Sleep deprivation induces changes in 5-HT actions and 5-HT1A receptor expression in the rat hippocampus.

A 12h sleep deprivation enhances the expression of 5-hydroxytryptamine (5-HT) 1A receptors in rat hippocampus that recedes with 48h sleep recovery. The depressant effect of applied 5-HT on the field excitatory postsynaptic potentials recorded in the CA1 area, is also enhanced in hippocampi of SD rats. Following a 24 or 48h sleep recovery, the increase in the 5-HT effect subsided. These results have implications for therapeutics treating clinical depression.

Genome sequence of classical Swine Fever virus genotype 1.1 with a genetic marker of attenuation detected in a continuous porcine cell line.

The complete genome sequencing and analysis of a classical swine fever virus (CSFV) detected in a porcine kidney cell line revealed a close relationship with genotype 1.1 viruses circulating in India and China. The presence of consecutive T insertions in the 3' untranslated region (UTR), as seen in vaccine strains of CSFV, suggested some degree of attenuation.

Genetic characterisation and phylogenetic analysis of PCV2 isolates from India: indications for emergence of natural inter-genotypic recombinants.

Porcine circovirus type 2 (PCV2), the necessary agent in pathogenesis of porcine circovirus diseases (PCVDs), has a worldwide distribution and is considered as one of the most important emerging viral pathogens of economic importance. PCV2 has been divided into four major genotypes namely PCV2a with five clusters or subtypes (2A-2E), PCV2b with three clusters (1A-1C), PCV2c and PCV2d, based on capsid (cap) gene analysis. PCV2 genome is rapidly evolving through events of recombination and mutation. Though, PCV2a was the predominant genotype initially, PCV2b shared majority of PCV2 sequences submitted to GenBank since 2003. In India, data regarding molecular characterisation of PCV2 is scant or absent. In the present study, we thoroughly analysed genetic heterogeneity of PCV2 strains circulating in Indian pig population. The results revealed that pigs in this region harboured PCV2 viruses of different genotypes including PCV2a-2D, PCV2b-1C and PCV2d. More interestingly, two isolates (PCV2Izn-89-13 and PCV2Izn-218-13) were classified as recombinant strains. Further detailed analysis suggested that these strains evolved from inter-genotypic recombination between PCV2a-2C and PCV2b-1C genotypes within cap gene. This study reports for the first time, the emergence of recombinant PCV2 strains in the Indian pig population.

Detection of bovine papilloma viruses in wart-like lesions of upper gastrointestinal tract of cattle and buffaloes.

In present investigation, etiopathological characterization of upper gastrointestinal tract (GIT) tumours of cattle and buffaloes was undertaken. A total of 27 GIT wart-like lesions in rumen, reticulum, mouth and oesophagus of cattle and buffaloes revealed the presence of small nodular to larger spherical or slender growths with thin base present on mucosa and ruminal pillar. Histopathologically, these cases were diagnosed as fibropapilloma/papilloma. This is the first world record on ruminal papillomatosis in buffaloes. Ruminal warts of cattle and buffaloes revealed the presence of BPV-5, -1 & -2, which is the first report of presence of these BPVs in the ruminal warts from India. Quantitative real-time PCR revealed that DNA samples of different GIT wart-like lesions contained varying amount of BPV DNA copy numbers. Immunohistochemistry revealed that the PCNA and Ki67 immunopositivity was present in the basal and spinosum layer of the fibropapilloma/papilloma, indicating these as the cellular proliferation site. In conclusion, the present investigation revealed that BPV-5, -1 & -2 are associated with certain ruminal wart-like lesions/growths in cattle and buffaloes, and the basal and spinosum layer of the ruminal fibropapilloma/papilloma were cellular proliferation sites.

Glutamate dysregulation in the trigeminal ganglion: a novel mechanism for peripheral sensitization of the craniofacial region.

