Profiling of virulence associated genes of Pasteurella multocida isolated from cattle

Pasteurella multocida is a causative agent of many major diseases of which haemorrhagic septiciemia (HS) in cattle & a buffalo is responsible for significant losses to livestock sector in India and south Asia. The disease outcome is affected by various host- and pathogen-specific determinants. Several bacterial species-specific putative virulence factors including the capsular and virulence associated genes have been proposed to play a key role in this interaction. A total of 23 isolates of P. multocida were obtained from 335 cases of various clinically healthy and diseased cattle. These isolates were examined for capsule synthesis genes (capA, B, D, E and F) and eleven virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) by PCR. A total of 19 P. multocida isolates belonging to capsular type B and 4 of capsular type A were isolated. All isolates of capsular type B harboured the virulence associated genes: tbpA, pfhA, hgbA, sodC and nanH, coding for transferrin binding protein, filamentous hemagglutinin, haemoglobin binding protein, superoxide dismutase and neuraminidases, respectively; while isolates belonging to capsular type A also carried tbpA, pfhA, hgbA and nanH genes. Only 50 % of capsular type A isolates contained sodC gene while 100 % of capsular type B isolates had sodC gene. The gene nanB and toxA were absent in all the 23 isolates. In capsular type A isolates, either sodA or sodC gene was present & these genes did not occur concurrently. The presence of virulence associated gene ptfA revealed a positive association with the disease outcome in cattle and could therefore be an important epidemiological marker gene for characterizing P. multocida isolates.     

In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA

Pasteurella multocida is a pathogenic, Gram-negative bacterium that is commonly found as normal flora in nasopharynx of variety of wild and domestic animals. Numerous virulence factors have been described for P. multocida isolates which include adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide (LPS), capsule and a variety of outer membrane proteins (Omp). OmpA has a significant role in stabilizing the cell envelope structure by providing physical linkage between the outer membrane & peptidoglycan. It has been shown to mediate P. multocida -host cells interaction via heparin and/or fibronectin binding and therefore act as an important invasive molecule which could determine the final outcome of initial infection. Comparative nucleotide sequence analysis of ompA gene of P. multocida has revealed that despite extensive genetic diversity in ompA of P. multocida, most sequences could be classified into two major allele classes namely ompA allele (I) and allele (II). The P. multocida recovered from nasal cavity of bovine and belonging to two ompA classes were tested for their differential virulence. In vitro pathogenicity studies on Madin Darby Bovine Kidney (MDBK) cell line employing adhesion and invasion assays indicated that P. multocida strain with ompA (I) is more invasive than P. multocida strain with ompA (II). In vivo studies in mice further reiterated that the isolates harbouring ompA(I) were comparatively more virulent to isolates harbouring ompA (II).     

Induction of <Emphasis Type="Italic">Fusarium</Emphasis> wilt (<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis>) resistance in garden pea using induced mutagenesis and in vitro selection techniques

Wilt caused by Fusarium oxysporum f. sp. pisi is a serious production constraint for peas worldwide. An attempt was made to isolate wilt-resistant mutants in two susceptible pea genotypes, Arkel and Azad P-1, employing induced mutagenesis and in vitro selection techniques. Two thousand seeds of each genotype were mutagenized either with ethyl methane sulfonate (EMS, 0.2% and 0.3%) or gamma rays (5-22.5 kR) in ⁶⁰Co gamma cell for three consecutive years. Screening of different mutagenized populations under wilt-sick plots resulted in the isolation of 25 mutants exhibiting complete or enhanced wilt resistance compared to parental genotypes. Five of these wilt-resistant mutants also outperformed the susceptible background genotypes in terms of yield and other horticultural traits. Efforts were also made to isolate wilt-resistant regenerants from callus cultures exhibiting insensitivity to culture filtrate (CF) of F. oxysporum f. sp. pisi. A total of 250 regenerants (R ₀) were obtained from CF-insensitive calli selected from medium supplemented with 20% culture filtrate. When evaluated in artificially inoculated sick plots, only five R ₂ lines obtained from the regenerants exhibited enhanced wilt resistance compared to parental cultivars. However, the selected lines did not exhibit resistance levels equivalent to those shown by wilt-resistant lines isolated through in vivo mutagenesis. To conclude, induced mutagenesis through irradiation and EMS treatments exhibited superiority over in vitro selection for inducing wilt resistance in peas.     

