Bacterial Agglutination Assay Research Articles

Identification and functional analysis of Toll-like receptor 2 from razor clam Sinonovacula constricta

Toll-like receptor 2 (TLR2) is a member of TLR family that plays important roles in the innate immune system, such as pathogen recognition and inflammation regulation. In this study, the TLR2 homologue was cloned from razor clam Sinonovacula constricta (denoted as ScTLR2) and its immune function was explored. The full-length cDNA of ScTLR2 comprised 2890 nucleotides with a 5′-UTR of 218 bp, an open reading frame of 2169 bp encoding 722 amino acids and a 3′-UTR of 503 bp. The deduced amino acid of ScTLR2 showed similar structure to TLR2 homologue with a conserved signal peptide, four LRR domains, one LRR-TYP domain, one LRR-CT domain, one transmembrane domain and a conserved TIR domain. ScTLR2 mRNA was detected in all examined tissues with the highest expression in the gill. After Vibrio parahaemolyticus challenge, the mRNA expression of ScTLR2 was significantly induced both in gill and haemocytes. The recombinant ScTLR2-LRR protein could bind all tested PAMPs including LPS, PGN and MAN. Bacterial agglutination assay showed that rScTLR2 could agglutinate the six tested bacteria with a calcium dependent manner. More importantly, ScTLR2 silencing by siRNA transfection could significantly depress the mRNA expression of Myd88, NF-κB, Tollip, IRF1, and IRF8. The survival rate of S. constricta was markedly decreased after V. parahaemolyticus challenge under this condition. Our current study demonstrated that ScTLR2 served as a pattern recognition receptor to induce immune response against invasive pathogen.

Insight into the molecular structure and function of peptidoglycan recognition protein SC2 (PGRP-SC2) from Amphiprion clarkii: Investigating the role in innate immunity

Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.

Identification and functional analysis of Mannose receptor in Asian swamp eel (Monopterus albus) in response to bacterial infection

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely MaMR). The open reading frame of MaMR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that MaMR shared the highest similarity with that of Paralichthys olivaceus. The expression of MaMR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda, the transcript level of MaMR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of MaMR was significantly induced post stimulated with LPS and LTA. Then the MaMR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that rMaMR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that MaMR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1β and tnf-α in EPC cells. Taken together, these results reveal that MaMR plays an important role in the immune response of fish to pathogen infections.

Characterization of a C-Type Lectin Domain-Containing Protein with Antibacterial Activity from Pacific Abalone (Haliotis discus hannai).

Genes that influence the growth of Pacific abalone (Haliotis discus hannai) may improve the productivity of the aquaculture industry. Previous research demonstrated that the differential expression of a gene encoding a C-type lectin domain-containing protein (CTLD) was associated with a faster growth in Pacific abalone. We analyzed this gene and identified an open reading frame that consisted of 145 amino acids. The sequence showed a significant homology to other genes that encode CTLDs in the genus Haliotis. Expression profiling analysis at different developmental stages and from various tissues showed that the gene was first expressed at approximately 50 days after fertilization (shell length of 2.47 ± 0.13 mm). In adult Pacific abalone, the gene was strongly expressed in the epipodium, gill, and mantle. Recombinant Pacific abalone CTLD purified from Escherichia coli exhibited antimicrobial activity against several Gram-positive bacteria (Bacillus subtilis, Streptococcus iniae, and Lactococcus garvieae) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). We also performed bacterial agglutination assays in the presence of Ca2+, as well as bacterial binding assays in the presence of the detergent dodecyl maltoside. Incubation with E. coli and B. subtilis cells suggested that the CTLD stimulated Ca2+-dependent bacterial agglutination. Our results suggest that this novel Pacific abalone CTLD is important for the pathogen recognition in the gastropod host defense mechanism.

