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Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and handling or consumption of contaminated poultry meat is the key source of infection. Glycoconjugate vaccines containing the C. jejuni N-glycan have been reported to be partially protective in chickens. However, our previous studies with subunit vaccines comprising the C. jejuni FlpA or SodB proteins with up to two or three C. jejuni N-glycans, respectively, failed to elicit significant protection. In this study, protein glycan coupling technology was used to add up to ten C. jejuni N-glycans onto a detoxified form of Pseudomonas aeruginosa exotoxin A (ExoA). The glycoprotein, G-ExoA, was evaluated for efficacy against intestinal colonisation of White Leghorn chickens by C. jejuni strains M1 and 11168H relative to unglycosylated ExoA. Chickens were challenged with the minimum dose required for reliable colonisation, which was 102 colony-forming units (CFU) for strain M1 and and 104 CFU for strain 11168H. Vaccine-specific serum IgY was detected in chickens vaccinated with both ExoA and G-ExoA. However, no reduction in caecal colonisation by C. jejuni was observed. While the glycan dose achieved with G-ExoA was higher than FlpA- or SodB-based glycoconjugates that were previously evaluated, it was lower than that of glycoconjugates where protection against C. jejuni has been reported, indicating that protection may be highly sensitive to the amount of glycan presented and/or study-specific variables.

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella Typhimurium is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two phylogenetically distinct variants, U288 and ST34, emerged in UK pigs around the same time but present different risk to food safety. Here we show using genomic epidemiology that ST34 accounts for over half of all S. Typhimurium infections in people while U288 less than 2%. That the U288 clade evolved in the recent past by acquiring AMR genes, indels in the virulence plasmid pU288-1, and accumulation of loss-of-function polymorphisms in coding sequences. U288 replicates more slowly and is more sensitive to desiccation than ST34 isolates and exhibited distinct pathogenicity in the murine model of colitis and in pigs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential., Mark Kirkwood et al. take an epidemiological, genomic, phylogenetic, and experimental approach to assess the evolutionary origins and virulence of the U288 and ST34 isolates of Salmonella enterica in affecting livestock and potentially humans. Their results indicate that the molecular evolution of these isolates is associated with altered pathogenicity in pigs and reduced zoonotic potential of U288.

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two variants, S. Typhimurium phage type U288 and monophasic S. Typhimurium (S. 4,[5],12:i:-) ST34 emerged and have accounted for the majority of isolates from pigs in the UK in the past two decades, but have distinct host range and risk to food safety. ST34 accounts for over 50% of all S. Typhimurium infections in people while U288 less than 2%. U288 and ST34 form distinct phylogenetic clusters within S. Typhimurium, defined by approximately 600 SNPs within their 5 Mbp genomes. Evolution of the U288 clade from an LT2-like ancestor was characterised by the acquisition of AMR genes, insertions and deletions in the virulence plasmid pU288-1, and the accumulation of polymorphisms, some of which resulted in truncation of coding sequences. U288 isolates exhibited lower growth rate and viability following desiccation compared to ST34 isolates, characteristics that could affect transmission through the food chain. U288 and ST34 isolates exhibited distinct outcomes of infection in the murine model of colitis, and colonised pigs in a manner that affected the disease symptoms and distribution in organs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential.ImportanceBacterial pathogens continually evolve to exploit new ecological niches as they emerge due to human activity including agricultural, medical or societal practice. The consequences of the emergence of new pathogens may affect outcome of infection and risk to human or animal health. Genome sequence can resolve the population structure, identify variants that are evolving as they enter a new niche, and pinpoint potential functional divergence. We report a variant S. Typhimurium that adapted to a unique niche distinct to that occupied by a second S. Typhimurium variant circulating in the same pig populations. Adaptation was accompanied by phenotypic and genotypic changes consistent with a more invasive lifestyle and a decreased zoonotic potential observed in the epidemiological record. Our findings suggest that pathogen genotypic variation may be useful in estimating zoonotic potential and threat to livestock welfare.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology&mdash, G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)&mdash, were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy.

