Calf Intestine Research Articles

Expression and Localization of Aquaporin-1 Along the Intestine of Colostrum Suckling Buffalo Calves

Aquaporin-1 (AQP1), a six-transmembrane domain protein, belongs to a highly conserved group of proteins called aquaporins known to regulate permeability across cell membranes. Although the role of AQP1 has been extensively studied, its specific activity along the gastrointestinal tract in animals during early postnatal development is poorly known. This study investigates the expression of AQP1 mRNA and protein in the small and large intestine of water buffalo calves after colostrum ingestion using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and cellular localization of AQP1 by immunohistochemistry. Our results revealed AQP1 immunoreactivity and the presence of the corresponding mRNA in all the examined tracts of the intestine but with a different cellular localization. Western blotting confirmed the presence of AQP1, with a more intense band in colostrum-suckling animals. These findings offer insights into AQP1 expression in the small and large intestine, suggesting its involvement in osmoregulation in gastrointestinal physiology particularly during the first week after birth in relation to specific maturation of intestinal structures.

Diet effects on glucose absorption in the small intestine of neonatal calves: Importance of intestinal mucosal growth, lactase activity, and glucose transporters

Colostrum (C) feeding in neonatal calves improves glucose status and stimulates intestinal absorptive capacity, leading to greater glucose absorption when compared with milk-based formula feeding. In this study, diet effects on gut growth, lactase activity, and glucose transporters were investigated in several gut segments of the small intestine. Fourteen male German Holstein calves received either C of milkings 1, 3, and 5 (d 1, 2, and 3 in milk) or respective formulas (F) twice daily from d 1 to d 3 after birth. Nutrient content, and especially lactose content, of C and respective F were the same. On d 4, calves were fed C of milking 5 or respective F and calves were slaughtered 2h after feeding. Tissue samples from duodenum and proximal, mid-, and distal jejunum were taken to measure villus size and crypt depth, mucosa and brush border membrane vesicles (BBMV) were taken to determine protein content, and mRNA expression and activity of lactase and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter (GLUT2) were determined from mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and BBMV were determined, as well as immunochemically localized GLUT2 in the intestinal mucosa. Villus circumference, area, and height were greater, whereas crypt depth was smaller in C than in F. Lactase activity tended to be greater in C than in F. Protein expression of SGLT1 was greater in F than in C. Parameters of villus size, lactase activity, SGLT1 protein expression, as well as apical and basolateral GLUT2 localization in the enterocytes differed among gut segments. In conclusion, C feeding, when compared with F feeding, enhances glucose absorption in neonatal calves primarily by stimulating mucosal growth and increasing absorptive capacity in the small intestine, but not by stimulating abundance of intestinal glucose transporters.

A terbium chelate based fluorescent assay for alkaline phosphatase in biological fluid

Determination of alkaline phosphatase (ALP) is of great importance because of its crucial roles in biomedical research and clinical diagnosis. In this work, a simple and sensitive fluorescent assay of studying dephosphorylation behavior of alkaline phosphatase (ALP) was developed by employing a terbium-guanosine monophosphate (Tb-GMP) chelate as fluorescent probe and calf intestine alkaline phosphatase (CIAP) as a model system. The removal of phosphate group from GMP by the hydrolysis of CIAP leads to the decrease in the fluorescence of Tb-GMP chelate. As a fluorescent probe for CIAP, Tb-GMP chelate exhibits excellent selectivity and high sensitivity up to 0.001U/L detection limit. Compared with other methods for the detection of ALP including CIAP, the Tb-GMP chelate probe not only has a simpler preparation procedure, but also possesses a long enough fluorescence lifetime for time-resolved fluorescent assays. The Tb-GMP chelate probe was also successfully applied to determine the CIAP in serum samples and showed a satisfactory result. Taking into account the advantages of natural biomolecule as substrate and long emission lifetime of Tb-GMP chelate, the proposed method was expected to have potential for assessing ALP in biological system and clinical samples.

