Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV preinfection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (,1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon c. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.

Capsular sialic acid of Streptococcus suis serotype 2 binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells.

Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.

Chicken Caecal Microbiome Modifications Induced by Campylobacter jejuni Colonization and by a Non-Antibiotic Feed Additive

Campylobacter jejuni is an important zoonotic foodborne pathogen causing acute gastroenteritis in humans. Chickens are often colonized at very high numbers by C. jejuni, up to 109 CFU per gram of caecal content, with no detrimental effects on their health. Farm control strategies are being developed to lower the C. jejuni contamination of chicken food products in an effort to reduce human campylobacteriosis incidence. It is believed that intestinal microbiome composition may affect gut colonization by such undesirable bacteria but, although the chicken microbiome is being increasingly characterized, information is lacking on the factors affecting its modulation, especially by foodborne pathogens. This study monitored the effects of C. jejuni chicken caecal colonization on the chicken microbiome in healthy chickens. It also evaluated the capacity of a feed additive to affect caecal bacterial populations and to lower C. jejuni colonization. From day-0, chickens received or not a microencapsulated feed additive and were inoculated or not with C. jejuni at 14 days of age. Fresh caecal content was harvested at 35 days of age. The caecal microbiome was characterized by real time quantitative PCR and Ion Torrent sequencing. We observed that the feed additive lowered C. jejuni caecal count by 0.7 log (p<0.05). Alpha-diversity of the caecal microbiome was not affected by C. jejuni colonization or by the feed additive. C. jejuni colonization modified the caecal beta-diversity while the feed additive did not. We observed that C. jejuni colonization was associated with an increase of Bifidobacterium and affected Clostridia and Mollicutes relative abundances. The feed additive was associated with a lower Streptococcus relative abundance. The caecal microbiome remained relatively unchanged despite high C. jejuni colonization. The feed additive was efficient in lowering C. jejuni colonization while not disturbing the caecal microbiome.

Actinobacillus pleuropneumoniae induces SJPL cell cycle arrest in G2/M-phase and inhibits porcine reproductive and respiratory syndrome virus replication

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. Methods: Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. Results: Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore, two G2/M-phase cell cycle inhibitors demonstrated the ability to inhibit PRRSV infection, indicating a potential key role for PRRSV infection. Finally, mass spectrometry lead to identify two molecules (m/z 515.2 and m/z 663.6) present only in the culture supernatant. Conclusions: We demonstrated for the first time that A. pleuropneumoniae is able to disrupt SJPL cell cycle resulting in inhibitory activity against PRRSV. Furthermore, two putative molecules were identified from the culture supernatant. This study highlighted the cell cycle importance for PRRSV and will allow the development of new prophylactic or therapeutic approaches against PRRSV.

Rôle de l'isoforme p35 de la chaîne invariante humaine dans la formation et le transport de complexes de CMH II

La présentation antigénique par les molécules de classe II du complexe majeur d’histocompatibilité (CMH II) est un mécanisme essentiel au contrôle des pathogènes par le système immunitaire. Le CMH II humain existe en trois isotypes, HLA-DP, DQ et DR, tous des hétérodimères composés d’une chaîne α et d’une chaîne β. Le CMH II est entre autres exprimé à la surface des cellules présentatrices d’antigènes (APCs) et des cellules épithéliales activées et a pour fonction de présenter des peptides d’origine exogène aux lymphocytes T CD4+. L’oligomérisation et le trafic intracellulaire du CMH II sont largement facilités par une chaperone, la chaîne invariante (Ii). Il s’agit d’une protéine non-polymorphique de type II. Après sa biosynthèse dans le réticulum endoplasmique (ER), Ii hétéro- ou homotrimérise, puis interagit via sa région CLIP avec le CMH II pour former un complexe αβIi. Le complexe sort du ER pour entamer son chemin vers différents compartiments et la surface cellulaire. Chez l’homme, quatre isoformes d’Ii sont répertoriées : p33, p35, p41 et p43. Les deux isoformes exprimées de manière prédominante, Iip33 et p35, diffèrent par une extension N-terminale de 16 acides aminés portée par Iip35. Cette extension présente un motif de rétention au réticulum endoplasmique (ERM) composé des résidus RXR. Ce motif doit être masqué par la chaîne β du CMH II pour permettre au complexe de quitter le ER. Notre groupe s’est intéressé au mécanisme du masquage et au mode de sortie du ER des complexes αβIi. Nous montrons ici que l’interaction directe, ou en cis, entre la chaîne β du CMH II et Iip35 dans une structure αβIi est essentielle pour sa sortie du ER, promouvant la formation de structures de haut niveau de complexité. Par ailleurs, nous démontrons que NleA, un facteur de virulence bactérien, permet d’altérer le trafic de complexes αβIi comportant Iip35. Ce phénotype est médié par l’interaction entre p35 et les sous-unités de COPII. Bref, Iip35 joue un rôle central dans la formation des complexes αβIi et leur transport hors du ER. Ceci fait d’Iip35 un régulateur clef de la présentation antigénique par le CMH II.

