Effect of ivermectin, amitraz and fipronil on midgut epithelium and digestive enzyme profile in Rhipicephalus microplus ticks (Acari: Ixodidae).

Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 µg/mL and 92.67 ± 3.72 µg/mL, respectively, as compared to the control group (150.0 ± 3.80 µg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 µmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 µmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.

Role of narL gene in the pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium (STM) is a facultative anaerobe and one of the causative agents of nontyphoidal salmonellosis (NTS). Its anaerobic metabolism is enabled under the hypoxic environment that is encountered inside macrophages and the gut lumen of the host. In both of these niches, free radicals and oxidative intermediates are released by neutrophils as an inflammatory response. These chemical species further undergo reactions to produce nitrate, which is preferably taken up by STM as an electron acceptor in the absence of oxygen. NarL, the response regulator of the two-component regulatory system NarX/L, and a transcription factor, gets activated under anaerobic nitrate-rich conditions and upregulates the nitrate reduction during anaerobic respiration of STM. To understand the role of NarL in the pathogenesis of STM, we generated a narL-knockout (STM:ΔnarL) as well as a narL-complemented strain of STM. Anaerobically, the mutant displayed no growth defect but a significant attenuation in the swimming (26%) and swarming (61%) motility, and biofilm-forming ability (73%) in vitro, while these morphotypes got rescued upon genetic complementation. We also observed a downregulation in the expression of genes associated with nitrate reduction (narG) and biofilm formation (csgA and csgD) in anaerobically grown STM:ΔnarL. As compared with wild STM, narL mutant exhibited a threefold reduction in the intracellular replication in both intestinal epithelial cells (INT- 407) and monocyte-derived macrophages of poultry origin. Further, in vivo competitive assay in the liver and spleen of the murine model showed a competitive index of 0.48 ± 0.58 and 0.403668 ± 0.32, respectively, for STM:ΔnarL.

MMP-7 derived peptides with MHC class-I binding motifs from canine mammary tumor tissue elicit strong antigen-specific T-cell responses in BALB/c mice.

Matrix Metalloproteinases (MMPs)-induced altered proteolysis of extracellular matrix proteins and basement membrane holds the key for tumor progression and metastasis. Matrix metalloproteinases-7 (Matrilysin), the smallest member of the MMP family also performs quite alike; thus serves as a potential candidate for anti-tumor immunotherapy. Conversely, being an endogenous tumor-associated antigen (TAA), targeting MMP-7 for immunization is challenging. But MMP-7-based xenovaccine can surmount the obstacle of poor immunogenicity and immunological tolerance, often encountered in TAA-based conventional vaccine for anti-tumor immunotherapy. This paves the way for investigating the potential of MMP-7-derived major histocompatibility complex (MHC)-binding peptides to elicit precise epitope-specific T-cell responses towards their possible inclusion in anti-tumor vaccine formulations. Perhaps it also ushers the path of achieving multiple epitope-based broad and universal cellular immunity. In current experiment, an immunoinformatics approach has been employed to identify the putative canine matrix matelloproteinases-7 (cMMP-7)-derived peptides with MHC class-I-binding motifs which can elicit potent antigen-specific immune responses in BALB/c mice. Immunization with the cMMP-7 DNA vaccine induced a strong CD8+ cytotoxic T lymphocytes (CTLs) and Th1- type response, with high level of gamma interferon (IFN-γ) production in BALB/c mice. The two identified putative MHC-I-binding nonameric peptides (Peptide32-40 and Peptide175-183) from cMMP-7 induced significant lymphocyte proliferation along with the production of IFN-γ from CD8+ T-cells in mice immunized with cMMP-7 DNA vaccine. The current observation has depicted the immunogenic potential of the two cMMP-7-derived nonapeptides for their possible exploitation in xenovaccine-mediated anti-tumor immunotherapy in mouse model.

Lentiviral-mediated delivery of classical swine fever virus Erns gene into porcine kidney-15 cells for production of recombinant ELISA diagnostic antigen.