Comparison of pathogenicities and nucleotide changes between porcine and bovine reassortant rotavirus strains possessing the same genotype constellation in piglets and calves

Although reassortment is one of the most important characteristics of group A rotavirus (RVA) evolution, the host range restriction and/or virulence of reassortant RVAs remain largely unknown. The porcine 174-1 strain isolated from a diarrheic piglet was identified as a reassortant strain, harboring the same genotype constellation as the previously characterized bovine strain KJ56-1. Owing to its same genotype constellation, the pathogenicity of the porcine strain 174-1 in piglets and calves was examined for comparison with that of the bovine reassortant KJ56-1 strain, whose pathogenicity has already been demonstrated in piglets and calves. The porcine 174-1 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas KJ56-1 had been reported to be virulent only in piglets, but not in calves. Therefore, full genomic sequences of 174-1 and KJ56-1 strains were analyzed to determine whether specific mutations might be associated with clinical and pathological phenotypes. Sequence alignment between the 174-1 and KJ56-1 strains detected one nucleotide substitution at the 3′ untranslated region of the NSP3 gene and 16 amino acid substitutions at the VP7, VP4, VP1, VP3, NSP1 and NSP4 genes. These mutations may be critical molecular determinants for different virulence and/or pathogenicity of each strain. This study presents new insights into the host range restriction and/or virulence of RVAs.

Binding of Clostridium botulinum C3 exoenzyme to intact cells

C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

Cytotoxicity and enzyme inhibition studies of polyoxometalates and their chitosan nanoassemblies

Polyoxometalates (POMs) have become very significant in biomedical research for their structural diversity which renders them highly active against bacterial, viral and cancer diseases. In this study three different POMs were synthesized and nanoassemblies were made with chitosan (CTS), a natural biodegradable polymer with excellent drug carrier properties. The compounds were tested on two isoenzymes of alkaline phosphatases including tissue specific calf intestine alkaline phosphatase (CIAP) and tissue non-specific alkaline phosphatase (TNAP). Compound [TeW6O24]6− (TeW6) showed the highest activity (45.4 ± 11.3 nM) among tested compounds against TNAP. Similarly, chitosan-[TeW6O24]6− (CTS-TeW6) was proved to be a potent inhibitor of CIAP with Ki value of 22 ± 7 nM. A comparative study was made to evaluate their cytotoxic potential against HeLa cells. Among all tested compounds, Chitosan-[NaP5W30O110]14− (CTS-P5W30) has showed higher percent cytotoxicity (88 ± 10%) at 10 μM when compared with the standard anticancer drug vincristine (72 ± 7%). The study revealed that selected POMs proved excellent anticancer potential and were equally effective against alkaline phosphatase enzyme, an increased level of which may indicate cancer metastasis.

Effect of method of delivery of sodium butyrate on maturation of the small intestine in newborn calves