Molecular and functional characterization of mitochondrial manganese superoxide dismutase from Macrobrachium rosenbergii during bacterial infection

Superoxide dismutases (SODs) are the main antioxidant enzymes involved in alleviating oxidative stress. Although mitochondrial manganese SOD (mMnSOD) has been reported to be correlated with the immune response in crustaceans, its biological properties and role in the immune response remain unclear. Here, we cloned the Macrobrachium rosenbergii mMnSOD (MrmMnSOD), analyzed its activity and expression pattern under Staphylococcus aureus and Vibrio parahaemolyticus infection, and further explored its possible mechanism during antibacterial immune response. The results showed that both enzyme activity and the expression of MrmMnSOD were significantly up-regulated by bacterial infection. MrmMnSOD knockdown made the prawn susceptible to Vibrio infection, which increased the mortality rate and the number of bacteria in haemocytes. The bacterial agglutination assay confirmed that MrmMnSOD decreases bacterial abundance via agglutination. Overall, this work identified antibacterial function of MrmMnSOD in the immune response. In addition to contributing to immunological theory, these findings aid disease prevention and control in crustacean aquaculture.

Immune function of cytosolic manganese superoxide dismutase from Macrobrachium rosenbergii in response to bacterial infection

An excess of reactive oxygen species (ROS) usually disrupts the balance of the antioxidant system and impairs the homeostasis of cells, resulting in serious oxidative damage. Therefore, a better understanding of how the immune system protects itself against such excessive oxidative stress is crucial. SODs play an indispensable role in antioxidant defense and the innate immune system. Among those, MnSOD is one of the most being. Previous studies using crustaceans have focused much on the gene expression changes underlying pathogenic infections, which generally indicate that MnSOD is involved in the antibacterial immune process of crustaceans. Yet the detailed biological properties of MnSOD and mechanism by which it counters pathogen infection in Macrobrachium rosenbergii remain unclear. Here we first cloned the cytosolic MnSOD from M. rosenbergii (MrcMnSOD), and then analyzed its distribution in tissues and expression patterns under bacterial infection with Staphylococcus aureus and Vibrio parahaemolyticus. The results revealed MrcMnSOD widely distributed in the tested tissues, whose expression level was highest in the hepatopancreas, followed by the stomach and intestine. Infection with S. aureus and V. parahaemolyticus significantly up-regulated both total SOD activity and transcription level of MrcMnSOD. To further investigate the possible mechanism of MrcMnSOD's participation in that antibacterial immune response, an RNA knockdown assay in vivo was performed. After knocking down MrcMnSOD, a higher mortality rate and more residual bacteria in haemocytes were recorded than for control group. Furthermore, an in vitro bacterial agglutination assay of MrcMnSOD demonstrated MrcMnSOD harbors broad bacterial agglutination to both gram-negative and gram-positive bacteria. Overall, our findings strongly suggest that MrcMnSOD is only capable of modulating the host's redox state but also exerting an antibacterial function in M. rosenbergii, thus shedding new light on the immune functioning of MrcMnSOD in crustaceans. Importantly, this work provides a new perspective for disease prevention and control in farmed crustaceans.

Molecular, transcriptional and functional delineation of Galectin-8 from black rockfish (Sebastes schlegelii) and its potential immunological role

Galectins are β-galactoside-binding lectins, which are involved in pattern recognition, cell adhesion, and stimulation of the host innate immune responses against microbial pathogens. In spite of several functional studies on different galectins isolated from vertebrates and invertebrates, this is the first report to present functional studies for galectin-8 from the marine teleost tissues. In the present study, we characterized galectin-8 homolog from black rockfish (Sebastes schlegelii), in molecular and functional aspects. Rockfish galectin-8 (SsGal8) was found to consist of a 969 bp long open reading frame (ORF), encoding a protein of 322 amino acids and the predicted molecular weight was 35.82 kDa. In silico analysis of SsGal8 revealed the presence of two carbohydrate binding domains (CRDs), at both N and C-termini and a linker peptide of 40 amino acids, in between the two domains. As expected, the phylogenetic tree categorized SsGal8 as a tandem-repeat galectin, and ultimately positioned it in the sub-clade of fish galectin-8. rSsGal8 was able to strongly agglutinate fish erythrocytes and the inhibition of agglutination was successfully exhibited by lactose and d-galactose. Bacterial agglutination assay resulted in agglutination of both Gram (+) and Gram (−) bacteria, including Escherichia coli, Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus parauberis, Lactococcus garvieae, Streptococcus iniae and Vibrio tapetis. The tissue distribution analysis based on qPCR assays, revealed a ubiquitous tissue expression of SsGal8 for the examined rockfish tissues, with the most pronounced expression in blood, followed by brain, intestine, head kidney and kidney. Furthermore, the mRNA transcription level of SsGal8 was significantly up-regulated in spleen, liver and head kidney, upon immune challenges with Streptococcus iniae, LPS and poly I:C, in a time dependent manner. Taken together, these findings strongly suggest the contribution of SsGal8 in regulating innate immune responses to protect the rockfish from bacterial infections.