Summary Aging has a profound effect on the immune system, termed immunosenescence, resulting in increased incidence and severity of infections and decreased efficacy of vaccinations. We previously showed that immunosurveillance in the intestine, achieved primarily through antigen sampling M cells in the follicle associated epithelium (FAE) of Peyer's patches, was compromised during aging due to a decline in M cell functional maturation. The intestinal microbiota also changes significantly with age, but whether this affects M cell maturation was not known. We show that housing of aged mice on used bedding from young mice, or treatment with bacterial flagellin, were each sufficient to enhance the functional maturation of M cells in Peyer's patches. An understanding of the mechanisms underlying the influence of the intestinal microbiota on M cells has the potential to lead to new methods to enhance the efficacy of oral vaccination in aged individuals., Graphical Abstract, Highlights • Exposure of aged mice to a young microbiota restores M cell density and function • Systemic flagellin treatment enhances M cell density and function in aged mice • Young microbiota exposure and flagellin treatment improve intestinal crypt function • Microbial stimulation can restore the aging-related decline in M cells, Microbiology; Microbiome

Additional file 1. Validation of pFPV25.1 for detection of Salmonella-infected bovine cells. Several in vitro and in vivo tests were performed to assess the suitability of pFPV25.1 for detection of Salmonella-infected cells. (A–C) Firstly, bovine peripheral blood-derived macrophages were infected in vitro with SD3246-GFP at a MOI of 100 and infected cells were identified using either the constitutive GFP-expression from pFPV25.1 or by staining with anti-Salmonella LPS. (A) While the majority of GFP+ (infected) cells were also LPS+, a proportion of infected cells were incorrectly identified by anti-LPS staining. (B) The GFP signal from infected cells was sensitive and specific. (C) Detection of infected cells by anti-LPS staining was less specific than GFP. (D) Following this, the effect of presence of pFPV25.1 on bacterial invasion of peripheral blood-derived macrophages was determined in vitro and it was found that SD3246-GFP was less invasive than wild-typec SD3246 nalR. (E) This phenotype was confirmed in vivo using the bovine ligated ileal loop model. While SD3246-GFP was less invasive than SD3246 nalR after 10 h of infection, the total bacterial numbers recovered were similar from loops inoculated with both strains. (F) Following oral challenge of calves with SD3246-GFP, the stability of pFPV25.1 was confirmed to inform the accuracy of identifying Salmonella-infected cells. It was found that pFPV25.1 was stably maintained within intracellular bacteria recovered from tissues of these calves 48 h post-infection, even in the absence of ampicillin selection, providing confidence that the GFP signal was used to accurately detect infected bovine cells. (G) Lastly, the effect of presence of pFPV25.1 on clinical signs was assessed and it was found that the pyrexia induced in SD3246-GFP-challenged calves was the same as that expected during wild-type S. Dublin infections, confirming that virulence of SD3246-GFP had not been adversely affected.

Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen., Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase. IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

Background Salmonella enterica subspecies enterica is an animal and zoonotic pathogen of global importance. Cattle are a significant reservoir of human non-typhoidal salmonellosis and can suffer enteric and systemic disease owing to the ability of Salmonella to survive within the bovine lymphatic system and intestines. Contamination of food can occur due to the incorporation of contaminated peripheral lymph nodes or by direct contamination of carcasses with gut contents. It is essential to understand the mechanisms used by Salmonella to enter and persist within the bovine lymphatic system and how they differ from those required for intestinal colonization to minimize zoonotic infections. Results Transposon-directed insertion site sequencing (TraDIS) was applied to pools of mutants recovered from mesenteric lymph nodes (MLNs) draining the distal ileum of calves after oral inoculation with a library of 8550 random S. Typhimurium mini-Tn5Km2 mutants in pools of 475 mutants per calf. A total of 8315 mutants representing 2852 different genes were detected in MLNs and their in vivo fitness was calculated. Using the same improved algorithm for analysis of transposon-flanking sequences, the identity and phenotype of mutants recovered from the distal ileal mucosa of the same calves was also defined, enabling comparison with previously published data and of mutant phenotypes across the tissues. Phenotypes observed for the majority of mutants were highly significantly correlated in the two tissues. However, 32 genes were identified in which transposon insertions consistently resulted in differential fitness in the ileal wall and MLNs, suggesting niche-specific roles for these genes in pathogenesis. Defined null mutations affecting ptsN and spvC were confirmed to result in tissue-specific phenotypes in calves, thus validating the TraDIS dataset. Conclusions This validation of the role of thousands of Salmonella genes and identification of genes with niche-specific roles in a key target species will inform the design of control strategies for bovine salmonellosis and zoonotic infections, for which efficacious and cross-protective vaccines are currently lacking. Electronic supplementary material The online version of this article (10.1186/s12864-018-5319-0) contains supplementary material, which is available to authorized users.