Activité antimicrobienne de peptides provenant d’hydrolysats de protéines de babeurre, de lactoferrine et de pois

Les antibiotiques sont fréquemment utilisés dans l’alimentation de la volaille afin de prévenir certaines maladies, dont l’entérite nécrotique, ce qui occasionne l’émergence de souches bactériennes résistantes aux antibiotiques. Une alternative prometteuse est l’utilisation de peptides antimicrobiens (AMPs) comme suppléments alimentaires, tels les AMPs provenant des produits laitiers. L’objectif du projet était de développer une méthode de production d’extraits peptidiques à partir de coproduits de la transformation alimentaire (babeurre, lactoferrine, isolat de protéines de pois), afin de tester si ces extraits peptidiques possédaient une activité antimicrobienne sur les pathogènes spécifiques aviaires suivants : Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli et Staphylococcus aureus. Les protéines ont été mises en suspension dans l’eau (5% p/p) et hydrolysées par la pepsine, 6 heures, pH de 2.5. Les peptides furent récupérés par ultrafiltration (< 10 kDa), puis fractionnés selon leur charge nette : totaux, cationiques, anioniques et non liés. L’effet antimicrobien a été évalué surmicroplaques, par la survie bactérienne en présence de concentrations croissantes d’extraits peptidiques. Les extraits cationiques de babeurre ont démontré une efficacité à une concentration inférieure ou égale à 5 mg/mL; perte de 3 log pour Escherichia coli O78 :H80. En comparaison, la lactoferrine cationique a été efficace à une concentration inférieure ou égale à 0.6 mg/mL; perte de 6 log pour E. coli O78 :H80. Les extraits peptidiques du pois ont démontré une efficacité faible. Cette méthode s’avère prometteuse pour le développement d’une alternative ou d’un complément pour la réduction de l’utilisation des antibiotiques dans l’alimentation de la volaille.

Kinetics of Bacterial Colony Growth by Laser Induced Fluorescence

The growth kinetics of an aerial bacterial colony on solid agar media was studied using laser induced fluorescence technique. Fluorescence quenching of Rhodamin B by the bacterial colony was utilized for the study. The lag phase, log phase, and stationary phase of growth curve of bacterial colony was identified by measuring peak fluorescence intensity of dye doped bacterial colony.

Semiconductor Oxides Mediated Photocatalytic Removal of Chemical and Bacterial Pollutants from Wastewater