Cloning and functional analysis of scavenger receptor B gene from the sea cucumber Apostichopus japonicus

Scavenger receptor (SR) class B (SR-B) is a transmembrane protein that belongs to the SR family with a wide range of functions in innate immunity. Here, an SR-B homologue, designated as AjSR-B, was cloned from the sea cucumber Apostichopus japonicus. AjSR-B comprised 2519 nucleotides with a 5′-untranslated region (UTR) of 153 bp, an open reading frame of 1581 bp encoding a 526 amino acid protein, and a 3′-UTR of 785 bp. SMART analysis indicated that AjSR-B has two transmembrane regions and a cluster determinant 36 domain. Multiple alignments and phylogenetic analysis supported that AjSR-B is a novel member of the SR-B protein family. Moreover, AjSR-B was constitutively expressed in all detected tissues, with the highest levels recorded in the intestine. Both were significantly induced in coelomocytes and the intestine after Vibrio splendidus challenge. Functionally, the recombinant rAjSR-B that corresponds to a large extracellular loop can bind pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan, and mannan, with a high binding affinity to LPS. Bacterial agglutination assay showed that rAjSR-B can agglutinate the four tested bacteria (Gram-negative and Gram-positive bacteria) with calcium dependence. However, the agglutination ability for Gram-negative bacteria completely disappeared in the presence of PAMPs but a weak ability to bind Gram-positive bacteria (Micrococcus luteus) was still exhibited, suggesting there might exist a competition between Gram-positive bacteria and PAMPs under same condition. Our current study indicated that AjSR-B is a PAMP that plays important roles in the innate immune process of sea cucumbers.

Immunostimulation of Cyprinus carpio using phage lysate of Aeromonas hydrophila

Over the last 50 years, various approaches have been established for the development of antigens for immunostimulation. We used phage lysate (PL), composed of inactivated antigens by the lytic bacteriophage pAh 6-c for Aeromonas hydrophila JUNAH strain to develop a vaccine for the prevention of A. hydrophila infection in Cyprinus carpio (common carp). We also assessed the poly D,L lactide-co-glycolic acid (PLGA) microparticles encapsulation method to increase the efficiency of the vaccine. Six groups of vaccines involving encapsulated by PLGA, formalin killed cells, or phage lysate at low or high concentration were prepared for intraperitoneal injection in C. carpio. Blood specimens and head kidney samples were collected at various time points for bacterial agglutination assay and to assess relative expression of immune-related genes interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), lysozyme C, and serum amyloid A (SAA). The vaccine groups using high dose phage lysate antigen showed significantly higher agglutination titers than all other groups at 4- and 6-weeks post vaccination (wpv), with the titer of the PLGA encapsulated vaccine group being highest from 10 wpv to the end of the experiment. The survival rate of fish immunized with the phage lysate vaccines were higher than that of fish immunized with the formailin killed cells vaccine in the challenge experiment conducted 6 wpv. Additionally, the PLGA-encapsulated high dose phage lysate antigen vaccinated groups showed the best protective efficacy in the challenge experiment 12 wpv. Vaccines using the phage lysate antigen also showed higher IL-1β and lysozyme C gene expression at 7 days post vaccination (dpv) and 2 wpv, and higher TNF-α gene expression was seen at 7 dpv. Higher SAA gene expression was seen in these groups at 1 dpv. These results suggest that phage lysate antigen has the potential to induce robust immune responses than formalin killed cells-based vaccines, and could be more effective as a novel inactivated antigen in preventing A. hydrophila infection in C. carpio.

Effects of effluent from electoplating industry on the immune response in the freshwater fish, Cyprinus carpio

The present study was designed to assess the effect of sublethal concentrations of electoplating industry effluent (EIE) on the non-specific and specific immune responses in the freshwater fish, Cyprinus carpio. Sublethal concentrations of electroplating industry effluent such as 0.004, 0.007, 0.010 and 0.013% were chosen based on the LC50 values. Experimental fish were exposed to these sublethal concentrations of EIE for 28 days. After 7, 14, 21 and 28 days of treatment, non-specific immune response by serum lysozyme activity, myeloperoxidase activity and antiprotease activity and specific immune response by antibody response to Aeromonas hydrophila using bacterial agglutination assay and ELISA were assessed. The results showed that chronic exposure of fish to 0.004, 0.007, 0.010 and 0.013% EIE, dose-dependently decreased the non-specific and specific immune responses on all the days tested compared to control fish whereas statistically significant suppressive effects were observed in fish exposed to 0.013% of EIE on all activities tested.

