Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay

A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A. A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies. This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P. multocida serogroup-A. A nested PCR method yielded a single 374 bp product. All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII.     

Prevalent Serotypes of Pasteurella multocida Isolated from Different Animal and Avian Species in India

Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused by P. multocida, a total of 206 bacterial cultures were identified as P. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping of P. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.     

Protection of mice against <Emphasis Type="Italic">Brucella abortus 544</Emphasis> challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides

Brucella abortus, a gram negative, facultative intracellular pathogen causes brucellosis in many animal species and humans. Although live, attenuated vaccines are available against this infection, they suffer from certain limitations. Therefore, the development of an effective subunit vaccine against brucellosis is an area of intense research. The outer membrane proteins (OMPs) of Brucella species have been extensively studied for its immunogenicity and protective ability. We have investigated the potential of CpG ODN to enhance the immunogenicity and protective efficacy of recombinant 28 kDa outer membrane protein (rOMP28) of Brucella melitensis. The study demonstrated vigorous immunoglobulin G (IgG) response of OMP28. The administration of rOMP28 with CpG caused increased cell mediated immune response in terms of induced IgG2a, T-cell proliferation and up-regulation of type I cytokine expression. In contrast, the free antigen suppressed the interferon gamma (type I cytokine) production on in-vitro stimulation of spleenocytes. The result indicates the role of OMP28 in the down regulation of IFN-γ production. Moreover, the B. abortus S-19 vaccinated mice showed highest production of IL-4 and IFN-γ. The protective ability of the antigen was evaluated by systemic bacterial clearance after challenging the mouse with B. abortus 544 pathogen. The level of protection was significant in rOMP28+CpG treated mice but was lower than the required level. The results of the present study indicate that rOMP28 could be an immunogen capable of inducing both humoral and cellular immune response. The humoral response was biased towards Th1 type when it was co-administered with CpG.     

Comprehensive transcriptional profiling and functional study of lymphangiogenic growth factor in placentome of early-stage pregnant water buffalo

The aim of the present study was to evaluate the expression and localization of lymphangiogenic factors (VEGF-C and VEGF-D), their receptor (VEGFR3) and lymphatic endothelial marker (LYVE1) in buffalo placenta during early pregnancy [EP], and to investigate the functional role of lymphangiogenic growth factors in placental lymphangiogenesis. The mRNA and protein expression of VEGF-C, VEGF-D, their receptor VEGFR3 and LYVE1 showed significant expression in EP1 (29–42 days) and EP2 stages (51–82 days) both in caruncle (maternal part) and cotyledon (foetal part) of the buffalo placenta. Immunoreactivity of VEGF-C, VEGF-D and LYVE1 was observed around the endometrial gland, in lymphatics and trophoblast cells, whereas VEGFR3 mainly localized in lymphatics of the caruncle and cotyledons. Cultured trophoblast cells were treated with VEGF-C/VEGF-D (50, 100 and 150 ng/ml) and combined doses of VEGF-C and VEGF-D (150 ng/ml) each for different time durations (24, 48 and 72 h). The mRNA expression of LYVE1 and PCNA was significantly (p < .001) upregulated with VEGF-C and VEGF-D and combined treatment (@150 ng/ml), as well as significantly downregulating Caspase-3 at 48 and 72 h. Thus, the present study provides evidence that lymphangiogenic factors are expressed in buffalo placental compartments and they may play a significant role in the regulation of placental function in water buffaloes.     

