Immune response of BALB/c mouse immunized with recombinant MSPs proteins of Anaplasma marginale binding to immunostimulant complex (ISCOM)

Anaplasmosis, caused by Anaplasma marginale, results in significant economic losses of cattle in tropical and subtropical regions worldwide. Six major surface proteins (MSPs) were well characterized and designated as MSP1, MSP2, MSP3, MSP4, and MSP5. The objective of this study was to evaluate the humoral immune response of BALB/c mice against the recombinant MSPs, incorporated into immunostimulating complex (ISCOM). The recombinant proteins purified by Ni-NTA columns were incorporated into ISCOM and ISCOMATRIX by the lipid film hydration method. BALB/c mice immunized with ISCOM/rMSPs and ISCOMATRIX/rMSPs vaccines produced whole IgG, IgG1, and IgG2a, in contrast to the negative groups (PBS and ISCOMATRIX adjuvant). All groups that received antigen responded specifically against the rMSPs by Western blotting, showing the rMSP1a (60-105kDa), rMSP1b (100kDa), rMSP4 (47kDa), and rMSP5 (29kDa). Additional studies will have to be performed in cattle to evaluate the humoral and cellular mechanisms of this subunit vaccine and their possible use as protective vaccines against homologous and heterologous strains of A. marginale.     

Isolation of Lawsonia intracellularis specific single-chain Fv antibody fragments from phage display library

Single-chain antibodies (scFv) exhibiting specific binding to Lawsonia intracellularis were isolated from a phagemid library expressing scFvs molecules on the surface of filamentous bacteriophages. For scFv selection whole bacterial cells were used and individual clones were tested in ELISA test. The total of seven unique clones with different fingerprint profiles was isolated. All clones were able to bind specifically in immunofluorescence assay. This is the first report of species specific recombinant antibodies against L. intracellularis.     

Effect of a saponin-based surfactant and aging time on ruminal degradability of flaked corn grain dry matter and starch

The objectives of this study were to investigate the effect of a saponin-based surfactant, Grain Prep surfactant (GP), and hot flake aging time on starch characteristics and ruminal DM and starch degradability of steam-flaked corn grain. In 2 experiments, the moisture content of incoming corn was automatically adjusted using the Grain Prep Auto Delivery System to 19.8% (Exp. 1) and 18.5% (Exp. 2). The application rate of GP was 22 mg/kg (as-is basis). Control corn was treated with water alone. Processed corn in Exp. 2 was stored in insulated containers for 0, 4, 8, or 16 h. Flaked corn samples were incubated in the rumen of lactating dairy cows for 0, 2, 4, 6, 16, or 24 h. In Exp. 1, GP increased, compared with the control, the soluble fraction and effective degradability (ED) of DM by 17.2 and 8.6%, respectively. The ED of cornstarch was increased by 6.7%. In Exp. 2, the concentration of soluble DM and starch were increased by GP by 15 and 24% compared with the control. The ED of DM and starch were also increased by 3 and 4%, respectively. No differences in gelatinization temperatures were observed due to treatment, except that GP-treated grain had a slightly greater mean gelatinization enthalpy in Exp. 2. In a pilot study, DM degradability parameters were not affected by germination of the corn kernels. Aging of the hot flakes for up to 16 h resulted in a quadratic decrease in DM and starch ruminal degradability. The aging process affected starch gelatinization enthalpy values of flaked grain in a manner opposite to that observed for ruminal DM and starch degradation. This phenomenon was most likely explained by increased starch intramolecular associations or crystallinity associated with starch annealing, or both. This study confirmed our previous observations that Grain Prep surfactant increases flaked corn DM and starch degradability in the rumen. Because the rate of degradation was not affected by the surfactant, the increase in degradability was attributed mainly to increases in DM and starch solubility.     