Newly identified invertebrate-type lysozyme (Splys-i) in mud crab (Scylla paramamosain) exhibiting muramidase-deficient antimicrobial activity

Lysozymes are widely distributed immune effectors exerting muramidase activity against the peptidoglycan of the bacterial cell wall to trigger cell lysis. However, some invertebrate-type (i-type) lysozymes deficient of muramidase activity still exhibit antimicrobial activity. To date, the mechanism underlying the antimicrobial effect of muramidase-deficient i-type lysozymes remains unclear. Accordingly, this study characterized a novel i-type lysozyme, Splys-i, in the mud crab Scylla paramamosain. Splys-i shared the highest identity with the Litopenaeus vannamei i-type lysozyme (Lvlys-i2, 54% identity) at the amino acid level. Alignment analysis and 3D structure comparison show that Splys-i may be a muramidase-deficient i-type lysozyme because it lacks the two conserved catalytic residues (Glu and Asp) that are necessary for muramidase activity. Splys-i is mainly distributed in the intestine, stomach, gills, hepatopancreas, and hemocytes, and it is upregulated by Vibrio harveyi or Staphylococcus aureus challenge. Recombinant Splys-i protein (rSplys-i) can inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio alginolyticus, Vibrio parahemolyticus, and Escherichia coli), Gram-positive bacteria (S. aureus, Bacillus subtilis, and Bacillus megaterium), and the fungus Candida albicans to varying degrees. In this study, two binding assays and a bacterial agglutination assay were conducted to elucidate the potential antimicrobial mechanisms of Splys-i. Results demonstrated that rSplys-i could bind to all nine aforementioned microorganisms. It also exhibited a strong binding activity to lipopolysaccharide from E. coli and lipoteichoic acid and peptidoglycan (PGN) from S. aureus but a weak binding activity to PGN from B. subtilis and β-glucan from fungi. Moreover, rSplys-i could agglutinate these nine types of microorganisms in the presence of Ca2+ at different protein concentrations. These results suggest that the binding activity and its triggered agglutinating activity might be two major mechanisms of action to realize the muramidase-deficient antibacterial activity. In addition, rSplys-i can hydrolyze the peptidoglycan of some Gram-positive bacteria because it exhibits weak isopeptidase activities in salt and protein concentration-dependent manner. This result indicates that such an isopeptidase activity may contribute to the muramidase-deficient antimicrobial activity to a certain degree. In conclusion, Splys-i is upregulated by pathogenic bacteria, and it inhibits bacterial growth by binding and agglutination activities as well as isopeptidase activity, suggesting that Splys-i is involved in immune defense against bacteria through several different mechanisms of action.

Purification, characterization, and analysis of antibacterial activity of a serum lectin from the grub of rhinoceros beetle, Oryctes rhinoceros

Abstract Lectins, also recognized as hemagglutinins, are multivalent proteins, and their desirable quality of fine sugar-binding specificity plays an important role in defense functions in invertebrates. In the present study, a galactose-specific lectin was detected, purified, and characterized from the hemolymph of the grub of rhinoceros beetle, Oryctes rhinoceros . The lectin was purified by affinity column chromatography with acid-treated Sepharose 6B as the matrix. The purified lectin was heat-labile, cation independent, and insensitive to EDTA. It revealed the presence of three distinct polypeptides with molecular weights of 90, 78, and 45 kDa. Thus, the native molecular weight of the purified lectin was calculated to be approximately 213 kDa, as revealed by a single band in native-PAGE. Studies on antibacterial activity and bacterial agglutination assays revealed that the purified lectin possess both these properties against two bacterial species, Bacillus subtilis and B. pumilus , isolated from the native larval habitat of O. rhinoceros .

Characterization and functional analysis of a tandem-repeat galectin-9 in large yellow croaker Larimichthys crocea.

