Liver proteomics of gilthead sea bream (Sparus aurata) exposed to cold stress.

The gilthead sea bream (Sparus aurata, L.) is very sensitive to low temperatures, which induce fasting and reduced growth performances. There is a strong interest in understanding the impact of cold on fish metabolism to foster the development and optimization of specific aquaculture practices for the winter period. In this study, an 8 week feeding trial was carried out on gilthead sea bream juveniles reared in a Recirculated Aquaculture System (RAS) by applying a temperature ramp in two phases of four weeks each: a cooling phase from 18 °C to 11 °C and a cold maintenance phase at 11 °C. Liver protein profiles were evaluated with a shotgun proteomics workflow based on filter-aided sample preparation (FASP) and liquid chromatography-mass spectrometry (LC-ESI-Q-TOF MS/MS) followed by label-free differential analysis. Along the whole trial, sea breams underwent several changes in liver protein abundance. These occurred mostly during the cooling phase when catabolic processes were mainly observed, including protein and lipid degradation, together with a reduction in protein synthesis and amino acid metabolism. A decrease in protein mediators of oxidative stress protection was also seen. Liver protein profiles changed less during cold maintenance, but pathways such as the methionine cycle and sugar metabolism were significantly affected. These results provide novel insights on the dynamics and extent of the metabolic shift occurring in sea bream liver with decreasing water temperature, supporting future studies on temperature-adapted feed formulations. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011059.

Relationship between milk cathelicidin abundance and microbiologic culture in clinical mastitis.

The availability of reliable tools to enable the sensitive and specific detection of mastitis in dairy cows can assist in developing control strategies and promote the more rational use of antibiotics. We have developed a milk cathelicidin ELISA that shows high sensitivity and specificity for dairy cow mastitis, based on latent class analysis. In this study, we investigated the effect of microbial agents on cathelicidin abundance in the milk of cows with clinical mastitis. We subjected 535 quarter milk samples (435 from quarters showing signs of clinical mastitis and 100 from healthy quarters as a control) to milk cathelicidin ELISA, somatic cell count (SCC), and microbiologic culture. Of the 435 clinical mastitis samples, 431 (99.08%) were positive for cathelicidin, 424 (97.47%) had SCC >200,000 cells/mL, and 376 (86.44%) were culture-positive. Of the 59 culture-negative samples, 58 (98.30%) were positive for cathelicidin and 55 (93.22%) had SCC >200,000 cells/mL. The abundance of cathelicidin and the extent of SCC increase depended on the causative agent: Streptococcus agalactiae and coagulase-negative staphylococci showed the highest and lowest changes, respectively. We also observed differences in behavior between the 2 markers depending on the pathogen: Streptococcus agalactiae induced the highest cathelicidin abundance, and Serratia spp. induced the highest SCC. Nevertheless, the different ability of microorganisms to induce cathelicidin release in milk did not compromise its value as a mastitis marker, given its higher sensitivity compared to SCC or microbiologic culture. All 100 negative control samples (collected from healthy quarters with SCC <100,000 cells/mL and culture-negative) were also negative for cathelicidin, corresponding to 100% specificity in the evaluated sample cohort. This study confirmed the value of the milk cathelicidin ELISA for detecting bovine mastitis, and highlighted the influence of mastitis-causing microorganisms on cathelicidin abundance. This influence did not compromise diagnostic performance; instead, it may have better reflected disease severity and evolution than SCC.

Evaluation of milk cathelicidin for detection of bovine mastitis.