School of Environmental Studies, Cochin University of Science and Technology

Studies on Production of Bacterial Xylanases

Xylanases with hydrolytic activity on xylan, one of the hemicellulosic materials present in plant cell walls, have been identified long back and the applicability of this enzyme is constantly growing. All these applications especially the pulp and paper industries require novel enzymes. There has been lot of documentation on microbial xylanases, however, none meeting all the required characteristics. The characters being sought are: higher production, higher pH and temperature optima, good stabilities under these conditions and finally the low associated cellulase and protease production. The present study analyses various facets of xylanase biotechnology giving emphasis on bacterial xylanases. Fungal xylanases are having problems like low pH values for both enzyme activity and growth. Moreover, the associated production of cellulases at significant levels make fungal xylanases less suitable for application in paper and pulp industries.Bacillus SSP-34 selected from 200 isolates was clearly having xylan catabolizing nature distinct from earlier reports. The stabilities at higher temperatures and pH values along with the optimum conditions for pH and temperature is rendering Bacillus SSP-34 xylanase more suitable than many of the previous reports for application in pulp and paper industries.Bacillus SSP-34 is an alkalophilic thertmotolerant bacteria which under optimal cultural conditions as mentioned earlier, can produce 2.5 times more xylanase than the basal medium.The 0.5% xylan concentration in the medium was found to the best carbon source resulting in 366 IU/ml of xylanase activity. This induction was subjected to catabolite repression by glucose. Xylose was a good inducer for xylanase production. The combination of yeast extract and peptone selected from several nitrogen sources resulted in the highest enzyme production (379+-0.2 IU/ml) at the optimum final concentration of 0.5%. All the cultural and nutritional parameters were compiled and comparative study showed that the modified medium resulted in xylanase activity of 506 IU/ml, 5 folds higher than the basal medium.The novel combination of purification techniques like ultrafiltraton, ammonium sulphate fractionation, DEAE Sepharose anion exchange chromatography, CM Sephadex cation exchange chromatography and Gel permeation chromatography resulted in the purified xylanase having a specific activity of 1723 U/mg protein with 33.3% yield. The enzyme was having a molecular weight of 20-22 kDa. The Km of the purified xylanase was 6.5 mg of oat spelts xylan per ml and Vmax 1233 µ mol/min/mg protein.Bacillus SSP-34 xylanase resulted in the ISO brightness increase from 41.1% to 48.5%. The hydrolytic nature of the xylanase was in the endo-form.Thus the organism Bacillus SSP-34 was having interesting biotechnological and physiological aspects. The SSP-34 xylanase having desired characters seems to be suited for application in paper and pulp industries.

Ecophysiology of nontuberculous Mycobacteria in marine aquaculture ponds

The world demand for fish and fishery products is increasing steadily and it is generally accepted that it will not be possible to meet the heavy demand with resources exploited from capture fishery alone. Now aquaculture is well established and fastdeveloping industry in many countries and is a major focus sector for development. During recent decades, aquaculture has gained momentum, throughout the world especially in developing countries. According to Food and Agricultural Oganisation (FAO, 2000), global aquaculture production was 26.38 tones in 1996 have reached 32.9 million tonnes during 1999. Only marine aquaculture sector has contributed 13.1 million tonnes during 1999.India is a major fish producing country. About one half of lndia’s brackish water lands are currently being utilized for farming in order to reduce the gap between supply and demand for fish. Aquaculture has become a major source of livelihood for people and its role in integrated rural development, generation of employment and earning foreign exchange, thereby alleviating poverty is being greatly appreciated around the world.Among the infectious agents, bacteria are becoming the prime causal organisms for diseases in food fishes and other marine animals. Sindermann, (1970) reported that bacterial fish pathogen most commonly found among marine fishes is species of Pseudomonas, Vibrio and Mycobacterium. These can be categorized into primary pathogens; secondary invaders that may cause systemic disease in immunocompromised hosts; and normal marine flora which are not pathogenic but may occur on body surfaces or even within the tissues of the host. I-Iigh density of animals in hatchery tanks and ponds is conducive to the spread of pathogen and the aquatic environment with regular application of protein rich feed, is ideal for culturing bacteria. Bacteria, which are normally present in seawater or on the surface of fish, can invade and cause pathological effects in fishes, which are injured or subjected to other environmental stresses.Mycobacteria except parasites are known as nontuberculosis mycobacteria (NTM), atypical mycobacteria or mycobacteria other than tuberculosis(MO'l'l"). This group of mycobacteria includes opportunistic pathogens and saprophytes. Environmental mycobacteria are ubiquitous in distribution and the sources may include soil, water, warm-blooded as well as cold-blooded animals. Disease caused by environmental mycobacterial strains in susceptible humans (Goslee & Wolinsky, 1976; Grange, 1987), animals and fishes are increasingly attracting attention. Greatest importance of environmental mycobacteria is believed to be their role in immunological priming of humans and animals, thereby modifying their immune responses to subsequent exposure to pathogenic species.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