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45 kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.

Partial replacement of dietary buffalo meat on the bone with chicken carcass improves serum antioxidant profile of zoo-housed Indian leopards (Panthera pardus fusca).

This experiment was conducted to study the effect of gradual replacement of dietary buffalo meat on the bone (BMB) with chicken carcass (CC) on nutrient utilization, serum cortisol, and total serum antioxidant profile of zoo-housed Indian leopard. Twelve adult leopards were randomly distributed into a replicated Latin square design comprising three treatments, three periods, four animals, and three sequences. Leopards in group T1 were fed normal zoo diet of BMB. On the basis of dry matter, 10% and 20% of BMB was replaced with CC in groups T2 and T3 , respectively. Each experimental period comprised 21 days. During each period, a digestion trial of 4-day collection period was conducted after an adaptation period of 17 days. On Day 21 of each experimental period, blood was collected from all the animals by puncturing the ventral coccygial vein. Intake and apparent digestibility of major nutrients were similar among the groups. Replacement of 20% BMB with addition of CC increased (p < 0.001) the calculated supply of I, niacin, and vitamin A. Carotenoid intake increased (p < 0.01) with increased level of CC in the diet. Serum concentration of cortisol decreased (p < 0.01) whereas serum concentration of total carotenoids increased (p < 0.001) with increased level of CC in the diet. Serum concentration of antioxidant enzymes increased (p < 0.001) with increased level of CC in the diet. It was concluded that replacement of 20% of BMB with CC increased antioxidant profile. This may reduce oxidative stress in zoo-housed Indian leopards without any adverse effect on nutrient utilization.

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains.

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (Tm) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.

Effect of feeding graded levels of crude protein on nutrient utilization and feather growth in Lady Amherst's pheasants.

In order to find out the optimum level of crude protein (CP) in the diet of captive Lady Amherst's pheasants (LAP) on molt, 18 male birds were randomly distributed into three groups of six each in an experiment based on completely randomized block design. The CP content of the diets of birds in groups I, II, and III was 13.4, 16.5, and 19.1%, respectively. Intake and apparent balance of nitrogen increased linearly (P < 0.001) as CP content of the diet increased. Intake and utilization of energy, calcium, and phosphorous were similar among groups. Body mass change and growth rate of feathers were significantly (P < 0.01) lower in group I as compared to groups II and III. There was a positive co-relationship between ME intake and change in body weight (R(2) = 0.89, F = 126.4, P < 0.001). Regression analysis indicates that LAP can maintain body mass when ME supply is 122.2 Kcal/kg BW(0.75)/d. Linear relationships between intake and apparent retention of N, Ca, and P as expressed on mg/kg BW(0.75)/d were all significant. Apparent nitrogen retention, and mean feather growth rate was lower in birds fed diet containing 13.4% CP. Feeding of the diets containing 16.5% CP resulted in improved retention of nitrogen, and mean feather growth rate. Further increase in dietary concentration of CP to 19.1% showed no further improvement. It was concluded that a diet containing 16.5% CP would be optimum for Lady Amherst's pheasants during molt.

Increasing the dietary supply of carotenoids through forage supplementation: Effect on nitrogen and mineral retention in captive Golden pheasants (Chrysolophus pictus).

This experiment was conducted to evaluate the effects of feeding different levels of green forages on feed consumption, nutrient and mineral utilization in Golden pheasants (GP). Twenty-seven female GP (BW 617-635 g) were randomly distributed into three groups of nine birds each in an experiment based on completely randomized design (CRD). Birds in group T1 were fed a conventional zoo diet containing 1.4% green forages; however, the diets of the birds in groups T2 and T3 contained 2.7% and 5.0% of green forages, respectively. Intake of total carotenoids increased with increased level of green forages in the diet. Apparent retention of N, Ca, and Zn was higher in GP laying hens fed diet containing 5.0% green forages as compared to those fed conventional diet containing 1.4% green forages. Results of the present study indicate that inclusion of 5% green forage in the diet of GP would improve the utilization of N, Ca, and Zn without any adverse effect on intake and utilization of other nutrients. Data related to nutrient intake, basal endogenous losses (BEL) and coefficient of retention (COR) of N, Ca, P, Mg, Fe, Cu, Mn, and Zn are novel and may be of use for future research. Zoo Biol. 35:522-532, 2016. © 2016 Wiley Periodicals, Inc.