Mastitis due to intramammary infection is one of the most economically relevant diseases in dairy cows, causing reductions in milk quality and quantity. Currently, mastitis monitoring is based on somatic cell count (SCC) and bacteriologic culture (BC) of milk. Nevertheless, inflammation-specific protein markers might provide more sensitive and reliable assays, enabling immunoassay-based screening strategies. Cathelicidin is an inflammatory protein released in milk that has recently demonstrated fair reliability and diagnostic potential for ewe mastitis. To assess its performance in cows, 531 quarter milk samples from 2 herds were tested using cathelicidin ELISA, SCC, and BC. We found that 29.0% of samples were positive for cathelicidin, 18.8% had SCC >200,000 cells/mL, and 13.7% were BC-positive. Cathelicidin showed a strong positive correlation with SCC as demonstrated by receiver operating characteristics curve analysis and by the clustering of cathelicidin-negative and cathelicidin-positive samples in association with low and high SCC values, respectively. For evaluating the diagnostic performance of a novel test, BC cannot be considered a reliable gold standard for true disease status because of its known limitations. Therefore, we assessed the sensitivity (Se) and specificity (Sp) of the milk cathelicidin ELISA using a latent class analysis approach together with BC and SCC by considering different diagnostic thresholds to identify the preferred Se/Sp combination. We modeled conditional dependence of cathelicidin and SCC to account for their close association. The cathelicidin ELISA showed higher Se than SCC and BC for almost all threshold combinations. In fact, at the best-performing threshold combination, the Se of cathelicidin was 80.6%, 6.2 percentage points higher than that of SCC >200,000 cells/mL (74.4%) and similar to that of SCC >100,000 cells/mL (80.2%). Most importantly, this Se was obtained with a loss in Sp of only 1.4 percentage points compared with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity.

Evaluation of milk cathelicidin for detection of dairy sheep mastitis.

Mastitis due to intramammary infections is one of the most detrimental diseases in dairy sheep farming, representing a major cause of reduced milk productions and quality losses. In particular, subclinical mastitis presents significant detection and control problems, and the availability of tools enabling its timely, sensitive, and specific detection is therefore crucial. We have previously demonstrated that cathelicidins, small proteins implicated in the innate immune defense of the host, are specifically released in milk of mastitic animals by both epithelial cells and neutrophils. Here, we describe the development of an ELISA for milk cathelicidin and assess its value against somatic cell counts (SCC) and bacteriological culture for detection of ewe mastitis. Evaluation of the cathelicidin ELISA was carried out on 705 half-udder milk samples from 3 sheep flocks enrolled in a project for improvement of mammary health. Cathelicidin was detected in 35.3% of milk samples (249/705), and its amount increased with rising SCC values. The cathelicidin-negative (n=456) and cathelicidin-positive (n=249) sample groups showed a clear separation in relation to SCC, with median values of 149,500 and 3,300,000 cells/mL, respectively. Upon bacteriological culture, 20.6% (145/705) of the milk samples showed microbial growth, with coagulase-negative staphylococci being by far the most frequent finding. A significant proportion of all bacteriologically positive milk samples were positive for cathelicidin (110/145, 75.9%). Given the lack of a reliable gold standard for defining the true disease status, sensitivity (Se) and specificity (Sp) of the cathelicidin ELISA were assessed by latent class analysis against 2 SCC thresholds and against bacteriological culture results. At an SCC threshold of 500,000 cells/mL, Se and Sp were 92.3 and 92.3% for cathelicidin ELISA, 89.0 and 94.9% for SCC, and 39.4 and 93.6% for bacteriological culture, respectively. At an SCC threshold of 1,000,000 cells/mL, Se and Sp were 93.3 and 91.9% for cathelicidin ELISA, 80.0 and 97.1% for SCC, and 39.4 and 93.5% for bacteriology, respectively. In view of the results obtained in this study, the measurement of cathelicidin in milk by ELISA can provide added Se while maintaining a high Sp and may therefore improve detection of subclinical mastitis.

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

A new magnetic resonance imaging approach for discriminating Sardinian sheep milk cheese made from heat-treated or raw milk.

The evaluation of milk heat treatment on dairy products via reliable analytical methods is a challenging issue that involves both industrial and fundamental research. We describe a new magnetic resonance imaging (MRI) protocol for discriminating Sardinian sheep milk cheese originating from heat-treated or raw milk. Thirty-six samples (18 pecorino cheeses manufactured from heat-treated milk and 18 Fiore Sardo cheeses made from raw milk) were investigated by means of MRI and bi-exponential signal decay analysis. The protocol is capable of discerning cheeses by virtue of the different distribution of the transversal (T2) relaxation time constant. Cheeses from heat-treated milk showed a significantly higher area fraction (≈70-80%), corresponding to the fast relaxing water protons (T2 ≈ 9 ms), compared with raw milk cheeses, whereas the opposite was observed for the long T2 (T2 ≈ 35 ms) proton population. The MRI protocol described is rapid and nondestructive, and it provides statistically significant discrimination between ewe milk cheeses made from heat-treated and raw milk.