The effect of sodium butyrate (SB) supplementation in milk replacer (MR), starter mixture (SM), or both on small intestine maturation in newborn calves was investigated. Twenty-eight male calves with a mean age of 5 (±1) d were randomly allocated into 1 of 4 groups (7 animals per group) and fed (1) MR and SM, without SB (MR− and SM−, respectively; MR−/SM−); (2) MR− and SM supplemented with SB encapsulated within triglyceride matrix (SM+, 0.6% as fed; MR−/SM+); (3) MR supplemented with crystalline SB (MR+, 0.3% as fed) and SM− (MR+/SM−); or (4) MR+ and SM+ (MR+/SM+). The MR was offered in amounts equal to 10% of initial body weight of the calf. The SM was blended with whole corn grain (50/50; wt/wt) and offered ad libitum as a starter diet. Calves were slaughtered at 26d (±1) of age and small intestine development was investigated. Treatment with MR+ decreased villus height in the proximal jejunum and decreased villus height, crypt depth, and tunica mucosa thickness in the middle jejunum, whereas treatment with SM+ tended to increase small intestine weight and crypt depth in the proximal jejunum, and increased villus height in the distal jejunum. In the duodenum, crypt depth and tunica mucosa thickness were greater for the MR−/SM+ group compared with MR−/SM−, MR+/SM−, and MR+/SM+ groups. In the ileum, crypt depth was less for MR−/SM+ compared with MR−/SM−. Supplementation with SB in both MR and SM enhanced cell proliferation and decreased apoptosis in the middle jejunum mucosa. Regarding brush border enzyme activities, addition of SB to MR increased lactase activity in the middle jejunum and maltase activity in the distal jejunum, and tended to increase lactase activity in the distal jejunum, aminopeptidase A activity in the middle jejunum and ileum, and aminopeptidase N activity in the ileum. In contrast, SM+ increased dipeptidylpeptidase IV activity in the distal jejunum and tended to increase aminopeptidase N in the distal jejunum. In conclusion, both MR+ and SM+ affected small intestine development in newborn calves. This effect depended on the method of SB delivery but MR+ generally had a more pronounced effect. No synergistic effect of SB supplementation into MR and SM was found.

Different virulence of porcine and porcine-like bovine rotavirus strains with genetically nearly identical genomes in piglets and calves

Direct interspecies transmissions of group A rotaviruses (RVA) have been reported under natural conditions. However, the pathogenicity of RVA has never been directly compared in homologous and heterologous hosts. The bovine RVA/Cow-tc/KOR/K5/2004/G5P[7] strain, which was shown to possess a typical porcine-like genotype constellation similar to that of the G5P[7] prototype RVA/Pig-tc/USA/OSU/1977/G5P9[7] strain, was examined for its pathogenicity and compared with the porcine G5P[7] RVA/Pig-tc/KOR/K71/2006/G5P[7] strain possessing the same genotype constellation. The bovine K5 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas the porcine K71 strain caused diarrhea and histopathological changes in the small intestine of piglets, but not in calves. Furthermore, the bovine K5 strain showed extra-intestinal tropisms in both piglets and calves, whereas the porcine K71 strain had extra-intestinal tropisms in piglets, but not in calves. Therefore, we performed comparative genomic analysis of the K71 and K5 RVA strains to determine whether specific mutations could be associated with these distinct clinical and pathological phenotypes. Full-length sequencing analyses for the 11 genomic segments for K71 and K5 revealed that these strains were genetically nearly identical to each other. Two nucleotide mutations were found in the 5′ untranslated region (UTR) of NSP5 and the 3′ UTR of NSP3, and eight amino acid mutations in VP1-VP4 and NSP2. Some of these mutations may be critical molecular determinants for RVA virulence and/or pathogenicity.

Intracellular deoxyribonucleotide pool imbalance and DNA damage in cells treated with hydroxyurea, an inhibitor of ribonucleotide reductase

Imbalance in the nucleotide pool of mammalian cells has been shown to result in genotoxic damage. The goal of this study was to devise a sensitive, reproducible and simple method for detection of nucleotide pool changes in mammalian cells that could be used for problem-solving activities in drug development, e.g. mechanistic explanation of a positive response in a mammalian in vitro genotoxicity test. The method evaluated in this study is based on ethanol extraction of the total nucleotide pool, heat treatment and filtration, treatment with calf intestine alkaline phosphatase to convert nucleotides to nucleosides and analysis of the nucleosides by high-performance liquid chromatography with ultraviolet detection. The method was applied to measure the intracellular levels of deoxyribonucleotides in mouse lymphoma (ML) L5178Y cells treated with various concentrations of a model compound, hydroxyurea (HU), a ribonucleotide reductase inhibitor. DNA strand breakage and micronuclei formation were assessed in the same experiments. Imbalance of nucleotide pool (i.e. changes in the relative ratios between individual nucleotide pools) in HU-treated ML cells has been observed already at a concentration of 0.01 mmol/l, whereas genotoxic effects became apparent only at higher concentrations of HU (i.e. 0.25 mmol/l and higher) as indicated by formation of DNA strand breaks and micronuclei.