Objectives: This study was designed to evaluate the dose-dependent toxic effects of imidacloprid on the female ratsthat were treated through three generations (F0, F1, and F2). F2 female rats were sacrificed at the end of the experiment to see the long-term effect of imidacloprid. Materials and Methods: Rats were divided into three groups of 6 each. Group I served as control. Group II served as treated I and given 1/45th LD50 (10 mg/kg/day) of imidacloprid. Group III served as treated II and given 1/22th LD50 (20 mg/kg/day) of imidacloprid. After 60 days, oral administration of imidacloprid females were mated with normal males to get F1 and F2 generation. F2 generation female rats were sacrificed at the end of the experiment. Biochemical and a histopathological investigation was done for three groups of F2 generation and statistically analyzed by ANOVA. Results: Average feed intake of F2 female rats was significantly reduced (P < 0.01) at 20 mg/kg/day dose of imidacloprid. There was a significant increase in the activity of alanine aminotransferase, AKP, and glucose 6-phosphate dehydrogenase in Group III rats of F2 generation. There was a significant decrease in acetylcholine esterase activity in plasma and brain of both the imidacloprid treated groups. Tissue samples of liver, kidney, and brain of females of F2 generation showed histopathological condition. Conclusion: The results indicated that imidacloprid at a dose of 20 mg/kg bw/day exerts significant toxicological effects on biochemical and histological studies of F2 generation females as compare to 10 mg/kg bw/day.

The Millennium Declaration committed the 193 member states of the United Nations to end poverty by 2015. Despite the efforts of the UN and World Health Organisation, and the G8 commitment to spend a fixed proportion of gross national income on overseas aid, more than 2.6 billion people still lack access to proper sanitation. The absence of effective public health strategies in developing countries results in significant health burdens following gastrointestinal infections. Diarrhoea associated with infections resulting from oral-faecal contamination is the second leading cause of death in children under 5 years of age, primarily in Africa and South Asia. Currently there are no appropriate vaccines that could be easily administered on a global scale to prevent these infections. Synthetic biology has the potential to contribute to development of such vaccines. Our work is directed at developing a range of multivalent oral vaccines against the most common diarrhoea-causing bacteria, e.g., Escherichia coli, Shigella and Salmonella. If synthetic biology is to avoid the suspicion and possible revulsion of the public, scientists need to demonstrate that this new field has something real to offer.

Strains and plasmids used in this study; Primers used in this study for reverse transcriptase analysis of RNA extracts from A. pleuropneumoniae HS143, complementation of ngt and agt mutants and construction of A. pleuropneumoniae deletion mutants; Expression analyses of ngt and sodC. Reverse transcriptase PCR results of RNA extracted from A. pleuropneumoniae serotype 15 at various time points and in various mutants. 1, 1.5 hr cDNA template; 2, 1.5 hr RNA template; 3, 3.0 hr cDNA template; 4, 3.0 hr RNA; 5, 5.0 hr cDNA template; 6, 5.0 hr RNA template; 7, Δngt overnight culture cDNA template; 8, Δngt overnight culture RNA template; 9, Δngt complemented with pMKExpressNGT overnight culture cDNA template; 10, Δngt complemented with pMKExpressNGT overnight culture RNA template; 11, genomic DNA template (positive PCR control).; Predicted transcriptional promoter and Rho-independent terminators identified by bioinformatics analysis. V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p. 61-78

