luteinizing hormone (LH), oestradiol, cholesterol and ovarian functions in layer poultry birds (Rhode Island Red: Gallus domesticus) fed a diet containing various concentrations of furazolidone (FZ). A total of 40 birds were randomly assigned to receive FZ 0, 200, 400 or 800 mg/kg feed (ppm) daily during the pre-laying age, i.e. 13-18 weeks (for 5 weeks). Blood samples were collected at weekly intervals. Concentrations of LH and oestradiol in serum were estimated at alternate weeks using radioimmunoassays. Serum cholesterol levels were analysed by an enzymatic calorimetric method. Furazolidone administration was terminated at the 18th week of age. The birds were sacrificed at 22nd week of age and ovarian tissues were processed for morphometric studies. Serum LH, oestradiol and cholesterol levels were affected by age (p < 0.001) and FZ dose (p < 0.001). Serum LH and oestradiol levels were lower (p < 0.05) in birds receiving FZ 800 mg/kg feed daily compared with the controls, whereas serum cholesterol profiles were lower (p < 0.05) in all FZ-administered groups than in the control group. The mean weight of ovaries having no yolky follicles observed in the group receiving FZ 400 or 800 mg/kg feed per day was reduced (p < 0.05) compared with the control group. Dosing FZ at 800 mg/kg feed per day reduced (p < 0.05) the mean volume of ovaries having no yolky follicles compared with the control group. In birds receiving FZ 800 mg/kg feed per day, the mean length of the oviduct was reduced (p < 0.05) as compared with the control group. Morphometric studies revealed that the mean number of oocytes with diameter in the range 401-800 microm decreased (p < 0.05) in birds fed FZ 400 or 800 mg/kg feed per day. Initial egg production was affected by age (p < 0.001) and dose (p < 0.001) of FZ. The mean number of eggs laid by different groups revealed that egg production was reduced (p < 0.05) in birds receiving FZ 800 mg/kg feed per day as compared with the controls. The present data suggest that FZ causes suppression in serum profiles of LH, oestradiol, cholesterol and ovarian functions in Rhode Island Red layer poultry birds. Therefore, great care must be taken with use of FZ in layer poultry birds (Gallius domesticus) with regard to dosage and duration of administration.     

Detection of specific hydatid antigens and antibodies in serum and urine of experimentally infected sheep

Immunodiagnostic confirmation of cystic human hydatidosis is frequently required before surgical intervention or of chemotherapy. However, it remains inadequate to detect specific antibodies or antigens in some confirmed cases of echinococcosis. This study was carried out to investigate the accuracy of three different immunodiagnostic tests for detection of specific circulating antigens or antibodies in the serum and urine of 13 experimentally infected sheep. For this purpose, Echinococcus granulosus were collected from small intestine of experimentally infected dogs, and 2000 taenid eggs were orally administered to each of the 13 sheep. There were six other sheep, which were kept as the control group. Biweekly serum and urine samples were collected from all the sheep for 4 months after infection. The sera were subjected to indirect hemagglutination test and the concentrated urine samples were subjected to coagglutination and counter immunoelectrophoresis tests. The results revealed that the sensitivity of these tests in detecting the hydatid antigens in the urine or antihydatid antibodies in the serum of the infected sheep reached their maximum in 12th and 13th week after infection; then it decreased in the following weeks. Examination of the non-infected sheep samples throughout the experiment showed that the aforesaid findings were specific only to the infected sheep. It seems that the appearance of specific hydatid antigen in urine and its antibodies in the serum were simultaneous. Although these tests are highly specific, false negative outcomes were encountered in their detection of cystic echinococcosis. In general, it seems rational to establish some series of diagnostic procedures in order to reveal antibodies and antigen of metacestode in serum and urine of the patients.     

Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.

METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.

RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.

CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.

CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.     

Oral vaccination of brushtail possums (Trichosurus vulpecula) with BCG: immune responses,persistence of BCG in lymphoid organs and excretion in faeces

AIMS: To determine immune responses, and the localisation and persistence of Mycobacterium bovis bacille Calmette-Guérin (BCG) in gut-associated lymphoid tissues (GALT) and other organs in possums vaccinated orally with lipid-formulated BCG vaccine. To determine the duration of excretion and longevity of survival of BCG in the faeces of vaccinated animals.