Genetic diversity in Acorus calamus L. as revealed by RAPD markers and its relationship with β-asarone content and ploidy level

β-Asarone content in Acorus calamus is a paramount issue because it limits the usage of plant for medicinal purpose. In the present study A. calamus L. accessions based on RAPD marker, ploidy level and β-asarone content were characterized and correlated on the basis of β-asarone content/ploidy level. Of the 40 random primers used, 6 primers generated polymorphism. Genetic relatedness among accessions evaluated by a similarity matrix based on Dice's coefficient ranged from 0.72 to 0.97. A phenetic dendrogram based on UPGMA analysis grouped accessions into two clusters. A. calamus L. accessions were found to be triploid and tetraploid and their β-asarone content was found in two ranges 6.92–8.0% and 73–88%. The study clustered the accessions as per their ploidy level, β-asarone content and geographical locations. This study would have extensive application in quality control of raw materials.     

Synthetic pyrethroid resistance in Rhipicephalus (Boophilus) microplus ticks from north-western Himalayas,India

In the present study, adult immersion test (AIT) was used for evaluation of resistance against synthetic pyrethroids (deltamethrin and cypermethrin) in Rhipicephalus (Boophilus) microplus ticks collected from nine districts of three agro-climatic zones of north-western Himalayan region of India. Resistance factors (RFs) were calculated between 0.94 to 50.71 for deltamethrin and 0.32 to 13.18 for cypermethrin. Resistance to deltamethrin was detected at level I in two, level II in four, level III and level IV in one isolate each while one isolate was susceptible. Against cypermethrin, resistance at levels I and II was detected in three isolates each while three isolates were found susceptible. The low altitude sub-tropical zone revealed higher density of resistant ticks where intensive animal husbandry practices were followed and the synthetic pyrethroid usage was common. Data generated on pyrethroid resistance status of ticks in north-western Himalayan region will provide new insights in acaricidal resistance particularly from remote areas of this region and will help in formulating suitable control measures.

     

Effect of trikatu pretreatment on the pharmacokinetics of pefloxacin administered orally in mountain Gaddi goats

The pharmacokinetics of orally administered pefloxacin were studied to evaluate the bio-enhancing effect of the herbal bio-enhancer, trikatu, in mountain Gaddi goats (n = 6). The findings of the study revealed a decreased plasma concentration (p > 0.05) of pefloxacin following trikatu administration during the absorption phase (10, 15, 20 min post pefloxacin administration). In contrast, the plasma concentrations of pefloxacin were significantly higher at 4, 6, 8 and 12 h (during the elimination phase) of the pefloxacin administration. The findings of the investigation revealed higher values for the area under the curve, the area under the first moment of the plasma drug concentration time curve, the mean residential time, the total duration of pharmacological action and bioavailability. Trikatu treatment, however, significantly reduced the elimination half life (t_1/2β) and zero time intercept of the elimination phase. The apparent volume of distribution based on the total area under the plasma drug concentration curve [(Vd_(area)] and the apparent volume of distribution based on the zero time plasma concentration intercept of the elimination phase [Vd_(B)] were significantly higher in trikatu treated animals indicating a better penetration of the drug. Based on the MIC of 0.8 µg/ml of pefloxacin, a priming dose of 6.0 mg/kg and a maintenance dose of 2.21 mg/kg is required to be administered at 8 h intervals. For practical purposes in goats this would mean a priming dose of 6 mg/kg and a maintenance dose of 2 mg/kg given by the oral route, to be repeated at 8 h intervals.     

N-Methyl-6, 7-dimethoxyisoquinolone in Annona squamosa twigs is the major immune modifier to elicit polarized Th1 immune response in BALB/c mice

Annona squamosa (AS) has traditionally been used as ethnomedicine. We have earlier extracted and fractionated the twigs of AS based upon its bioactivity and observed its immune potentiating activity that was localized in its three fractions. Present communication deals with the phytochemical analysis and pharmacological investigation of the most active chloroform fraction that led to isolation and identification of a number of compounds whose structures were elucidated using 1D and 2D NMR spectroscopic analysis. Amongst the twelve pure compounds isolated, five compounds Lanuginosine (1), (+) -O- methylarmepavine (2), (+)-anomuricine (3), Isocorydine (4), and N-methyl-6, 7-dimethoxyisoquinolone (5) were evaluated in vivo for their immune modifier activities in BALB/c mice after oral administration at three log doses of 0.3, 1.0 and 3.0 mg/kg for 14 consecutive days. Of these, three compounds (1, 2 and 5) showed dose dependent immune stimulating activity. However, the uppermost activity was noted in the compound N-methyl-6, 7-dimethoxyisoquinolone at the 3.0 mg/kg oral dose. The activity was assessed in the form of increased splenic T and B cellular proliferation, up-regulated CD4+, CD8+ and CD19+ cell population and accentuation in the peritoneal macrophage function. The compound possibly acted modifying the expression of Th1- and Th2- cytokines via stimulation of pro-inflammatory Th1 cytokines IL-2 and IFN-γ. These results warrant the use of the above compounds as an efficient immune-stimulant or immune-adjuvant against diseases with immune suppression. The analogs of the compound may further be chemically synthesized to achieve desired immune modifying activity.