The pathology of enterotoxemia by Clostridium perfringens type C in calves

The pathology of Clostridium perfringens type C infection has been described with detail only in foals and piglets. The current report describes the diagnostic workup and detailed pathology of 3 cases of C. perfringens type C infection in calves. A 2-day-old Jersey calf and fresh and fixed tissues from a 4-week-old Angus calf and from a 1-week-old Jersey calf were received at the California Animal Health and Food Safety Laboratory System with a history of digestive disease and death. The gross changes in the gastrointestinal tract of 1 calf consisted of multifocal subserosal hemorrhages of the rumen, diffuse congestion and multifocal hemorrhages of the small intestinal mucosa, and dilated cecum with bloody liquid contents. In a second calf, a large segment of small intestine was hemorrhagic. The small intestine of the third calf was dilated and filled with abundant yellow fluid content. Microscopically, all 3 calves had diffuse coagulation necrosis of the intestinal mucosa. Clostridium perfringens type A was isolated from the intestinal content of 2 calves. In addition, enzyme-linked immunosorbent assay for Bovine rotavirus was positive on colonic content of 1 calf. Small numbers of cryptosporidia were seen in smears of colonic content of 2 calves, and Salmonella sp. group E was detected in the small intestinal content of another calf. Clostridium perfringens beta toxin was detected in the intestinal content of the 3 animals. A diagnosis of C. perfringens type C infection was confirmed based on pathological findings and detection of beta toxin in the intestinal content of the 3 animals.

ОЧИЩЕННЯ І ХАРАКТЕРИСТИКА НОВОГО МАНОЗОСПЕЦИФІЧНОГО ЛЕКТИНУ ІЗ ЦИБУЛИН ГІАЦИНТИКА ГОСТРОЛОПАТЕВОГО (Hyacinthella аcutiloba K. Perss.)

A new lectin with 6.5 mg/kg yield was purified from fresh bulb Hyacinthella аcutiloba K. Perss. by combination of affinity chromathography on yeast mannan and by ion exchange chromatography on DEAE-toyopearl. The best lectin inhibitor among tested monoand disaccharides was D-turanose (Glcp α1-3 Fruf). Very powerful inhibitor of lectin activity was yeast mannan. The lectin revealed weak affinity to α-methyl-Dmannoside, D-fructose and 2-acetamido-D-galactopyranoside. The lectin interacted also with pig liver glycogen, starch and mannose-containing glycoproteins (ovoalbumin, ovomucoid and calf thyroglobulin), but don’t interacted with horsera dish peroxidase and calf intestine alkaline phosphatase. According to electrophoresis, in 20% SDS-PAAG containing SDS-Na the lectin consists with subunits of molecular weight 12 kDa. Molecular weight of the lectin is 48 kDa according to gel-chromatography on Toyopearl HW-55. The lectin agglutinated best of all rabbit erythrocytes and worse agglutinated guinea-pig, very weak — rat erythrocytes and did not agglutinate human and goat erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin did not lose hemaglutinating activity and endured heating to +65 °C during one hour.

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges(1,2). These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production(3). In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (λ=532 nm, Fluence~110 J/cm(2), spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 ± 6 kPa, (n=30). The adhesion strength drops to 2 ± 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1.