METHODS: Possums (n=28) were vaccinated with lipid-formulated BCG (1 x 10 ⁸ colony forming units (cfu) of formulated BCG) by the oral route. Control possums (n=17) were fed oral bait pellets containing formulation medium only. Possums were sacrificed at 3 days and at 1, 3, 6 and 8 weeks after vaccination or ingestion of bait. Proliferation responses to bovine purified protein derivative (PPD) were measured in lymphocytes from blood and mesenteric lymph nodes (MLN) and samples of lung, spleen, liver, MLN and Peyer's patches (PP) were cultured for the presence of BCG. The number of BCG organisms excreted in faeces and the duration of excretion were determined in eight vaccinated possums and eight control possums over a 3-week period. In a separate experiment, a further six possums were vaccinated with oral BCG vaccine (5–10 x 10 ⁸ cfu BCG/possum) and their faeces collected over 48–72 h, for culture of BCG. The longevity of survival of BCG in these faeces was determined by storing faecal samples (n=12) under three different conditions: in an incubator (22.5°C), and conditions which simulated the forest floor and open pasture. A proportion (1–2 g) of these faecal samples was collected after storage for 1, 3, 5, 8 or 20 weeks, and cultured for BCG.

RESULTS: Possums vaccinated orally with BCG vaccine showed strong proliferation responses to bovine PPD in peripheral blood lymphocytes at 6–8 weeks post-vaccination (p.v.). Positive lymphocyte proliferation assay (LPA) responses to bovine PPD were first evident in MLN at 3 weeks p.v. BCG was cultured from MLN and PP in a proportion of animals at 3–8 weeks p.v. BCG was not cultured from sections of spleen, lung or liver at any time p.v. BCG was recovered in low to moderate numbers from the faeces of vaccinated possums for up to 7 days, and maximal numbers were cultured in faeces collected 48–72 h p.v. After storage for 1 week, BCG was cultured from all faecal samples placed in the incubator and from a proportion of faeces exposed to conditions similar to those on the forest floor and pasture. With the exception of one faecal sample stored under forest floor conditions which was culture-positive for BCG at 3 and 5 weeks, BCG was not cultured from any other faecal sample stored for more than 1 week.

CONCLUSIONS: Ingestion of oral BCG vaccine by possums was associated with the development of strong cell-mediated immunity in both blood and MLN. Following oral vaccination with BCG, the organisms were localised and persisted in GALT but did not spread to the spleen, liver or lungs. BCG was shed in low to moderate numbers in the faeces for up to 7 days p.v. The viability of BCG excreted in faeces decreased rapidly, particularly when faeces were exposed to an open pasture environment. Oral vaccination of possums with formulated BCG is unlikely to result in undue contamination of the environment with BCG.     

Oral vaccination of brushtail possums with BCG: Investigation into factors that may influence vaccine efficacy and determination of duration of protection

AIMS: To determine factors that may influence the efficacy of an oral pelleted vaccine containing Mycobacterium bovis bacille Calmette-Guérin (BCG) to induce protection of brushtail possums against tuberculosis. To determine the duration of protective immunity following oral administration of BCG.

METHODS: In Study 1, a group of possums (n=7) was immunised by feeding 10 pellets containing dead Pasteur BCG, followed 15 weeks later with a single pellet of live Pasteur BCG. At that time, four other groups of possums (n=7 per group) were given a single pellet of live Pasteur BCG orally, a single pellet of live Danish BCG orally, 10 pellets of live Pasteur BCG orally, or a subcutaneous injection of live Pasteur BCG. For the oral pelleted vaccines, BCG was formulated into a lipid matrix, and each pellet contained approximately 10⁷ colony forming units (cfu) of BCG, while the vaccine injected subcutaneously contained 10⁶ cfu of BCG. A sixth, non-vaccinated, group (n=7) served as a control. All possums were challenged by the aerosol route with a low dose of virulent M. bovis 7 weeks after vaccination, and killed 7–8 weeks after challenge. Protection against challenge with M. bovis was assessed from pathological and bacteriological findings.

In Study 2, lipid-formulated live Danish BCG was administered orally to three groups of possums (10–11 per group), and these possums were challenged with virulent M. bovis 8, 29 or 54 weeks later. The possums were killed 7 weeks after challenge, to assess protection in comparison to a non-vaccinated group.

RESULTS: The results from Study 1 showed that vaccine efficacy was not adversely affected by feeding dead BCG prior to live BCG. Feeding 10 vaccine pellets induced a level of protection similar to feeding a single pellet. Protection was similar when feeding possums a single pellet containing the Pasteur or Danish strains of BCG. All vaccinated groups had significantly reduced pathological changes or bacterial counts when compared to the non-vaccinated group. In Study 2, oral administration of Danish BCG induced protection against challenge with M. bovis, which persisted for at least 54 weeks after vaccination. Some protection was observed in possums challenged 54 weeks after vaccination, but this protection was significantly less than that observed in groups vaccinated 29 or 8 weeks prior to challenge. There was a strong relationship between the proportion of animals producing positive lymphocyte proliferation responses to M. bovis antigens and protection against challenge with M. bovis.