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins that it produces (TcdA and TcdB) are responsible for the characteristic pathology of C. difficile infection (CDI), while its spores persist in the environment, causing its widespread transmission. Many different strains of C. difficile exist worldwide and the epidemiology of the strains is ever-changing: in Scotland, PCR ribotype 012 was once prevalent, but currently ribotypes 106, 001 and 027 are endemic. This study aimed to identify the differences among these ribotypes with respect to their growth, and toxin and spore production in vitro. It was observed that the hypervirulent ribotype 027 produces significantly more toxin than the other ribotypes in the exponential and stationary phases of growth. Further, the endemic strains produce significantly more toxins and spores than ribotype 012. Of note was the observation that tcdC expression did not decrease into the stationary phase of growth, implying that it may have a modulatory rather than repressive effect on toxin production. Further, the increased expression of tcdE in ribotype 027 suggests its importance in the release of the toxins. It can thus be concluded that several genotypic and phenotypic traits might synergistically contribute to the hypervirulence of ribotype 027. These observations might suggest a changing trend towards increased virulence in the strains currently responsible for CDI.

In present study the effect of oral administration of imidacloprid over two generations on the ovary of female Wistar rats was assessed. Female rats were divided into 3 groups. Group 1, as control, was given corn oil only while imidacloprid was administered in group 2 and 3 @ 10 and 20 mg kg−1 bw day1, respectively. A disruption in estrous cyclicity was observed with a prolonged diestrous in both F1 and F2 generation. The weight of ovary decreased significantly in treated female rats of F2 generation. Histopathology of ovary of groups 2 and 3 revealed different stages of follicles. Percentage atresia of follicles was found higher in treated rat ovaries as compared to their respective control rats in both the generations. The results indicate that no observed effect level of imidacloprid for two generations female reproductive toxicity is 10 mg kg−1 bw day−1.