In the trigeminal ganglion (TG), satellite glial cells (SGCs) form a functional unit with neurons. It has been proposed that SGCs participate in regulating extracellular glutamate levels and that dysfunction of this SGC capacity can impact nociceptive transmission in craniofacial pain conditions. This study investigated whether SGCs release glutamate and whether elevation of TG glutamate concentration alters response properties of trigeminal afferent fibers. Immunohistochemistry was used to assess glutamate content and the expression of excitatory amino acid transporter (EAAT)1 and EAAT2 in TG sections. SGCs contained glutamate and expressed EAAT1 and EAAT2. Potassium chloride (10 mM) was used to evoke glutamate release from cultured rat SGCs treated with the EAAT1/2 inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)ben zoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA) or control. Treatment with TFB-TBOA (1 and 10 µM) significantly reduced the glutamate concentration from 10.6 ± 1.1 to 5.8 ± 1.4 µM and 3.0 ± 0.8 µM, respectively (p<0.05). Electrophysiology experiments were conducted in anaesthetized rats to determine the effect of intraganglionic injections of glutamate on the response properties of ganglion neurons that innervated either the temporalis or masseter muscle. Intraganglionic injection of glutamate (500 mM, 3 µl) evoked afferent discharge and significantly reduced muscle afferent mechanical threshold. Glutamate-evoked discharge was attenuated bythe N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovalerate (APV) and increased by TFB-TBOA, whereas mechanical sensitization was only sensitive to APV. Antidromic invasion of muscle afferent fibers by electrical stimulation of the caudal brainstem (10 Hz) or local anesthesia of the brainstem with lidocaine did not alter glutamate-induced mechanical sensitization. These findings provide a novel mechanism whereby dysfunctional trigeminal SGCs could contribute to cranial muscle tenderness in craniofacial pain conditions such as migraine headache.

Spontaneous cutaneous papillomatosis in yaks and detection and quantification of bovine papillomavirus-1 and -2.

Seven clinical cases of cutaneous papillomatosis in yaks were studied in Arunachal Pradesh, India. Sporadic, single or a chain of multiple varying size warts appeared around the eyes or on the body. Predominant site of warts was around eyes. Histopathologically, these cases were diagnosed as fibropapilloma. It was confirmed by the detection of BPV-1 and BPV-2 or their mixed infection by PCR and sequencing. Quantitative SYBR Green real-time PCR detected comparatively lower viral DNA copy number in cutaneous warts (CWs). Cases of CWs and its causative agent as bovine papillomavirus (BPVs) are reported for the first time in yaks.

Detection and quantification of bovine papilloma virus type 2 (BPV-2) by real-time PCR in urine and urinary bladder lesions in enzootic bovine haematuria (EBH)-affected cows.

This study was conducted with the objectives of detecting bovine papillomavirus type 2 (BPV-2) in urine samples and urinary bladder lesions in bovines using polymerase chain reaction (PCR) and real-time PCR-based molecular diagnostic tests, and quantifying BPV-2 in urinary bladder lesions especially in enzootic bovine haematuria (EBH)-affected animals. BPV-2 viral DNA was detected in urine samples (50%) and urinary bladder tissue (68.6%). Cloning and sequencing results showed a close homology with other Indian BPV-2 sequences. Quantitative real-time PCR (SYBR Green assay) showed that the BPV-2 load was low and similar irrespective of inflammatory or neoplastic lesions in the bladder. It was concluded that BPV-2 DNA is frequently present in urine and urinary bladder lesions in cows in an EBH endemic region and virus load was low in urinary bladder lesions.

Preliminary assessment of binary ethylenimine inactivated and saponized cutaneous warts (BPV-2) therapeutic vaccine for enzootic bovine haematuria in hill cows.