Liver proteome dataset of Sparus aurata exposed to low temperatures.

We report the proteomic dataset of livers from Sparus aurata exposed to low temperature during growth. Gilthead sea bream juveniles were reared in Recirculating Aquaculture Systems (RAS) and exposed to a temperature ramp made of two phases of four weeks each: a Cooling phase from 18 °C (t0) to 11 °C (t1) and a Cold Maintenance phase at 11 °C (t1-t2) in a 8 week feeding trial. At the end of the experiment, sea bream livers were collected and analyzed with a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry, peptide identification carried out using Sequest-HT as search engine within the Proteome Discoverer informatic platform, and label-free differential analysis. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011059 (Vizcaíno et al., 2016; Deutsch et al., 2017; Perez-Riverol et al., 2016). The dataset described here is also related to the research article entitled "Liver proteomics of gilthead sea bream (Sparus aurata) exposed to cold stress" (Ghisaura et al., 2019).

Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model.

The intestinal epithelium represents the major barrier to absorption of orally administered drugs and peptides into the systemic circulation. Entry of molecules through the paracellular pathway is restricted by tight junctions. We have previously reported that these structures can be modulated by Zonula occludens toxin (Zot). In the present report, we show that Zot reversibly increases rabbit intestinal permeability to insulin by 72% (P = 0.034) and immunoglobulins by 52% (P = 0.04) in vitro. When tested in vivo, Zot induced a 10-fold increase of insulin absorption in both the rabbit jejunum and ileum, whereas no substantial changes were detected in the colon. Similar results were obtained with immunoglobulins, whereby Zot induced twofold and sixfold increases of IgG absorption in the jejunum and ileum, respectively. In diabetic rats, bioavailability of oral insulin coadministered with Zot was sufficient to lower serum glucose concentrations to levels comparable to those obtained after parenteral injection of the hormone. The survival time of diabetic animals chronically treated with oral insulin + Zot was comparable to that observed in parenterally treated rats. These studies offer an innovative strategy for the oral delivery of drugs and proteins normally not absorbed through the intestine.

Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function.

Proteomic changes in the ileum of sheep infected with Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, ß-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.

The bovine milk microbiota: insights and perspectives from -omics studies.

Recent significant progress in culture-independent techniques, together with the parallel development of -omics technologies and data analysis capabilities, have led to a new perception of the milk microbiota as a complex microbial community with great diversity and multifaceted biological roles, living in an environment that was until recently believed to be sterile. In this review, we summarize and discuss the latest findings on the milk microbiota in dairy cows, with a focus on the role it plays in bovine physiology and health. Following an introduction on microbial communities and the importance of their study, we present an overview of the -omics methods currently available for their characterization, and outline the potential offered by a systems biology approach encompassing metatranscriptomics, metaproteomics, and metametabolomics. Then, we review the recent discoveries on the dairy cow milk microbiome enabled by the application of -omics approaches. Learning from studies in humans and in the mouse model, and after a description of the endogenous route hypothesis, we discuss the role of the milk microbiota in the physiology and health of both the mother and the offspring, and report how it can be changed by farming practices and during infection. In conclusion, we shortly outline the impact of the milk microbiota on the quality of milk and of dairy products.

Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines.

In the present study, we report the preliminary characterization of the epithelial cell receptor for Vibrio cholerae zonula occludens toxin (Zot). Zot receptor was purified by ligand-affinity chromatography. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis revealed a protein of ca. 66 kDa. Partial N-terminal sequence obtained from purified murine and human Zot receptor revealed homology between the two proteins and with human alpha-1-chimaerin. Zot protein domain(s) involved in receptor binding were also analyzed by constructing several in frame deletion derivatives of a recombinant fusion Zot protein tagged with maltose binding protein. Our results suggest that Zot binding to its cellular membrane receptor requires a sequence that spans between amino acids 118 and 299.

Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice.

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv(-) derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.

Sequence, cloning, expression and characterisation of the 81-kDa surface membrane protein (P80) of Mycoplasma agalactiae.

Mycoplasma agalactiae, the causative agent of contagious agalactia in small ruminants, produces a protein, named P80, that is detectable in all wild-type isolates examined to date and that appears expressed during the early phase of infection. We describe here the identification, cloning and expression of the gene encoding P80 (ma-mp81). The deduced amino acid sequence is consistent with a hydrophobic and basic protein that possesses a lipoprotein signal peptide. Sequence analysis of gene ma-mp81 suggests that P80 is a membrane lipoprotein that shows significant homology with other putative lipoproteins of M. pneumoniae. An internal 1-kb fragment of ma-mp81 was expressed in Escherichia coli as a 6xHis-tagged protein. The purified recombinant protein greatly reacted with polyclonal anti-P80 sera raised in lamb.

Tracking of clinical and environmental Vibrio cholerae O1 strains by combined analysis of the presence of toxin cassette, plasmid content and ERIC PCR.

Clinical and environmental Vibrio cholerae O1 strains associated with the cholera epidemic in the Luanda province of Angola from 1991 to 1994 were tracked by toxin distribution, plasmid content and chromosomal polymorphism of the enterobacterial repetitive intergenic consensus (ERIC) sequences by PCR fingerprinting. To follow the distribution of ace, zot and ctxA toxin genes, 6 specific PCR tests were applied to 100 Vibrio strains, after preliminary hybridization experiments. Clinical isolates of Vibrio cholerae O1 were characterized by high stability of the toxigenic cassette and the presence of a large conjugative multi-resistant plasmid of incompatibility class C. Such characteristics were present in all isolates during the four years of the epidemic. Environmental strains, isolated from the river supplying water to the Luanda population showed three different genetic profiles: the presence of both cassette and plasmid, the presence of cassette only or absence of both. To assess the clonal relationship between the clinical isolates and the three groups of environmental strains, the strains were analyzed by PCR ERIC polymorphism. This analysis, supported by the toxin and plasmid content, suggested the stability of the epidemic strain in clinical cases during the epidemic and led to the finding that there was a strict genetic relationship of the epidemic strain with the environmental ones as characterized by the presence of the toxin cassette. The role of the water supply from Bengo River as a reservoir of the Vibrio epidemic strain is discussed.

Draft Genome Sequence of the Host-Restricted Salmonella enterica Serovar Abortusovis Strain SS44.

Salmonella enterica serovar Abortusovis is a pathogen strictly adapted to ovines, in which it causes abortion. To enhance our understanding of this pathogen, we assembled the first draft sequence of an S. Abortusovis genome (strain SS44). The obtained genomic data might facilitate the study of S. enterica evolution and host adaptation.

Proteomic characterization of hepatitis C eradication: enzyme switch in the healing liver.

Lipid pathway impairment, decrease in the antioxidant pool and downregulation in amino-acid metabolism are just some of the metabolic variations attributed to chronic HCV infection. All of them have been studied separately, mainly in animal models. Thanks to proteomic analysis we managed to describe (for the fist time to the best of our knowledge), in vivo and in humans, the metabolic alterations caused by HCV, and the recovery of the same alterations during HCV treatment. We performed proteomic analysis on liver specimens of a 28-year-old woman affected by hepatitis C genotype 1a, alcoholism and diabetes mellitus type 1, before and after antiviral treatment with pegylated interferon alpha 2b and ribavirin. The subject, thanks to a patient-tailored therapy, reached Sustained Virological Response. Throughout the treatment period the patient was monitored with subsequent biochemical, clinical and psychological examinations. The data obtained by the patient's close monitoring suggest a direct interaction between insulin resistance and an active HCV genotype 1 infection, with a leading role played by the infection, and not by insulin resistance, as demonstrated by the sharp fall of the insulin units needed per day during treatment. The proteomic analysis showed that after therapy, a downregulation of enzymes involved in amino acid metabolism, glycolysis/gluconeogenesis and alcohol catabolism takes place, the latter probably due to cessation of alcohol abuse. On the contrary, the metabolic pathways linked to metabolism of the reactive oxygen species were upregulated after therapy. Finally, a significant alteration in the pathway regulated by peroxisome proliferator-activated receptor alpha (PPARA), a major regulator of lipid metabolism in the liver, was reported. These "real time" data confirm in vivo, in humans, that during HCV infection, the pathways related to fatty acids, glucose metabolism and free radical scavenging are inhibited. The same enzyme deficit is completely recovered after HCV eradication.