Imidacloprid was given orally to female albino rats for 60 days. Rats were divided into three groups. The control animals (first group) were given corn oil. Second group treated with 1/45th LD50 of imidacloprid and third group with 1/22th LD50 of imidacloprid to assess the alterations in the level of some biochemical prameters in tissues. Liver alanine aminotransferase (ALT), aspartate aminotransferase (AST) increased significantly at high dose of imidacloprid. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activity increased significantly at 1/22th LD50 of imidacloprid in liver and ovary. Significant decrease was observed in plasma 3βhydroxysteroid dehydrogenase (3β HSDH) activity at 1/22th LD50 of imidacloprid. Histological examination of ovary of 1/22th LD50 of imidacloprid showed atretic follicles. Imidacloprid at 1/22 th LD50 was found to be toxic to rats. Introduction Imidacloprid, a relatively new, systemic insecticide related to nicotine, was introduced to the market in 1991 as the first chloronicotinyl insecticide and has become largest selling insecticide worldwide (Menscke, 2002). In agriculture, it is most commonly used on rice, cereal, maize, potatoes, vegetables, sugar beets, fruits, cotton and hops for control of sucking insects, coleopteran and others (Cox, 2001). Imidacloprid and its analogs are remarkably potent neurotoxic insecticides, which act as nicotinic acetylcholine receptor (nAChRs) agonists (Matsuda, 2005). nAChRs play a central role in rapid cholinergic synaptic transmission and are important targets of insecticides (Sattelle, 1990). It is moderately toxic and its acute oral LD50 is 450 mg/kg for rats and 150 mg/kg for mice (Tomlin, 1997). Recently imidacloprid has raised concern because of reports of egg shell thinning; reduced egg production and hatching time which are considered as signs of possible endocrine disruption (Berny et al., 1999 and Matsuda et al., 2001). Aminotransferases and phosphatases are important and critical enzymes in the liver metabolic activity and are responsible for detoxificiation processes. So any interference in various enzyme levels lead to biochemical impairment and lesions of the tissue. The liver is the principal target of imidacloprid toxicity, as demonstrated by its elevated serum transaminase, alkaline phosphatase and/or glutamate dehydrogenase activities; and alterations of other clinical parameters(Rahman et al.,2000). Many pesticides having endocrine disruptors properties are known to adversely impair reproductive competence of male and females (Yousef, 2010). Although it is widely used worldwide, there is still less work done related to its toxicity in female rats. Therefore, the present study has been designed to evaluate the effect of imidacloprid on transaminases and phosphatases in female albino rats. Materials and methods Commercial product of imidacloprid (Confidor, 17.8%, SL) used in this study was purchased from the local market in Ludhiana, India. The study was conducted on sexually mature female Wistar albino rats, 3 months of age, weighing 100– 150 g obtained from Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana. The animals were housed in groups of two rats per cage. The rats were acclimatized for one week before using them for experimentation. The rats were maintained under controlled conditions of temperature (22 ± 20C) and humidity (30–70%) with 12 h light and dark cycle. The animals were given standard diet containing pelleted food and water ad libitum. The experimental protocol met the National guidelines on the proper care and use of animals in the laboratory research. The Institutional Animal Ethics Committee (IAEC) approved this experimental protocol. Adult females were divided into three groups. Group 1 served as Control and given corn oil through oral intubation. Group 2 served as Treated 1 given 1/45th LD50 of imidacloprid. Group 3 served as Treated 2 given 1/22th LD50 of imidacloprid for 60 days. Biochemical Measurements After completion of 60 days rats were sacrificed, blood samples were drawn into heparinized tubes and plasma was separated by centrifuging at 3000 rpm for 10 minutes at room temperature. The tissue sample of ovary was homogenized in the phosphate buffer saline. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) were estimated by the method of Reitman and Frankel as described by Bergmeyer (1974) and acid phosphatase (ACP), alkaline phosphatase (ALP) was estimated by method of Bessey et al (1946). The enzyme 3 β-hydroxy steroid dehydrogenase was estimated in ovary and plasma by the method of Angular et al 1992. Lactate dehydrogenase (LDH) was estimated by method of King (1965) and urea by method of Hawk (1968) and total proteins by Lowry et al (1951). Representative samples from the ovaries were collected in 10% neutral formalin. After washing in running water and dehydration in alcohol, tissues were embedded and 5 μm paraffin sections cut and stained with haematoxylin and eosin as per the method of Luna (1968). Biochemical analyses were presented as the mean ± standard error of means (S.E.M). Comparisons were made between control and treated groups using ‘‘Analysis of Variance (ANOVA)’’ as a statgraphics statistical package. Results and Discussion Daily oral administration of imidacloprid at 1/45th LD50 imidacloprid and 1/22th LD50 of imidacloprid produced a significant rise in the levels of ALT, AST, ALP and ACP in liver and ovary tissue. Table 1 shows the relative organ weight data of female rats orally administered imidacloprid. There was nonsignificant decrease in weight of oviduct. Table 2 shows that there was significant increase in ALT and AST activity in the liver. Bhardwaj et al (2010) also observed that oral adminis

Carbon tetrachloride (CCl4) intoxication in rodents is a commonly used model of both acute and chronic liver injury. Carbon tetrachloride induced hepatic injury as measured by alterations in serum enzymes and histopathological changes were investigated in Swiss Albino mice (Musmusculus). Mice were divided into two groups: control group and carbon tetrachloride administered group. A single dose of CCl4 (0.8 ml/kg body weight, sc) was given and induced hepatotoxicity was investigated, when sacrificed at 1, 3, 7 days of treatment and 1, 3 and 6 months post treat - ment. The hepatotoxicity was manifested biochemically as significant elevation of the enzyme activities of serum alanine aminotransferase (ALT, EC: 2.6.1.2), aspartate aminotransferase (AST, EC: 2.6.1.1), alkaline phosphatase (ALP, EC: 3.1.3.1) and bilirubin after 1, 3 and 7 days post treatment. While after 1, 3 and 6 months, levels of serum enzymes and bilirubin were reached to their normal or near normal values. The toxicity was further evidenced by histopathological changes as hydropic degeneration, congestion of sinusoids, vacuolation of cytoplasm and centrilobular necrosis in the liver of mice. It is concluded that CCl4 is a potent hepatotoxin with a dose of 0.8ml/kg body weight.