A preliminary therapeutic vaccine trial was conducted in hill cows to evaluate the therapeutic potential of binary ethylenimine (BEI) inactivated and saponized bovine papillomavirus-2 (BPV-2) for enzootic bovine haematuria (EBH). Although the vaccine failed to show favorable clinical vaccine results in treatment of EBH affected cows at 120 days post-vaccination but immunopathological responses were encouraging. A significant difference was observed in humoral (against Brucella abortus strain 19S) and cell-mediated (in vivo phytohaemagglutination delayed type hypersensitivity (PHA DTH) test and CD4+/CD8+ T-cells ratio by FACS analysis) immune responses following vaccination. The vaccinated animals grossly failed to show regression of bladder tumours but microscopically engorgement and marked perivascular infiltration of mononuclear cells was observed which are indicative of the induction of initial stages of tumour regression. Overall results indicated that the therapeutic vaccine developed can have potentials for treating EBH in cows, for which further modifications in vaccine dose and field trial is required.

Papillomatosis in buffaloes: a less-known disease.

Scant information is available on papillomatosis in buffaloes, and it is an almost unknown disease. It has been described from India, Italy and Turkey. Buffalo papillomatosis occurs in cutaneous and mucosal forms. Cutaneous papillomatosis is manifested as cutaneous wart (CW) and teat papilloma types. The condition is known to be caused by bovine papillomaviruses (BPV)-1 and 2 and their mixed infections. Buffalo CWs are experimentally transmissible to hamsters, cattle as well as buffaloes. Once BPV establishes infection in buffaloes, infection spreads from buffalo to buffalo, without cattle intermediary. Histologically, CWs are mostly diagnosed as fibropapillomas. The mucosal form occurs as urinary bladder tumours similar to enzootic bovine haematuria which is also associated with bracken fern infested areas. BPVs are yet to be demonstrated in teat papillomas and urinary bladder tumours of buffalo cases. Papillomatosis in buffaloes is a little-known disease, but it is a separate infectious ailment of buffaloes and deserves more attention by researchers.

Haematology, blood biochemistry and tissue histopathology of lambs maintained on diets containing an insect controlling protein (Cry1Ac) in Bt-cottonseed.

This experiment assessed the effect of feeding genetically modified cottonseed (Bt) containing an insect controlling protein (Cry1Ac) on haematology, blood biochemistry and histopathology of lambs. Haemato-biochemicals were estimated at periodic intervals, and histopathology at termination of experiment. Thirty three weaner lambs were fed a composite feed mixture (CFM) ad libitum individually, in three groups for 123 days. The isonitrogenous CFM had roughage (Perl Millet Stover) and concentrate ratio of 350:650. Diet fed to control lambs contained groundnut oil meal as protein source while other two groups received diet containing either whole cottonseed (N-Bt) or Bt-cottonseed (Bt-CS). Daily feed intake and average daily gain were similar among lambs of three groups. Lambs fed N-Bt diet had higher (p < 0.05) serum protein and globulin compared to control and Bt diets, while albumin content was higher (p = 0.018) in Bt diet fed lambs. Serum urea and creatinin content, alkaline phosphatase (ALP) and serum glutamate pyruvic transaminase (SGPT) activities were not different among lamb groups, while urea and creatinin content and ALP activities increased linearly (p < 0.001) with increased feeding period. Blood haemoglobin (Hb), haematocrit (Hc), white blood cells (WBC), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) ranged from 11.1% to 11.2%, 31.8% to 32.8%, 7.0 to 8.3 (× 10(3) /µl), 19.1 to 22.5 fl and 33.2% to 35.5%, respectively, were similar among lamb groups. Erythrocyte sedimentation rate (ESR) (p = 0.008) and red blood cell counts (p = 0.006) were higher in Bt diet fed lambs. Control and N-Bt diet fed lambs had mild fatty infiltration in liver and/or micro-calculi in renal cortex, and such lesions were not seen in Bt diet fed lambs. Growth, haemato-biochemical and histopathology did not change by Bt-CS feeding in growing lamb. However, before recommending the use of Bt-CS in routine feed formulations prolonged feeding experiments of Bt-cotton seed require.

Influenza A H1N1 virus in Indian pigs & its genetic relatedness with pandemic human influenza A 2009 H1N1.