Galectins are a family of endogenous lectins with β-galactosides affinity, playing significant roles in the innate immunity of vertebrates and invertebrates. In this report, a new galectin-9 cDNA was identified and characterized in large yellow croaker Larimichthys crocea (designated as LcGal-9). The complete cDNA sequence of LcGal-9 was 1795 bp, with an open reading frame (ORF) of 1032 bp encoding 343 amino acids. The putative LcGal-9 protein contained two carbohydrate recognition domains (CRDs) connected by a linker peptide, with each carrying two conserved β-galactoside binding motifs H-NPR and WG-EE-, and it possessed neither a signal peptide nor a transmembrane domain. LcGal-9 protein shared 43-74% identity with galectin-9 sequences from other species. The qRT-PCR analysis revealed that LcGal-9 mRNA was constitutively expressed in all tissues examined, predominately expressed in liver, spleen, gill, kidney, head-kidney and intestine. Western blot analysis showed that LcGal-9 protein was highly expressed in liver, spleen, intestine, kidney, head-kidney, skin, gill, and heart, but not detected in muscle and plasma. LcGal-9 mRNA transcripts were induced by poly I:C in the liver (from 6 h to 48 h), spleen (at 12 h) and head-kidney (at 12 h and 24 h). In contrast, Vibrio parahaemolyticus caused a significant down-regulation in these three tissues, except for in spleen of 48 h and head-kidney of 3 h. Post-infection with Cryptocaryon irritans, the transcripts were dramatically up-regulated in gill, skin, spleen and head-kidney during initial infection period, while significant down-regulation afterward was also observed both in spleen and head-kidney. The recombinant LcGal-9 (named as rLcGal-9) purified from Escherichia coli BL21 (DE3) demonstrated hemagglutination against human, rabbit and L. crocea in a Ca(2+)-independent manner, which was inhibited by α-Lactose and LPS. The results of bacterial agglutination assays showed that rLcGal-9 was able to agglutinate Gram-negative bacteria V. alginolyticus and Aeromonas hydrophila in a Ca(2+)-independent manner. By immunohistochemistry assay, significant increases of LcGal-9 protein appeared in the spleen stimulated with poly I:C (for 12 h) and V. parahaemolyticus (for 48 h) compared with the control. Based on the collective data, LcGal-9 might play an important role in innate immune responses, especially defense against Gram-negative bacteria in L. crocea.

Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus.

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S.hominis at a dose of 2mg, 20mg and 200mgkg(-1) body weight. Commercial surfactant surfactin (sigma) at 20mgkg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S.hominis stimulates the immunity of finfish thereby could enhance aquaculture production.

PtLGBP, a pattern recognition receptor in Portunus trituberculatus involved in the immune response against different challenges.

Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) is a pattern recognition receptor that can recognize and bind LPS and b-1,3-glucan. LGBP has crucial roles in innate immune defense against Gram-negative bacteria and fungi. In this study, LGBP functions in Portunus trituberculatus innate immunity were analyzed. First, the mRNA expression of PtLGBP in hemocytes, hepatopancreas, and muscle toward three typical pathogen-associated molecular patterns (PAMPs) stimulations were examined using real-time PCR. Results show that the overall trend of relative expressions of the LGBP gene in three tissues is consistent, showing up-down trend. In each group, the highest expression of the LGBP gene was at 3 and 12 h post-injection. The LGBP gene is also expressed significantly higher in the hemocytes and hepatopancreas than in the muscle. The highest level of LGBP was in the lipopolysaccharides (LPS) and glucan-injected group, whereas the lowest level was in the PGN-injected group. Furthermore, bacterial agglutination assay with polyclonal antibody specifically for PtLGBP proved that the recombinant PtLGBP (designated as rPtLGBP) could exhibit obvious agglutination activity toward Gram-negative bacteria Escherichia coli, Vibrio parahaemolyticus, and V. alginolyticus; Gram-positive bacteria Bacillus subtilis; and fungi Saccharomyces cerevisiae. LGBP in Portunus trituberculatus possibly served as a multi-functional PRR. In addition, LGBP is not only involved in the immune response against Gram-negative and fungi, as manifested in other invertebrates, but also has a significant role in anti-Gram-positive bacteria infection.

Orally administered P22 phage tailspike protein reduces salmonella colonization in chickens: prospects of a novel therapy against bacterial infections.

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans.

Changes in biochemical, histological and specific immune parameters in Catla catla (Ham.) by Cynodon dactylon (L.)