Imidacloprid, a neonicotinoid the newest class of major insecticide has outstanding potency and systemic action for crop protection against piercing and sucking insects pests and also highly effective for control of flea on cats and dogs. The effect of oral administration of two doses of imidacloprid 10 and 20 mg/kg/day for 60 days on biochemical parameters, histopathology and protein profile of female albino rat was assessed. Average feed intake was significantly reduced (P < 0.01) at 20 mg/kg/day. Relative weight of heart and spleen decreased significantly (P < 0.05) at higher dose level. Non significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP), alkaline phosphatase (AKP) activity was observed in both the imidacloprid treated groups. There was significant decrease (P < 0.01, P < 0.05) in acetyl cholinesterase (AChE) activity in plasma and brain of both the imidacloprid treated groups. Microscopically, liver tissue of rats treated with higher dose of imidacloprid showed marked dilation and congestion of central vein and degeneration of hepatocytes. The exposure to imidacloprid produced histopathological changes that could be correlated with changes in the biochemical profile of female albino rats. The blood plasma proteins were examined by SDS PAGE. There was no diagnostic difference in the pattern of plasma protein profile of control and treated rats. Based on the present physiological, biochemical and histological studies it is evident that imidacloprid did not produce any significant effects at 10 mg/kg/day dose but induced toxicological effects at 20 mg/kg/day to female rats.

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses, replicating vectors have generally been used but the use of such vectors in humans may be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide) (PLG) microparticles as a delivery system for a CTL epitope representing residues 51–59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freund's Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins produced by C. difficile are responsible for the characteristic pathology observed in C. difficile disease, but several surface-associated proteins of C. difficile are also recognized by the immune system and could modulate the immune response in infection. The aim of this study was to assess the induction of cytokines in a macrophage cell line in response to different antigens prepared from five C. difficile strains: the hypervirulent ribotype 027, ribotypes 001 and 106 and reference strains VPI 10463 and 630 (ribotype 012). PMA-activated THP-1 cells were challenged with surface-layer proteins, flagella, heat-shock proteins induced at 42 and 60 °C and culture supernatants of the five C. difficile strains. The production of the pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8 and IL-12p70 was observed in response to the surface-associated proteins, and high levels of TNF-α, IL-1β and IL-8 were detected in response to challenge with culture supernatants. The immune response triggered by the surface-associated proteins was independent of the strain from which the antigens were derived, suggesting that these proteins might not be related to the varying virulence of the hypervirulent ribotype 027 or ribotypes 001 and 106. There was no interstrain difference observed in response to the culture supernatants of the tested C. difficile strains, but this was perhaps due to toxicity induced in the macrophages by large amounts of toxin A and toxin B.

Clostridium difficile is a common nosocomial pathogen transmitted mainly via its spores. These spores can remain viable on contaminated surfaces for several months and are resistant to most commonly used cleaning agents. Thus, effective decontamination of the environment is essential in preventing the transmission of C. difficile in health-care establishments. However, this emphasis on decontamination must also be extended to laboratories due to risk of exposure of staff to potentially virulent strains. Though few cases of laboratory-acquired infection have been reported, the threat of infection by C. difficile in the laboratory is real. Our aim was to test the efficacy of four disinfectants, Actichlor, MicroSol 3+, TriGene Advance and Virkon, and one laboratory decontaminant, Decon 90, against vegetative cells and spores of C. difficile. Five strains were selected for the study: the three most commonly encountered epidemic strains in Scotland, PCR ribotypes 106, 001 and 027, and control strains 630 and VPI 10463. MICs were determined by agar dilution and broth microdilution. All the agents tested inhibited the growth of vegetative cells of the selected strains at concentrations below the recommended working concentrations. Additionally, their effect on spores was determined by exposing the spores of these strains to different concentrations of the agents for different periods of time. For some of the agents, an exposure of 10 min was required for sporicidal activity. Further, only Actichlor was able to bring about a 3 log(10) reduction in spore numbers under clean and dirty conditions. It was also the only agent that decontaminated different hard, non-porous surfaces artificially contaminated with C. difficile spores. However, this too required an exposure time of more than 2 min and up to 10 min. In conclusion, only the chlorine-releasing agent Actichlor was found to be suitable for the elimination of C. difficile spores from the environment, making it the agent of choice for the decontamination of laboratory surfaces.