Effect of the seaweed Ascophyllum nodosum on lambs during forced walking and transport

The objective of this study was to determine the effect of feeding Ascophyllum nodosum (ANOD) to lambs at 0 (control), 0.5, 1, or 2% of DMI/d for 2 wk on lamb physiology in response to forced walking and transport during hot weather. Forty-four lambs (26 kg +/- 4.3) were used, and each lamb swallowed 3 gelatin capsules filled with ANOD or their normal 16% CP, pelleted grain ration twice daily, with the amount of ANOD dependent on the treatment. The amount of ANOD did not affect ear canal temperature or cortisol concentrations during 60 min of forced walking. The range between the minimum and maximum ear canal temperature for each lamb during 12 h of transport was narrower in lambs receiving the 2% ANOD than the control group (P = 0.05), and the 2% ANOD group also had lower (P = 0.05) ear canal temperatures than the control group during hot periods of transport. After 4 (P = 0.09) and 8 h (P = 0.05) of transport, the control group tended to have greater cortisol concentrations than the 2% ANOD group. Many differences among treatments were found in plasma protein, albumin, calcium, phosphorus, glucose, blood urea nitrogen, aspartate aminotransferase, magnesium, sodium, potassium, and chloride concentrations posttransport; mainly, the control and 0.5% ANOD groups had greater (P < 0.05) concentrations than the other 2 treatments. Aldosterone concentrations were greater in the control and 0.5% ANOD group than in the 1 and 2% ANOD groups before transport, whereas the concentrations were not different after transport, suggesting pretransport concentrations were lowered by supplementation. The 1 and 2% ANOD groups lost more BW than the control group as a result of transport (P = 0.04). After transport, no differences were observed in the latency for lambs to drink, eat, or lay. There was a suppression of the IgG and IgM antibody responses at 4 and 7 d after administration of ovalbumin, with greater ANOD supplementation rates suppressing antibody response the greatest. Although ANOD decreased ear canal temperature in hot periods of transport, stabilized electrolyte concentrations, and decreased cortisol throughout transport, it also suppressed the antibody response indicating that the effect of ANOD on immune function merits further investigation.     

Detection of genes with moderate effects on disease resistance using ovine mhc and resistance to nematodes as an example

Detecting some of the genes that influence disease resistance would improve our understanding of the processes that cause disease and also simplify disease control. Genes within the major histocompatibility complex (mhc) are strong candidates for disease resistance and they have been intensely studied for the last 30 years. Recently, several groups working independently have reported the existence of alleles within the mhc that are associated with enhanced resistance to nematode infection. This article uses hindsight to describe some of the potential pitfalls that hinder the search for valid disease resistance genes. The search requires a good understanding of disease biology, molecular genetics, statistical genetics and especially, the design and analysis of experiments. The power to detect mhc effects is quite low and is quite sensitive to the frequency of the putative resistance alleles.     

Validation of endogenous reference genes for expression profiling of RAW264.7 cells infected with Mycobacterium avium subsp. paratuberculosis by quantitative PCR

Reference genes are frequently used to normalize between different biological samples the levels of mRNA measured using quantitative PCR (qPCR). The expression level of many commonly used reference genes has been shown to vary between tissues or cells, or following exposure to various treatments including infection with microbes. The selection of an appropriate reference gene for an individual experiment is therefore a crucial step in the process of accurately determining changes in gene expression. For this purpose, we analyzed the expression of nine commonly used reference genes in a murine macrophage cell line, RAW264.7, for their potential use in the analysis of differential gene expression by quantitative polymerase chain reaction (qPCR) following experimental infection with Mycobacterium avium subsp. paratuberculosis. Only one of nine putative reference genes tested, casc3a, was found to be suitable, and combinations of two or more reference genes were disadvantageous. Based on data from the study, we recommend an approach for selection of reference genes, conducting assays with technical replicates in duplicate rather than triplicate, determining decision-limit quality control criteria for technical replicates and assessing the significance of gene expression fold differences using DeltaDeltaC(t) based on knowledge of the variation in the reference gene.     