Indian major carp, Catla catla (Ham.), was fed with ethanolic extract of Cynodon dactylon (L.) mixed diet with 0.05% (group A), 0.5% (group B) and 5% (group C) extract for 60days with an interval of 10days. Significant (P>0.05) alterations were observed in the haematological and biochemical parameters such as white blood cell counts, red blood cell counts, haemoglobin content, serum glucose, cholesterol, protein, albumin, globulin, albumin/globulin ratio and RNA/DNA ratio in the experimental diet and control fishes. Non-specific immune response was assessed by the anti-protease activity, which was significantly increased in the experimental diet groups. The specific immune response of fish was evaluated by antibody response against heat-killed Aeromonas hydrophila for 28days by using ELISA and bacterial agglutination assay. Aggregation of melanomacrophage centres (MMC) and considerable modifications were observed in the histological analysis of the spleen of experimental diet groups. These results suggested that C. dactylon (L.) could combat the microbial infection by stimulating the immune response in fish.

Surfactant Protein D Interacts with α2-Macroglobulin and Increases Its Innate Immune Potential

Surfactant protein D (SP-D) is an innate immune collectin that recognizes microbes via its carbohydrate recognition domains, agglutinates bacteria, and forms immune complexes. During microbial infections, proteases, such as elastases, cleave the carbohydrate recognition domains and can inactivate the innate immune functions of SP-D. Host responses to counterbalance the reduction of SP-D-mediated innate immune response under these conditions are not clearly understood. We have unexpectedly identified that SP-D could interact with protein fractions containing ovomucin and ovomacroglobulin. Here, we show that SP-D interacts with human alpha(2)-macroglobulin (A2M), a protease inhibitor present in the lungs and serum. Using enzyme-linked immunosorbent assays, surface plasmon resonance, and carbohydrate competition assays, we show that SP-D interacts with A2M both in solid phase (K(D) of 7.33 nM) and in solution via lectin-carbohydrate interactions under physiological calcium conditions. Bacterial agglutination assays further show that SP-D x A2M complexes increase the ability of SP-D to agglutinate bacteria. Western blot analyses show that SP-D, but not A2M, avidly binds bacteria. Interestingly, intact and activated A2M also protect SP-D against elastase-mediated degradation, and the cleaved A2M still interacts with SP-D and is able to enhance its agglutination abilities. We also found that SP-D and A2M can interact with each other in the airway-lining fluid. Therefore, we propose that SP-D utilizes a novel mechanism in which the collectin interacts with protease inhibitor A2M to decrease its degradation and to concurrently increase its innate immune function. These interactions particularly enhance bacterial agglutination and immune complex formation.

Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent

The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45–50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC 50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC 50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments.

Immune response and disease resistance of Oreochromis mossambicus to Aeromonas hydrophila after exposure to hexavalent chromium

The objective of this study was to investigate the effect of chronic exposure to sublethal concentrations of hexavalent chromium (K2Cr2O7) on the immune response and disease resistance of Oreochromis mossambicus (Peters) to bacterial Aeromonas hydrophila infection. Fish (45 to 50 g) were exposed to 0.005, 0.05, 0.5, and 5 mg l(-1) [0.01, 0.1, 1, and 10% LC50, respectively] of hexavalent chromium Cr (VI) for 28 d. The specific immune response was assessed by antibody response to A. hydrophila by bacterial agglutination assay, and to sheep red blood cells (SRBC) by plaque forming cell (PFC) assay. In addition, nonspecific immune mechanisms were assessed by serum lysozyme activity and reactive nitrogen intermediates, the latter in terms of nitric oxide (NO) production by peripheral blood leucocytes. Overall immunity was assessed by disease resistance against live virulent A. hydrophila. The study clearly indicated that chronic exposure of fish to 0.5 and 5 mg l(-1) of chromium (VI) decreased both nonspecific and specific parameters of the immune system, which resulted in a lower disease resistance to A. hydrophila. Interestingly, 0.05 mg l(-1) of Cr (VI) enhanced disease resistance and both nonspecific and specific immune responses to A. hydrophila. Our study revealed a concentration-dependent modulation of the immune system by chromium (VI), as demonstrated by suppressive or stimulatory effects on lymphocytes, lysozyme, phagocytic killing mechanisms, and disease resistance in O. mossambicus.