LGI1 microdeletion in autosomal dominant lateral temporal epilepsy.

OBJECTIVES: To characterize clinically and genetically a family with autosomal dominant lateral temporal epilepsy (ADLTE) negative to LGI1 exon sequencing test. METHODS: All participants were personally interviewed and underwent neurologic examination. Most affected subjects underwent EEG and neuroradiologic examinations (CT/MRI). Available family members were genotyped with the HumanOmni1-Quad v1.0 single nucleotide polymorphism (SNP) array beadchip and copy number variations (CNVs) were analyzed in each subject. LGI1 gene dosage was performed by real-time quantitative PCR (qPCR). RESULTS: The family had 8 affected members (2 deceased) over 3 generations. All of them showed GTC seizures, with focal onset in 6 and unknown onset in 2. Four patients had focal seizures with auditory features. EEG showed only minor sharp abnormalities in 3 patients and MRI was unremarkable in all the patients examined. Three family members presented major depression and anxiety symptoms. Routine LGI1 exon sequencing revealed no point mutation. High-density SNP array CNV analysis identified a genomic microdeletion about 81 kb in size encompassing the first 4 exons of LGI1 in all available affected members and in 2 nonaffected carriers, which was confirmed by qPCR analysis. CONCLUSIONS: This is the first microdeletion affecting LGI1 identified in ADLTE. Families with ADLTE in which no point mutations are revealed by direct exon sequencing should be screened for possible genomic deletion mutations by CNV analysis or other appropriate methods. Overall, CNV analysis of multiplex families may be useful for identifying microdeletions in novel disease genes.

SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium are synthesized at late stages of infection in mice.

Salmonella pathogenicity island (SPI)-1 is essential for invasion of non-phagocytic cells, whereas SPI-2 is required for intracellular survival and proliferation in phagocytes. Some SPI-1 effectors, however, are induced upon invasion of both phagocytic and non-phagocytic cells, suggesting that they may also be required post-invasion. In the present work, the presence was analysed of SipA, SopA, SopB, SopD and SopE2 effector proteins of Salmonella enterica serovar Typhimurium in vitro and in vivo during murine salmonellosis. Tagged (3xFLAG) strains of S. enterica serovar Typhimurium were inoculated intraperitoneally or intragastrically to BALB/c mice and recovered from the spleen and mesenteric lymph nodes of moribund mice. Tagged proteins were detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. In vitro experiments showed that SPI-1 effector proteins SipA, SopA, SopB, SopD and SopE2 were secreted under SPI-1 conditions. Interestingly, it was found that S. enterica serovar Typhimurium continued to synthesize SipA, SopB, SopD and SopE2 in colonized organs for several days, regardless of the route of inoculation. Together, these results indicate that SPI-1 effector proteins may participate in the late stages of Salmonella infection in mice.

Characterization of virulence factors in the newly described Salmonella enterica serotype Keurmassar emerging in Senegal (sub-Saharan Africa).

From 2000 to 2001, nine strains of Salmonella enterica belonging to the new serotype Keurmassar have been isolated from human and poultry samples at the Senegalese National Salmonella and Shigella Reference Laboratory at the Pasteur Institute, in Dakar. All strains carried virulence factors including Salmonella Pathogenicity Islands (SPI)-1, -2, -3 and -5 encoded genes. Strains did not harbour virulence plasmid. Ribotyping analysis revealed a single clone identical to Salmonella Decatur isolated in Zimbabwe. These data suggest that strains are closely related, and may have been spread clonally. In this new serotype, insertion sequence IS200 is not present.