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay.

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 × 10(1) CFU and 2.28 × 10(2) CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

Loop-Mediated Isothermal Amplification for Rapid Detection and Differentiation of Wild-Type Bovine Herpesvirus-1 and Glycoprotein E-Deleted Marker Vaccine Strain.

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen affecting cattle and causing numerous reproductive disorders leading to significant economic losses to the cattle industry. The control programs for BoHV-1 are widely based on the use of glycoprotein E-deleted marker vaccines, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In this study, we report rapid and simple loop-mediated isothermal amplification (LAMP) assays for detection and differentiation of gE-deleted BoHV-1 from wild-type virus under isothermal conditions. The assays could be completed in 90 mintes, including viral DNA isolation, target amplification and visual interpretation of results with naked eye. The analytical sensitivity of the assays was 10 times higher than conventional PCR and could detect as little as 100 fg of viral DNA per reaction. The applicability of LAMP for detection of BoHV-1 in bovine semen was assessed by testing semen samples collected from breeding bulls and compared with TaqMan real-time PCR (as gold standard). The LAMP assays had diagnostic specificity of 100%. The diagnostic sensitivity was 88.2% and 83.3% for gB- and gE-LAMP, respectively, when compared with TaqMan real-time PCR. Our results have shown that the LAMP method developed in this study is a potential tool for rapid, sensitive, specific, cost-effective, and user-friendly detection and differentiation of wild type BoHV-1 from gE-deleted marker vaccine.

Prokaryotic expression of ovis aries conglutinin encoding neck and carbohydrate recognition domain and its functional characterization.

Conglutinin, a soluble pattern recognition receptor of innate immune system in bovines is known for its potential defensive activity against microorganisms either by direct agglutination in the presence of calcium or by acting as opsonin. In the present study, sheep (Ovis aries) conglutinin encoding neck and carbohydrate recognition domain (rSCGN) was expressed in the E coli BL21 expression host. The recombinant conglutinin revealed molecular weight of 27 kDa in SDS PAGE and also in western blotting using antibuffalo conglutinin polyclonal serum. The protein was characterized further for its functional activity in various assays. In ELISA based sugar and LPS binding assay, the rSCGN revealed its high binding activity toward N-acetyl glucosamine and E. coli LPS in the presence and the absence of calcium ions, respectively. Hemagglutination of chicken red blood cells caused by Newcastle disease virus was not inhibited in the presence of rSCGN as it lacked complete collagenous region present in the native protein. In virus neutralization test, the recombinant protein was found to reduce multiplication of bovine herpes virus-1 propagated in MDBK cells. This prokaryotically expressed 27 kDa recombinant sheep conglutinin can serve as antigen in future studies to develop sandwich ELISA for assessing the level of native conglutinin in sheep serum.

Rapid detection of bovine herpesvirus 1 in bovine semen by loop-mediated isothermal amplification (LAMP) assay.

Bovine herpesvirus 1 (BoHV-1) is the most common viral pathogen found in bovine semen, causing numerous reproductive disorders leading to economic losses to the cattle industry. For rapid detection of BoHV-1 in bovine semen, in this study, we applied a loop-mediated isothermal amplification (LAMP) assay. The assay could be completed within 90 min, including total DNA isolation, target amplification, and visual interpretation of positive or negative results with the naked eye. The assay detected as little as 10 fg of BoHV-1 DNA per reaction. The analytical sensitivity of the assay was 0.2 TCID50 BoHV-1 per reaction, which was 100 times more sensitive than conventional PCR and comparable to TaqMan real-time PCR. The applicability of the assay was assessed by analysing 118 semen samples collected from breeding bulls. On comparison with TaqMan real-time PCR, the LAMP assay had a diagnostic sensitivity of 97 %, specificity of 100 %, and accuracy of 99.2 % for detection of BoHV-1 in bovine semen. The LAMP assay developed in this study is a rapid, sensitive, and cost-effective alternative for detection of BoHV-1 in bovine semen.

