Comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs

Two commercially available adjuvants, Gerbu LQ 3000 and Montanide ISA 50V, were assessed as potential replacements for Freund's adjuvant by evaluating their efficacy in the production of polyclonal antibodies to veterinary drugs in rabbits. The aim was to find an adjuvant that could produce a similar (or enhanced) immune response in the host animal without the undesirable side effects associated with Freund's complete and incomplete adjuvant. The assessment involved the examination of each injection site and the characterisation of the resultant antibodies with regards to antibody titre and sensitivity. It was found that the rabbits immunised with Gerbu adjuvant produced some of the most sensitive antibodies. However, titres were relatively low and adverse effects at injection sites were relatively common. Montanide adjuvant produced no adverse effects and the related antibodies were found to be of adequate sensitivity when compared to those from rabbits immunised with Freund's. It was concluded that Montanide ISA 50V could be considered as a suitable replacement to Freund's for the production of polyclonal antibodies, to low molecular weight compounds in rabbits. (C) 2007 Elsevier B.V. All rights reserved.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

The developmental and acute phases of insulin-induced laminitis involve minimal metalloproteinase activity

Metalloproteinases have been implicated in the pathogenesis of equine laminitis and other inflammatory conditions, through their role in the degradation and remodelling of the extracellular matrix environment. Matrix metalloproteinases (MMPs) and their inhibitors are present in normal equine lamellae, with increased secretion and activation of some metalloproteinases reported in horses with laminitis associated with systemic inflammation. It is unknown whether these enzymes are involved in insulin-induced laminitis, which occurs without overt systemic inflammation. In this study, gene expression of MMP-2, MMP-9, MT1-MMP, ADAMTS-4 and TIMP-3 was determined in the lamellar tissue of normal control horses (n = 4) and horses that developed laminitis after 48 h of induced hyperinsulinaemia (n = 4), using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Protein concentrations of MMP-2 and MMP-9 were also examined using gelatin zymography in horses subject to prolonged hyperinsulinaemia for 6 h (n = 4), 12 h (n = 4), 24 h (n = 4) and 48 h (n = 4), and in normal control horses (n = 4). The only change in gene expression observed was an upregulation of MMP-9 (p < 0.05) in horses that developed insulin-induced laminitis (48 h). Zymographical analysis showed an increase (p < 0.05) in pro MMP-9 during the acute phase of laminitis (48 h), whereas pro MMP-2 was present in similar concentration in the tissue of all horses. Thus, MMP-2, MT1-MMP, TIMP-3 and ADAMTS-4 do not appear to play a significant role in the pathogenesis of insulin-induced laminitis. The increased expression of MMP-9 may be associated with the infiltration of inflammatory leukocytes, or may be a direct result of hyperinsulinaemia. The exact role of MMP-9 in basement membrane degradation in laminitis is uncertain as it appears to be present largely in the inactive form.

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Antibody reactivity to the transmembrane protein of the caprine arthritis encephalitis virus correlates with severity of arthritis: No evidence for the involvement of epitope mimicry

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Mimicry of 21. 5 KDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKRPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TPC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogenesis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.

Immune responses to hemagglutinin-neuraminidase protein of peste des petits ruminants virus expressed in transgenic peanut plants in sheep

Peste des petits ruminants (PPR) is an acute, highly contagious disease of small ruminants caused by a morbillivirus, Peste des petits ruminants virus (PPRV). The disease is prevalent in equatorial Africa, the Middle East, and the Indian subcontinent. A live attenuated vaccine is in use in some of the countries and has been shown to provide protection for at least three years against PPR. However, the live attenuated vaccine is not robust in terms of thermotolerance. As a step towards development of a heat stable subunit vaccine, we have expressed a hemagglutinin-neuraminidase (HN) protein of PPRV in peanut plants (Arachis hypogea) in a biologically active form, possessing neuraminidase activity. Importantly. HN protein expressed in peanut plants retained its immunodominant epitopes in their natural conformation. The immunogenicity of the plant derived HN protein was analyzed in sheep upon oral immunization. Virus neutralizing antibody responses were elicited upon oral immunization of sheep in the absence of any mucosal adjuvant. In addition, anti-PPRV-HN specific cell-mediated immune responses were also detected in mucosally immunized sheep. (C) 2010 Elsevier B.V. All rights reserved.

Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection. (C) 2009 Elsevier B.V. All rights reserved.

Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2. Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus). Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels. (C) 2008 Elsevier B.V. All rights reserved.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.