Vibrio are important during hatchery rearing. aquaculture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp farmers and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases.With the progress in aquaculture, intensive systems used for shrimp aquaculture create an artificial environment that increases bacterial growth. To maintain the productivity of such an intensive aquaculture, high inputs of fish protein have to be employed for feeding together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence. The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose, water, feed, shrimp quality and aquaculture management.Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio.Of the l2 pathogenic Vibrio species, 8 species are known to be directly food associated. Strict quality guidelines have been laid by the importing nations, for the food products that enter their markets. The microbiological quality requirement for export of frozen shrimp products is that V.cholerae, V.parahaemolyticus and V. vulnificus should be absent in 25g of the processed shrimp (Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic vibrios, the shrimp aquaculture system.So the present study was undertaken to study the incidence of pathogenic Vibrio spp. in Penaeus monodon shrimp hatcheries and aquaculture farms, to carry out biochemical investigations of the pathogenic Vibrio spp isolated from P. monodon hatchery and. aquaculture environments, to assess the effect of salt (NaCl) on the growth and enzymatic activities of pathogenic Vibrio spp., to study the effect of preservatives, and chemicals on the growth of pathogenic Vibrio spp. and to employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.Samples of water (n=7) and post-larvae (n=7) were obtained from seven Penaeus monodon hatcheries and samples of water (n=5), sediment (n=5) and shrimp (n=5) were obtained from five P. monodon aquaculture farms located on the East Coast of lndia. The microbiological examination of water, sediment, post-larvae and shrimp samples was carried out employing standard methods and by using standard media.The higher bacterial loads were obtained in pond sediments which can be attributed to the accumulation of organic matter at the pond bottom which stimulated bacterial growth.Shrimp head. (4.78 x 105 +/- 3.0 x 104 cfu/g) had relatively higher bacterial load when compared to shrimp muscle 2.7 x 105 +/- 1.95 x 104 cfu/g). ln shrimp hatchery samples, the post-larvae (2.2 x 106 +/- 1.9 x 106 cfu/g) had higher bacterial load than water (5.6 x 103 +/- 3890 cfu/ml).The mean E.coli counts were higher in aquaculture pond sediment (204+/-13 cfu/g) and pond water (124+/-88 cfu/ml). Relatively lower Escherichia coli counts were obtained from shrimp samples (12+/-11 to 16+/-16.7 cfu/g). The presence of E.coli in aquaculture environment might have been from the source water. E.coli was not detected in hatchery waters and post-larvae.

Studies on the effect of the organophosphorus pesticide Eka(Rlu) x EC 25 on the bacterial flora of Villorita cyprinoides var. cochinensis (Han1ey)

The thesis is Studies on the Effect or the Obganophosphorus Pesticide Ekalux(R) EC 25 on the Bacterial Flora or Villorita Cyprinoides Var.Cochinensis (Hanley). For the present investigation, the black clam Villorita gyprinoides var. cochinensis (Hanley), a most common clam genus present in this estuarine system has been selected as test organaism and Ekalux (R) EC 25 as toxicant. The aspects dealt with are 1. Total heterotrophic bacterial population, 2. Generic composition, 3. Hydrolytic enzyme producing bacteria, 4. Antibiotic resistance, 5. Heavy metal resistance, 6. The effect of pesticide concentration on the growth of the bacteria and 7. Effect of temperature, pH and sodium chloride on the growth and phosphate release of selected isolates.The samples for the experiment were collected from the Vembanad Lake, near Kumbalam Island during the period of September 1985 to May '86. The THB of the estuarine water and clams contained 6.5 x I04/ml and 2.975 x l06/g respectively, immediately after collection. Untreated water and clam samples showed enormous increase in THB from 0 hr population. The treated samples (water and clams) contained higher THB than 0 hr. In general, THB was observed to increase tremendously in the samples treated with pesticide when compared to their native flora. With reference to various concentrations of pesticides, THB recorded an increase with increase of concentration in water and clam samples.

Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.