Postnatal Osteological Development of the Hyoid Bone in the New Zealand White Rabbit

This study augments knowledge of bone growth by observing the development of the hyoid bone in the New Zealand White rabbit. Preserved hyoid bones representing five different age periods, each period including five individuals and the total number of animals being 25, were fixed in 3.5% formaldehyde solution and 95% ethanol, followed by a pure acetone bath. They were then stained with an alcian blue–alizarin red combination. The mode of bone formation was intracartilaginous type ossification. While the basihyoideum and thyrohyoideum were observed to start ossifying first at prenatal stage, indicating that they are the main skeletal structures of the hyoid apparatus, the ceratohyoideum and lingual process began to ossify in the second and third periods. The separately occurring primary ossification centres fused completely among themselves in the fifth period. Because no further ossification centers were observed and the ossified parts continued the development and growth, the research was terminated after 10 weeks of age.     

Effects of Polyether Ionophores on the Protective Immune Responses of Broiler Chickens against Angara Disease and Newcastle Disease Viruses

Immunization against Angara disease virus (ADV), a serotype 4 avian adenovirus, and Newcastle disease virus (NDV), an avian paramyxovirus serotype 1, is the mainstay of a broiler vaccination programme, while polyether ionophores usually form an essential component of a broiler medication programme in most parts of India and Pakistan. The role of polyether ionophores in the protective immune responses of broiler chickens vaccinated and challenged with ADV and NDV was investigated. A total of 1600 birds were divided into eight groups of 200 birds each. First four groups were vaccinated against NDV and ADV, while the remaining four served as unvaccinated controls. The first 3 groups of birds were administered salinomycin, monensin and cyclophosphamide (CYP), respectively. The last group served as an untreated control. The same treatment schedule was also followed for the next four unvaccinated groups. The post-vaccination and post-challenge serological responses to NDV and ADV, body and lymphoid organ weight gains, post-challenge survival rate and detection of NDV and ADV in the tissues of infected birds were evaluated. Birds administered salinomycin showed a significant stimulation of protective immune responses against both NDV and ADV as compared to the untreated and CYP-treated birds. Monensin also enhanced the protective immune responses against both viruses but the effect was not statistically significant. Thus, it is concluded that monensin and salinomycin augment the anti-NDV and anti-ADV immune responses in broiler chickens, which supports their use in poultry flocks.     

Effects of Epidural Ketamine–Xylazine Combination on the Clinicophysiological and Haematobiochemical Parameters of Uraemic and Healthy Goats

Xylazine–ketamine combination was evaluated for its efficacy and safety after epidural administration in uraemic and healthy goats. The combination (xylazine 0.025 mg/kg and ketamine 2.5 mg/kg) was administered to uraemic (n = 6) and healthy (n = 6) animals in the lumbosacral epidural space. The combination was evaluated in terms of clinical, physiological, haematological and biochemical parameters. The onset of analgesia was faster in healthy animals than in uraemic animals. Xylazine and ketamine produced complete analgesia of tail, perineum, inguinal and thigh regions in all animals of both groups. However, healthy animals showed longer duration of complete analgesia than did uraemic animals. Greater ataxia was recorded in healthy animals than in uraemic animals. The heart rate showed a significant decrease in both groups; however, respiratory rate and rectal temperature did not show any significant changes. Haemoglobin, packed cell volume and total leukocyte count decreased non-significantly in both groups. Total leukocyte count was significantly higher in uraemic animals. A significantly higher value of urea nitrogen and creatinine was recorded in uraemic animals. The blood electrolytes (Na⁺, K⁺ and Cl⁻) and blood gases (P o ₂ and P co ₂) did not show any significant changes in both groups; however, base excess was significantly higher in uraemic animals. The effects produced by the combination on different systems were transient and values normal as the effect of the drugs wore off. The results suggest that the combination when used epidurally in uraemic goats produced effective and safe surgical analgesia.