Evaluation of single and dual siRNAs targeting rabies virus glycoprotein and nucleoprotein genes for inhibition of virus multiplication in vitro.

Small interfering RNAs (siRNAs) targeting rabies virus (RV) glycoprotein (G) and nucleoprotein (N) genes were evaluated as antiviral agents against rabies virus in vitro in BHK-21 cells. To select effective siRNAs targeting RV-G, a plasmid-based transient co-transfection approach was used. In this, siRNAs were expressed as short hairpin RNAs (shRNAs), and their ability to inhibit RV-G gene expression was evaluated in cells transfected with a plasmid expressing RV-G. The nine different siRNAs designed to target RV-G exhibited varying degrees of knockdown of RV-G gene expression. One siRNA (si-G7) with considerable effect in knockdown of RV-G expression also demonstrated significant inhibition of RV multiplication in BHK-21 cells after in vitro challenge with the RV Pasteur virus-11 (PV-11) strain. A decrease in the number of fluorescent foci in siRNA-treated cells and a reduction (86.8 %) in the release of RV into infected cell culture supernatant indicated the anti-rabies potential of siRNA. Similarly, treatment with one siRNA targeting RV-N resulted in a decrease in the number of fluorescent foci and a reduction (85.9 %) in the release of RV. As a dual gene silencing approach where siRNAs targeting RV-G and RV-N genes were expressed from single construct, the anti-rabies-virus effect was observed as an 87.4 % reduction in the release of RV. These results demonstrate that siRNAs targeting RV-G and N, both in single and dual form, have potential as antiviral agent against rabies.

Effect of concentrate supplementation on feed consumption, nutrient utilization and blood metabolite profile in captive spotted deer (Axis axis) fed oat (Avena sativa) and berseem (Trifolium alexandrinum) fodders based diet.

This experiment was conducted to determine the optimum level of a maize-soybean meal-wheat bran concentrate supplement fed to captive spotted deer fed an oat and berseem fodder-based diet. Twelve adult spotted deer [64-76 kg body weight (BW)] were distributed into three groups of four each and were housed individually. A diet consisting of 5 kg of oat fodder and 5.5 kg of berseem fodder was offered to each one of the experimental animals. The animal in group I received no supplementary concentrate, whereas, those in groups II and III received 0.5 and 1 kg of supplementary concentrate, respectively. A 60 days digestibility trial was conducted with a 5 days collection period on Days 55-59 of the trial. Blood samples were collected from all animals on Day 60 of the experiment. Average daily dry matter intake (DMI) was 1,224, 1,613, and 1,574 g/day in groups I, II, and III, respectively. Dry matter (DM) and organic matter (OM) intake was lowest (P < 0.01) in group I. Intake of P, Cu, and Zn was highest (P < 0.01) in group III, followed by groups II and I. Digestibility of neutral detergent fiber was highest (P < 0.05) in group II. Digestibility of OM and CP was lowest (P < 0.05) in group I. Digestibility of gross energy was highest (P < 0.01) in group III (74.9%), followed by groups II (69.3%) and I (66.2%). Digestible energy (DE) intake (kcal/kg BW(0.75) ) was highest (P < 0.01) in group III (195.4), followed by groups II (180.9) and I (129.8). Initial BW was 72.7, 72.5, and 71.0 kg, whereas, final BW was 71.0, 72.7, and 73.5 kg, in groups I, II and III, respectively. Average daily change in body mass was significantly (P < 0.01) different among the groups. The body mass was lost (-29.2 g/day), maintained (4.1 g/day) and gained (41.6 g/day) in groups I, II, and III, respectively. Blood glucose and cholesterol concentration was highest (P < 0.05) in group III, followed by groups II and I. Serum concentration of Cu and Zn was highest (P < 0.05) in group III, followed by groups II and I. Supplementation of forage only diet with 0.5 kg of concentrate mixture increased intake and digestibility of nutrients, without change in body mass. Animals fed 1 kg of supplementary concentrate received energy in excess of requirements, were consistently gaining body mass and were prone to obesity. Thus, it is a right strategy to supplement forage only diet of captive spotted deer with 0.5 kg of concentrate.

Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean-Congo Hemorrhagic Fever Virus.

Hyalomma anatolicum is the principal vector for Theileria annulata, T. equi, and T. Lestoquardi in animals and the Crimean-Congo hemorrhagic fever virus in humans. Due to the gradual loss of efficacy of the available acaricides against field tick populations, the development of phytoacaricides and vaccines has been considered the two most critical components of the integrated tick management strategies. In the present study, in order to induce both cellular and humoral immune responses in the host against H. anatolicum, two multi-epitopic peptides (MEPs), i.e., VT1 and VT2, were designed. The immune-stimulating potential of the constructs was determined by in silicoinvestigation on allergenicity (non-allergen, antigenic (0.46 and 1.0046)), physicochemical properties (instability index 27.18 and 35.46), as well as the interaction of constructs with TLRs by docking and molecular dynamics analysis. The immunization efficacy of the MEPs mixed with 8% MontanideTM gel 01 PR against H. anatolicum larvae was determined as 93.3% and 96.9% in VT1- and VT2-immunized rabbits, respectively. Against adults, the efficacy was 89.9% and 86.4% in VT1- and VT2-immunized rabbits, respectively. A significant (p < 0.001) reduction in the anti-inflammatory cytokine (IL-4) and significantly higher IgG response was observed in a VT1-immunized group of rabbits as compared with the response observed in the control group. However, in the case of the VT2-immunized rabbits, an elevated anti-VT2 IgG and pro-inflammatory cytokine (IL-2) (>30 fold) along with a decreased level of anti-inflammatory cytokine IL-4 (0.75 times) was noted. The efficacy of MEP and its potential immune stimulatory responses indicate that it might be useful for tick management.

Molecular characterization of lapinized classical Swine Fever vaccine strain by full-length genome sequencing and analysis.

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6-98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5'-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3'-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3'-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3'-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3'-UTR indicated that Indian CSFV strain requires further passage to obtain a 3'-UTR structure similar to most of the attenuated strains.

Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock.

Animal production has a key role in global economic development and food security. Ticks, specifically Rhipicephalus microplus cause substantial economic and health impacts on more than eighty percent of the world cattle population. Though synthetic acaricides play a major role in tick management, their injudicious usage has caused environmental pollution and also promote the establishment of multi-acaricide resistant tick populations which is a matter of great concern. To provide an effective tool for controlling these resistant ticks, the present work was aimed to develop safe and inexpensive antitick natural formulations. Our bioprospection studies of Ageratum conyzoides plant established it as a species potentially having strong acaricidal activity due to the presence of potent acaricidal phyto-chemicals. To develop a suitable antitick natural formulation, 41 samples/fractions/formulations were prepared from the dry powder of the whole aerial part of the A. conyzoides plant using different techniques and delivery matrices. The strongest antitick effect was recorded for formulation ACF6, which demonstrated 87 ± 6% mean mortality with 57 % inhibition of oviposition in treated female ticks. Ticks treated with the ACF6 formulation showed a significant (p < 0.001) reduction in cuticular protein (1.238 ± 0.01 mg/mL) as compared to control ticks (2.928 ± 0.01 mg/mL) but no significant difference in chitin content of treated ticks and control ticks was observed. The formulation was found safe in a rat model as no significant differences in biochemical and haematological parameters among treated and control rats were noted. Histopathological studies indicated no sign of hepatocellular necrosis and no significant changes in the weights of liver and spleen was recorded. The overall in vivo efficacy of the formulation was 85 % for experimentally infested cattle with direct mortality of more than 80 % within 96 h post-application. The lethal effect of the formulation was in the form of drying and dead ticks 1-2 d after application. The developed formulation has the potential to be adopted as an alternative tick control measure in an ecofriendly manner.