BACKGROUND & OBJECTIVES: With the emergence of a new reassortant influenza A H1N1 virus that caused the 2009 pandemic it was felt necessary that pigs should be closely monitored for early detection of any influenza virus infection. Therefore, we investigated disease outbreaks with clinical history suggestive for swine influenza reported to our laboratory by owners of affected pig farms in Uttar Pradesh. METHODS: Detection of swine influenza A virus (SIV) was attempted by isolation in embryonated chicken eggs. Presence of virus was detected by haemagglutination (HA) test and RT-PCR for amplification of different gene segments, cloning and sequencing. BLAST analysis of sequence data, phylogenetic analysis and mutation analysis based on HA, NA and matrix genes was done. RESULTS: SIV could be isolated from one farm and all eight gene segments amplified by RT-PCR. BLAST analysis of partial nucleotide sequences and phylogenetic analysis using nucleotide sequence of HA (601 nt), NA (671 nt) and M (1031 nt) genes indicated close genetic relationship of the Indian swine isolate (A/Sw/UP-India-IVRI01/2009) with human pandemic 2009 (H1N1). The HA gene showed close relationship with the viruses of "North American Swine" lineage, whereas the NA and M genes clustered with the viruses of "Eurasian Swine" lineage, indicating a novel HA-NA reassortant. The remaining of 5 genes (NP, PA, PB1, PB2 and NS) belonged to "North American Swine" lineage. INTERPRETATION & CONCLUSIONS: This is perhaps the first report describing swine influenza among Indian pigs caused by an influenza A H1N1 virus sharing close homology with the human pandemic (H1N1) 2009 virus. Further reassortment with circulating influenza viruses must be closely monitored.

Detection of BPV-1 and -2 and quantification of BPV-1 by real-time PCR in cutaneous warts in cattle and buffaloes.

Bovine cutaneous warts (CWs) were investigated in Northern India. Of 49 cases, 44 were recorded in cattle and 5 in buffaloes. These animals had mild to moderate grade infections. Grossly, cases of CWs appeared to be of exophytic type, however, different types of growth patterns were observed. A total of 26 biopsies (cattle 21 and buffaloes 5) from CWs-affected animals studied histopathologically were diagnosed as exophytic and cauliflower-like fibropapilloma 13, exophytic and dome-shaped fibropapilloma 5, occult and/or fibroblastic type papilloma 3, cauliflower-like papilloma 3, endophytic fibropapilloma 1 and fibroma 1. On PCR analysis, 11 CWs and 2 normal skin samples showed BPV-1, -2 mixed infections. A rapid, sensitive and reliable real-time SYBR Green PCR test to detect BPV-1, BPV-2 and to quantify BPV-1 was developed. Results of amplification and dissociation plot of real-time PCR revealed that six samples were BPV-1 positive, eight were BPV-2 positive and six were positive for both BPV-1 and -2. CWs samples from different dairy farms testing positive for BPV-1 by PCR assay were also positive using Quantitative real-time SYBR Green PCR assay. For the first time, mixed infection of BPV-1 and -2 was detected in India and BPV-1 load was quantified by real-time SYBR Green PCR assay.

Pathological studies on Bovine papilloma virus-fern interaction in hamsters.

Early pathological changes of Bovine papilloma virus (BPV-2)-fern (Pteridium aquilinum and Onychium contiguum fern) interaction in hamsters were studied. In bracken-exposed cattle, BPV induces malignancy in gastrointestinal and urinary bladder mucosa. Cutaneous warts were transmitted successfully in hamsters approximately after 3 months post inoculation while urinary bladder tumour of enzootic bovine haematuria cases were not transmitted. Histologically, tumour was diagnosed as fibroma. Onychium produced more pronounced effects than bracken fern which was characterized by significant reduction in body weight and testicular atrophy. BPV-fern interaction was not appreciable during early period of tumour induction and requires long-term studies for 12 to 18 months.

Specific binding of non-steroidal anti-inflammatory drugs (NSAIDs) to phospholipase A2: structure of the complex formed between phospholipase A2 and diclofenac at 2.7 A resolution.