A synthetic peptide representing a measles virus (MV) cytotoxic T cell epitope (CTL) when encapsulated in poly (D,L-lactide co-glycolide) (PLG) 50:50 microparticles induced a strong CTL response after a single intraperitoneal immunization of mice which was greater than that following administration of the peptide in Freund's complete adjuvant. A 100 micrograms dose of encapsulated peptide was shown to be more effective for CTL priming than 50 and 25 micrograms doses. A vaccine formulation prepared by simply mixing empty 50:50 PLG microparticles with the peptide resulted in the induction of CTL responses comparable to those induced by the encapsulated peptide. Moreover, a CTL response against MV-infected target cells was observed. These findings highlight the potential immunostimulatory effect of PLG microparticles for the induction of MV and peptide-specific CTL responses.

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

In this study, the potential of the amino-terminal sequence from the F 1 polypeptide responsible for the fusion of measles virus (MV) with cell membranes as a carrier system for a CTL epitope from the MV nucleoprotein was examined. The addition of the fusion sequence (FP) at either the amino or the carboxyl terminus of the CTL epitope peptide rendered it immunogenic after intraperitoneal immunization in mice. The CTLs induced were able to lyse target cells pulsed with the peptide or persistently infected with MV. After intranasal administration of a FP–CTL chimera with or without cholera toxin B subunit (CTB) as an adjuvant, CTL responses to the peptide pulsed and to MV-infected target cells were detected. Responses in groups of mice where CTB was used as an adjuvant were stronger. However, intranasal administration of the CTL epitope did not induce a protective response against intracranial challenge with a neuroadapted strain of MV. These findings highlight the potential of fusion sequences as a carrier system for CTL epitopes and the potential of the intranasal route for administration of synthetic peptides representing MV sequences.

A systemic antibody response was induced when a chimeric peptide containing two copies of a promiscuous T-cell epitope and one copy of a B-cell epitope (TTB) from the fusion protein of measles virus (MV) was administered to mice intranasally without adjuvant. A higher antibody titre was produced when the peptide was administered intranasally with cholera toxin B subunit (CTB) as an adjuvant and these antibodies crossreacted with the MV. Furthermore, splenocytes from intranasally immunized mice proliferated in vitro in the presence of the TTB peptide. The immune response following intranasal immunization with the peptide was influenced by the MHC haplotype of the strain of mice used. Thus CBA and BALB/c mice were high responders whereas C57BL/6 mice were low responders. Although peptide administered intranasally with CTB to CBA mice induced an immune response, no significant protection was observed against intra-cranial challenge with canine distemper virus which is antigenically related to MV.

Strains and plasmids used in this study; Primers used in this study for reverse transcriptase analysis of RNA extracts from A. pleuropneumoniae HS143, complementation of ngt and agt mutants and construction of A. pleuropneumoniae deletion mutants; Expression analyses of ngt and sodC. Reverse transcriptase PCR results of RNA extracted from A. pleuropneumoniae serotype 15 at various time points and in various mutants. 1, 1.5 hr cDNA template; 2, 1.5 hr RNA template; 3, 3.0 hr cDNA template; 4, 3.0 hr RNA; 5, 5.0 hr cDNA template; 6, 5.0 hr RNA template; 7, Δngt overnight culture cDNA template; 8, Δngt overnight culture RNA template; 9, Δngt complemented with pMKExpressNGT overnight culture cDNA template; 10, Δngt complemented with pMKExpressNGT overnight culture RNA template; 11, genomic DNA template (positive PCR control).; Predicted transcriptional promoter and Rho-independent terminators identified by bioinformatics analysis. V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p. 61-78