Surgical Correction of Uterine Torsion and Mare–Foal Survival in Advance Pregnant Equine Patients

This article describes the surgical management of uterine torsion by midline celiotomy and cesarean section on 12 mares presented with signs of colic to a teaching veterinary hospital. The mares were either in full term of gestation (n = 7) or in advanced stage of pregnancy (n = 5). Six mares were in first parity. Uterine torsion was diagnosed by per rectal and per vaginal examinations. For surgical intervention, mares were anesthetized using a combination of xylazine (1.1 mg/kg) and ketamine (2.2 mg/kg), intravenously. After intubation, the animals were maintained on halothane (n = 4) or isoflurane (n = 8) inhalation anesthesia. Midline celiotomy was performed, and foals were delivered by cesarean section. In 11 mares, before closing the abdominal wound, the uterus was detorted manually and confirmed for its normal position. Both anesthetic protocols using halothane and isoflurane were found satisfactory for surgical correction of uterine torsion. After long-term follow-up, the study reported 75.0% (9/12) survival rate for mares. One mare was euthanized because of devitalized, necrosed, and adhered uterus to the abdominal wall. Of the nine surviving mares, seven were successfully bred. Three foals were born alive, and only one could survive on long-term basis. Of the nine dead foals, two had umbilical cord torsion.     

Effect of a novel microbial phytase on production performance and tibia mineral concentration in broiler chickens given low-calcium diets

1. In a 42-d feeding trial, 264 one-d-old, as hatched, Cobb 400 broiler chickens (6 pens per group, n = 11 per pen in a 2?×?2 factorial arrangement) were fed on two concentrations of dietary calcium (Ca) (9.0 and 7.5 g/kg in starter, 7.5 and 6 g/kg in grower phases) and supplemental phytase (0 and 500 U/kg diet).

2. During d 0–21, the high Ca + phytase diet improved body weight. During d 0–42, feed intake was increased by the low Ca diet and decreased by phytase supplementation. Feed conversion ratio during d 0–21 was improved by the high Ca + phytase diet.

3. At d 42, Ca in duodenal digesta was reduced by low dietary Ca and supplemental phytase. High dietary Ca reduced P in duodenal and jejunal digesta. Phytase reduced digesta P and increased serum P concentration.

4. Relative tibia length decreased with low dietary Ca and increased with phytase. The robusticity index of tibia was improved by the low Ca diet and phytase supplementation. Phytase supplementation increased tibia ash and concentrations of Ca, magnesium (Mg), manganese (Mn), copper (Cu), zinc (Zn) and iron (Fe) in tibia. The low Ca diet increased Mg, Mn and Fe and reduced Cu and Zn in tibia.

5. It was concluded that 7.5 g Ca/kg during weeks 0–3 and 6 g Ca/kg during weeks 3–6 sustained broiler performance and bone ash, while phytase supplementation facilitated tibia mineralisation, particularly during the grower phase.     

Effect of immunization with bovine luteinizing hormone receptor on ovarian function in cats

OBJECTIVE: To determine the effect of immunization with bovine luteinizing hormone receptor (LH-R) on ovarian function of cats. ANIMALS: 9 adult female domestic cats. PROCEDURE: 7 cats were immunized with 0.5 mg of LH-R encapsulated in a silastic subdermal implant (3 x 10 mm); 2 served as control cats. Receptors had 80% specific binding to 125I-human chorionic gonadotropin with a binding capacity of 2,682 pM/mg. Cats received booster injections of LH-R. Cats were induced to ovulate with luteinizing hormone (LH) releasing hormone on day 345. Samples of venous blood and vaginal cells were collected through day 395. Observation of estrus behavior continued until day 516. Serum concentrations of estradiol, progesterone, thyroid gland hormones, LH, and LH-R antibody were determined. RESULTS: LH-R antibody was detected in the sera of immunized cats within 21 days after implantation. Detection of LH-R antibody was associated with suppression of serum progesterone to < or = 0.5 ng/mL during the study period, compared with concentrations of 5 to 10 ng/mL in control cats. Immunized cats did not display signs of estrus. Release of LH after administration of LH-releasing hormone indicated an intact hypothalamic-pituitary axis but poor corpus luteum function. Serum estradiol concentrations remained between 30 to 40 pg/mL in immunized and control cats. With the decrease antibody titers, hormone concentrations returned to a pattern consistent with that during fertility. CONCLUSIONS AND CLINICAL RELEVANCE: Active immunization with LH-R suppressed corpus luteum function in cats. The effect was reversible. An LH-R-based antifertility vaccine may have clinical application in other vertebrates.     

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.