Type IIA secretory phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids to release fatty acids and lysophospholipids. In order to elucidate the role of PLA2 in inflammatory disorders and to determine the mode of binding of non-steroidal anti-inflammatory drugs (NSAIDs) to PLA2, the detailed three-dimensional structure of a complex formed between a group IIA PLA2 from Daboia russelli pulchella and 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid (diclofenac) has been determined. The preformed complex was crystallized by equilibrating the protein solution against a mixture of 0.20 M ammonium sulfate and 30% PEG 4000. The crystals belong to space group P4(3), with unit-cell parameters a = b = 53.0, c = 48.4 A. The structure was solved by the molecular-replacement method and refined to R(cryst) and R(free) factors of 0.192 and 0.211, respectively, using reflections to 2.7 A resolution. The structure showed that diclofenac occupies a very favourable position in the centre of the substrate-binding hydrophobic channel that allows a number of intermolecular interactions. The binding mode of diclofenac involved crucial interactions with important residues for substrate recognition such as Asp49, His48 and Gly30. In addition, it included three new interactions involving its Cl atoms with Phe5, Ala18 and Tyr22. It also showed an extensive network of hydrophobic interactions involving almost all of the residues of the substrate-binding hydrophobic channel. The binding affinity of diclofenac was determined using surface plasmon resonance, which gave an equilibrium constant of 4.8 +/- 0.2 x 10(-8) M.

Proliferative urocystica and adenoma in a guinea-pig.

Proliferative urocystica and adenoma occurred in the urothelium of one clinically normal guinea-pig in a group of nine fed with a diet containing shade-dried Christella dentata (Forssk) fern. It was concluded that the lesions were due to prolonged feeding (285 days) of the fern.

Effects of branched beta-carbon dehydro-residues on peptide conformations: syntheses, crystal structures and molecular conformations of two tetrapeptides: (a) N-(benzyloxycarbonyl)-DeltaVal-Leu-DeltaPhe-Leu-OCH3 and (b) N-(benzyloxycarbonyl)-DeltaIle-Ala-DeltaPhe-Ala-OCH3.

The roles of branched beta-carbon dehydro-residues in the design of peptide conformations have not been systematically explored so far. In order to determine the effects of branched beta-carbon dehydro-residues on the peptide conformations, two N-protected tetrapeptides containing new combinations of DeltaVal and DeltaPhe in (a) N-(benzyloxycarbonyl)-DeltaVal-Leu-DeltaPhe-Leu-OCH(3) and DeltaIle and DeltaPhe in (b) N-(benzyloxycarbonyl)-DeltaIle-Ala-DeltaPhe-Ala-OCH(3) were synthesized by solution procedure. The crystal structures of these peptides were determined by X-ray diffraction methods. Single crystals of both peptides were grown by slow evaporation method from their solutions in acetone-water mixtures (80 : 20) at 25 degrees C. The crystals of these peptides belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 12.342(1) A, b = 15.659(1) A, c = 18.970(1) A for peptide (a) and a = 8.093(1) A, b = 15.791(1) A, c = 23.816(1) A for peptide (b) having Z = 4 in the unit cells of both peptides. The structures were refined by full-matrix least-squares procedure to R-factors of 0.076 and 0.052 respectively. Both peptides adopt the right-handed 3(10)-helical conformations stabilized by two intramolecular (i + 3-->i) hydrogen bonds between the CO of N-terminal benzyloxycarbonyl (Cbz) group and the NH of residue at position 3, and between the CO of residue at position 1 and NH of the residue at position 4. The two consecutive 10-membered rings formed by the hydrogen bonds have dihedral angles corresponding to the standard values for type III beta-turns. DeltaVal and DeltaIle in peptides (a) and (b) respectively are located at the (i + 1) position of the first beta-turn while DeltaPhe is located at the (i + 2) position of the second beta-turn. In the crystals, the molecules are linked head to tail by intermolecular hydrogen bonds to form long helical chains. The axes of helices are parallel to the b-axes while the neighbouring helices run in the opposite directions. The crystal packings are further stabilized by van der Waals forces between the columns of molecular packings.

Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India.

In the present study characterisation has been done for six group I fowl adenoviruses (FAV) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years 1994-98. These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values 0.93-0.96). Under the experimental conditions, we have been able to induce IHP using FAV serotype 4 isolate AD: 411 and were also able detect FAV antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FAV serotype 4 strain replicate in myocardial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FAV serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FAV serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, any one of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine.

Ultrasonographic evaluation of urinary bladder in normal, fern fed and enzootic bovine haematuria-affected cattle.

A total of 19 adult hill cattle of both sexes were subjected to trans-rectal ultrasound scanning of urinary bladder to evaluate bladder wall thickness and the presence of space-occupying lesions. The animals were divided into four groups. Eight apparently healthy hill cattle maintained under standard ration served as control (group I) and the remaining II animals were divided into three groups (II, III and IV). Group II animals (n = 8) were fed with different type of ferns which were further divided into subgroups II-P, -D and -B and fed with Polystichum squarrosom (n = 2). Dryopteris juxtaposita (n = 2) and Pteridium aquilinum (n = 4) ferns, respectively. The one animal in group III was a natural case of enzootic bovine haematuria (EBH) and the two animals in group IV were natural cases of microscopic EBH fed with Polystichum squarrosum fern. In group I animals, the average bladder wall thickness was 1.45 mm. The delineation of the bladder wall was uniformly smooth and the echo pattern of the bladder was homogeneously black, which was suggestive of clear urine content. In group II (P, D and B) the average bladder wall thickness of the six animals was 1.87 mm and the sonographic features were within normal limit when compared with controls. In two of the animals of group II-B, the bladder wall was apparently thick (4.36 mm) and there was no intraluminal mass except at one or two focal elevated points. Animals of groups III and IV showed the average bladder wall thickness of 4.86 mm and were characterized by the presence of irregular sessile masses extending into the bladder lumen. The homogeneous anechoic area was reduced centrally due to the presence of a hypoechoic soft tissue mass all around the bladder wall. Post-sonographic urinalysis, biopsy and necropsy of selected cases further confirmed the sonographic findings.

A preliminary study on the carcinogenicity of the common fern Onychium contiguum.

Onychium contiguum (Family Cryptogrammaceae) is a common terrestrial fern in the Himalayas and in many other parts of the world. It is also present on the pastures in areas where grazing animals suffer from bovine urinary bladder cancer. This fern is occasionally grazed by animals and in some areas it is present as a contaminant in grasses stored for winter feeding. Certain species of the genus Onychium are used in folk medicine. Long-term exposure of experimental animals to O. contiguum appeared to cause tumours of the ileum. urinary bladder and mammary glands.

Pathological evaluation of Polystichum squarrosum (D. Don) fern in laboratory rats.

Polystichum squarrosum fern fed (30% w/w) rats showed moderate mortality, decrease in body weight, less body fat and splenomegaly. On post-mortem examination, significant gross lesions were not seen in sacrificed animals. Histopathologically, Polystichum fed rats showed dilated Virchow Robin's space in brain, mild to moderate vascular changes likeoedema, engorgement of blood vessels and haemorrhages in most of the visceral organs, interstitial pneumonia in lungs, focal necrosis and generalised vacuolative degenerative changes in liver, more haemosiderin deposition and presence of higher number of megakaryocytes in spleen, shrunken glomeruli, more peri-glomerular space and more number of glomeruli per microscopic field in kidneys, focal hyperplasia of urinary bladder and moderate to marked depletion of germinal epithelium and spermatids in seminiferous tubules of testes. Pathologically, progressive changes were observed only in liver, urinary bladder and testes on 180 days post feeding (DPF). One fern fed rat sacrificed on 135 DPF showed hepatic tumour which was diagnosed as hepatocellular carcinoma. The results showed that P. squarrosum produced almost comparable pathological changes/preneoplastic lesions as reported in bracken fern fed animals. Long term exposure studies (i.e. 2 yrs) are desired.