Heterologous Expression of a Gene Encoding a 35 kDa Protein of Mycobacterium avium paratuberculosis in Escherichia coli

The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.     

Avian Reovirus Induces an Inhibitory Effect on Lymphoproliferation in Chickens

The cellular immune responses of chickens inoculated with the vaccine strain S-1133 and/or a field isolate VA-1 of avian reovirus (ARV) were studied. Both strains of virus caused inhibition of the phytohaemagglutinin (PHA)-induced lymphoproliferative response of peripheral blood mononuclear cells (PBMC) and splenic mononuclear cells (SMC) during the initial stage from day 4 up to day 10 post-inoculation (PI), with a later return to the normal value. The inhibition in the PHA-induced lymphoproliferation of SMC could be partially overcome by depletion of adherent cells. The supernatant of the PHA-stimulated SMC culture was also checked in vitro for the presence of suppressive factor(s) produced in response to ARV infection. The culture supernatant from chickens at day 5 PI caused significant inhibition of the PHA-induced lymphoproliferation of control birds, suggesting the presence of suppressive factor(s). ARV infection also significantly inhibited IL-2 production on day 5. There was a significant increase in nitric oxide production by the splenic mononuclear cells of chickens inoculated with either strain of ARV.     

Mycobacterium bovis AN5 antigens vary in their ability to induce nitric oxide production in blood monocytes of experimentally infected cattle

Reactive nitrogen intermediates (RNI) are the principal effector molecules of activated monocyte/macrophage populations, responsible for killing and inhibiting the growth of virulent mycobacteria. In vitro nitrite production by blood monocytes of cattle inoculated with live Mycobacterium bovis AN5 was assessed from 0 day through 45 weeks post inoculation (PI). High in vitro nitrite production was observed at the 8th and 12th weeks PI in sensitized cattle but reactivity had fallen by the 20th week PI. To assess the in vitro nitrite producing ability of monocytes induced by individual polypeptides within culture filtrate antigens (CFA) of M. bovis AN5, cellular blotting was performed using peripheral blood mononuclear cells (PBMC) at the 12th week PI. It was observed that polypeptides of MW 70, 65, 60, 25, 24 and 22 kDa of CFA induced high nitrite production by blood monocytes while many polypeptides had little or no effect.     

Changes in Mithun (Bos frontalis) spermatozoa during epididymal passage

Genital organs of 10 healthy, adult Mithun bulls (6-8 years old) that were slaughtered at the dwellings of tribal people for meat were collected. Immediately after collection, spermatozoa from 3 different regions of the epididymis, i.e. the head, body and tail, were obtained to study morphological changes of the spermatozoa during passage through these regions. The prevalence of proximal cytoplasmic droplets significantly decreased from the head to the tail of the epididymis. Conversely, the percentage of distal cytoplasmic droplets increased significantly from the head to the tail region. The incidence of tailless heads rose significantly from head to body and then reduced significantly in the tail region. The percentage of total head abnormalities did, however, not change markedly, but total mid-piece and tail abnormalities differed significantly between the three epididymal regions.     

Sensitivity of Ascochyta rabiei populations to prothioconazole and thiabendazole

Ascochyta rabiei causes Ascochyta blight, a yield-limiting disease of chickpea (Cicer arietinum) world-wide. In 2007, fungal populations of A. rabiei resistant to the QoI group of fungicides were detected in the Northern Great Plains of the United States. Assays were conducted to determine fungal sensitivity for two alternative fungicidal modes of action. A total of 78 isolates of A. rabiei collected between 1983 and 2007 were screened to determine baseline sensitivity to the demethylation-inhibiting foliar fungicide, prothioconazole, and 100 isolates collected between 1987 and 2007 were screened for sensitivity to the methyl benzimidazole carbamate (MBC) fungicide, thiabendazole. Isolates were tested using an in vitro mycelial growth assay to determine the effective fungicide concentration at which 50% of fungal growth was inhibited (EC₅₀) for each isolate-fungicide combination. Baseline EC₅₀ values of prothioconazole ranged from 0.0526 to 0.2958 μg/ml, with a mean of 0.1783 μg/ml. Isolates of A. rabiei collected from 2007 to 2009 from North Dakota chickpea fields exposed to prothioconazole, were screened for prothioconazole sensitivity using the same assay. Mean EC₅₀ values for these isolates were 0.3544 μg/ml, 0.3746 μg/ml, and 0.7820 μg/ml, respectively. These values represent an approximate 2.0 (2007-2008) and 4.4-fold (2009) decrease in sensitivity from the baseline mean. EC₅₀ values of thiabendazole ranged from 1.192 to 3.819 μg/ml, with a mean of 2.459 μg/ml. No significant decrease in fungicide sensitivity was observed for thiabendazole. To date, no loss of Ascochyta blight control has been observed with the use of either prothioconazole or thiabendazole.     

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm ) along with a control (0 mm ) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC‐I) (12–72 hpi) and IVC‐II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC‐I and IVC‐II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.     

Acellular dermal graft for repair of abdominal wall defects in rabbits

Sixteen clinically healthy New Zealand white rabbits of either sex were divided into 2 equal groups (I and II) of 8 animals each. Under thiopental sodium (2.5%) anaesthesia a 2 x 3 cm full-thickness abdominal wall defect in the mid-ventral abdominal wall was created and repaired with an acellular dermal graft (ADG) in all the animals of group I (test group). In animals of group II (control group) a full-thickness linear midline abdominal muscular wall incision was made and repaired with a continuous suture pattern using 2-0 nylon.