CONCLUSIONS: Factors considered potentially capable of interfering with vaccination, including feeding dead BCG to possums prior to feeding live BCG, feeding multiple doses of BCG at one time, and changing strains of BCG, were shown not to interfere with the acquisition of protective immune responses in possums. Protection against tuberculosis was undiminished up to 29 weeks after vaccination with BCG administered orally. It is concluded that vaccination of possums by feeding pellets containing BCG is a robust and efficient approach to enhance the resistance of these animals to tuberculosis.     

Ability of sorption–desorption experiments to predict potassium leaching from calcareous soils

When potassium (K⁺) fertilizers are applied to the soil, K⁺ is subject to displacement through the soil profile. Leaching can play an important role in agricultural K⁺ losses that can decrease groundwater quality. To avoid overfertilization, estimation of K⁺ leaching from soil is important. The ability of the soils to retain K⁺ against leaching varies according to the adsorption coefficient of the soils. The aim of this study was to relate the K⁺ leaching from a wide range of calcareous soils to the values obtained from a sorption–desorption experiment. The soil columns were leached with 10 mM CaCl₂ solution and the leachate was analyzed for K⁺. The breakthrough curves for K⁺ were different, and the amounts of K⁺ leached varied considerably between different soils. In these calcareous soils where crops are irrigated with water containing significant concentrations of Ca²⁺ and other cations, large amounts of K⁺ will be leached. Cumulative K⁺ leached after five pore volumes leaching with 10 mM CaCl₂ was significantly (r = 0.776, p < 0.01) related to the equilibrium K⁺ concentration. The results of this study enabled us in many cases to estimate the K⁺ leaching from soil without conducting column experiments, minimizing the laborotary work.     

Effects of supplemental dietary l‐carnitine and ractopamine on the performance of juvenile rainbow trout,Oncorhynchus mykiss

This study was carried out to investigate a possible protein‐sparing action of l ‐carnitine and ractopamine in rainbow trout, Oncorhynchus mykiss. An 8‐week feeding trial was carried out to evaluate the effects of supplementation of three levels of l ‐carnitine (0, 1 and 2 g kg^?1) and two levels of ractopamine (0 and 10 mg kg^?1) on growth performance, fillet fatty acid compositions and blood biochemical parameters in a 3 × 2 factorial experimental design. Ractopamine and 1 g kg^?1 carnitine improved the specific growth rate (1.03% and 1.05% day^?1), feed conversion ratio (FCR, 1.3 and 1.29), protein efficiency ratio (PER, 1.88 and 1.85) of fish and crude protein (73.5 and 73.8) content of fish fillet. l ‐carnitine and ractopamine increased the levels of albumin, total protein and globulin in the serum of fish. Apart from eicosapentaenoic acid and docosahexaenoic acid, other fatty acids of fish fillet were increased by ractopamine, while total saturated fatty acids were almost intact. However, the total n‐3 poly unsaturated fatty acids were reduced by l ‐carnitine supplementation (P<0.05). The present study showed that 1 g kg^?1l ‐carnitine and 10 mg kg^?1 ractopamine each can improve the performance of rainbow trout and their combination in diet could enhance the protein level and change the fatty acids profile in fillet muscle.     

Neuronal Cell Reconstruction with Umbilical Cord Blood Cells in the Brain Hypoxia-Ischemia

Background: Brain hypoxia-ischemia is a human neonatal injury that is considered a candidate for stem cell therapy. Methods: The possible therapeutic potential of human umbilical cord blood (HUCB) stem cells was evaluated in 14-day-old rats subjected to the right common carotid occlusion, a model of neonatal brain hypoxia-ischemia. Seven days after hypoxia-ischemia, rats received either saline solution or 4 × 10⁵ HUCB cells i.v. Rats in control group did not receive any injection. After two weeks, rats were assessed using two motor tests. Subsequently, rats were scarified for histological and immunohistochemical analyses. Results: Our immunohistochemical findings demonstrated selective migration of the injected HUCB cells to the ischemic area as well as reduction in infarct volume. Seven days after surgery, we found significant recovery in the behavioral performance in the test group (12.7 +/- 0.3) compared to the sham group (10.0 +/-0.05), a trend which continued to day 14 (15.3 ± 0.3 vs. 11.9 ± 0.5, P<0.05). Postural and motor asymmetries at days 7 and 14 in the test group showed a significant decrease in the percentage of right turns in comparison to the sham group (75% and 59% vs. 97% and 96%, P<0.05). Conclusion: The results show the potential of HUCB stem cells in reduction of neurologic deficits associated with neonatal hypoxia-ischemia. Key Words: Hypoxia-Ischemia, Nerve cell, Umbilical Cord Blood