Functional characterization of partial recombinant goat conglutinin: Its role as innate immunity marker and use as antigen in sandwich ELISA.

Conglutinin, a liver synthesized versatile innate immune marker consisting C-type lectin domain belongs to collectin superfamily of proteins. The protein, first detected in bovine serum as soluble pattern recognition receptor (PRR) has wide range of antimicrobial activities. In the present study, open reading frame (ORF) encoding neck and carbohydrate recognition domain (NCRD) of goat conglutinin gene ligated to the vector pRSET-A was expressed in E. coli BL-21(pLys) cells. The 27 kDa recombinant protein (rGCGN) purified by single step Ni+2 -NTA affinity chromatography was found to cross-react with recombinant anti-buffalo conglutinin antibody raised in poultry. Further, it displayed calcium-dependant sugar binding activity towards yeast mannan and calcium-independent binding activity towards LPS. The mannan binding activity of rGCGN was inhibited in the presence of N-acetyl-glucosamine because of higher affinity towards this sugar. The recombinant protein was found to stimulate production of superoxide ions and hydrogen peroxide in goat neutrophils, which are instrumental in stimulating phagocytic activity of cells. When used as antigen in Sandwich ELISA, straight line (Y = 0.299x + 0.067, R2 = 0.997) was observed within the concentration range of 200-1000 ng/100 µl of rGCGN. Using this equation, the native conglutinin concentration in goat sera was estimated to be 0.5-7.5 µg/ml. The results indicated that prokaryotically expressed functionally active rGCGN can be used as antigen to assess native serum conglutinin levels in Sandwich ELISA and as immunomodulator in therapeutic applications to sequester unwanted immune complexes from the circulation.

IL-18 immunoadjuvanted xenogeneic canine MMP-7 DNA vaccine overcomes immune tolerance and supresses the growth of murine mammary tumor.

The development of the tumorigenesis and angiogenesis through proteolytic cleavage of extracellular matrix protein and basement membranes is promoted by Matrix metelloproteinases-7 (MMP-7). Consequently, MMP-7 is presumed as potential target for mammary cancer immunotherapy. However, MMP-7 is an endogenous tumor associated antigen (TAA); therefore, immunization is challenging. In current study, a potent anti-tumor immune response has been elicited through recombinant bivalent plasmid pVIVO2.IL18.cMMP7 which subside the highly metastatic 4 T1 cell line induced mammary tumors and efficiently negate the existing challenge of using MMP-7 as immunotherapeutic target. Balb/c mice were immunized with canine MMP-7 (cMMP-7) using interleukine-18 (IL-18), as an immunoadjuvant, to explore the potential of the combination regarding elicitation of a potent anti-tumor immune response. Mice vaccinated with pVIVO2.IL18.cMMP7 DNA plasmid reduced the tumor growth significantly along with augmentation of the immune response to fight against tumor antigen as depicted by substantial enrichment of CD4+ and CD8+ population in splenocytes, infiltration of immune system cells in tumor tissue and enhanced survival time of mice. Further, splenocyte supernatant examination of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were remarkably up-regulated demonstrating the stimulation of cell-mediated immune response. Thus the current observations vividly portray that administration of xenogeneic MMP-7 DNA vaccine bypasses the tolerance barrier.

Removing the threat of diclofenac to critically endangered Asian vultures.

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genus Gyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captive Gyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vulture Gyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered Asian Gyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40 G. africanus. Subsequently, six G. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species (Gyps bengalensis, Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity to Gyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.