In vitro cell compatibility study of rose bengal–chitosan adhesives

Photochemical tissue bonding (PTB) using rose bengal (RB) in conjunction with light is an alternative technique to repair tissue without suturing. It was recently demonstrated that laser-irradiated chitosan films, incorporating RB, bonded firmly to calf intestine in vitro. It is thus required to investigate the possible cytotoxic effects of the RB-chitosan adhesive on cells before testing its application to in vivo models. Adhesive films, based on chitosan and containing ~0.1 wt% RB were fabricated. Their cytotoxicity was assessed by growing human and murine fibroblasts either in media in which adhesive strips had been incubated, or directly on the adhesive. The adhesive was either laser-irradiated or not. Cells were stained after 48 hours with Trypan blue and the number of live and dead cells was recorded for cell viability. Murine and human fibroblasts grew confluent on the adhesives with no apparent morphological changes or any exclusion zone. Cell numbers of murine fibroblasts were not significantly different when cultured in media that was extracted from irradiated (86 ± 7%) and non-irradiated adhesive (89 ± 4%). A similar result was obtained for the human fibroblasts. These findings support that the RB-chitosan films induced negligible toxicity and growth retardation in murine and human fibroblasts.

Label-free, nucleotide-mediated dispersion of magnetic nanoparticles for “non-sandwich type” MRI-based quantification of enzyme

In the present work, we demonstrate that nucleotide can adsorb efficiently on the surface of carboxylic acid-functionalized nanoparticles and stabilize the particles against aggregation. In the present study we take magnetic nanoparticles (MNPs), manganese oxide nanoparticles (MnO), and upconversion nanophosphors (UCNPs) as models. The result shows that not only MNPs, but also other kinds of nanoparticles that have similar surface properties can be dispersed and stabilized by nucleotides. Interestingly, adenosine bearing different numbers of phosphate groups has distinct stabilizing effect. On the basis of this observation, we developed a magnetic relaxation-based enzyme assay for quantitative analysis of alkaline phosphatase. A detection limit of 0.002U/μL for calf intestine alkaline phosphatase (CIAP) could be obtained, which is lower than the gold nanoparticle-based colorimetric method. In contrast to the conventional magnetic relaxation switches (MRSw), this assay was achieved without covalent modification and separation steps, sandwich type binding was not required as well, which would potentially expand the application of magnetic relaxation-based analysis.

P3‐034: Rapamycin downregulates tau phosphorylation in SH‐SY5Y cells

P3‐034: Rapamycin downregulates tau phosphorylation in SH‐SY5Y cells

A small scale study on the effects of oral administration of the β-glucan produced by Aureobasidium pullulans on milk quality and cytokine expressions of Holstein cows, and on bacterial flora in the intestines of Japanese black calves

BackgroundThe β–(1 → 3),(1 → 6)-D-glucan extracellularly produced by Aureobasidium pullulans exhibits immunomodulatory activity, and is used for health supplements. To examine the effects of oral administration of the β–(1 → 3),(1 → 6)-D-glucan to domestic animals, a small scale study was conducted using Holstein cows and newborn Japanese Black calves.FindingsHolstein cows of which somatic cell count was less than 3 x 105/ml were orally administered with or without the β-(1 → 3),(1 → 6)-D-glucan-enriched A. pullulans cultured fluid (AP-CF) for 3 months, and the properties of milk and serum cytokine expression were monitored. Somatic cell counts were not significantly changed by oral administration of AP-CF, whereas the concentration of solid non fat in the milk tended to increase in the AP-CF administered cows. The results of cytokine expression analysis in the serum using ELISA indicate that the expressions of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in all cows which were orally administered with AP-CF became slightly lower than that of control cows after the two-month treatment. On the other hand, IL-8 expression tended to indicate a moderately higher level in all treated cows after the three-month administration of AP-CF in comparison with that of the control cows. Peripartum Japanese Black beef cows and their newborn calves were orally administered with AP-CF, and bacterial flora in the intestines of the calves were analyzed by T-RFLP (terminal restriction fragment length polymorphism). The results suggest that bacterial flora are tendentiously changed by oral administration of AP-CF.ConclusionsOur data indicated the possibility that oral administration of the β–(1 → 3),(1 → 6)-D- glucan produced by A. pullulans affects cytokine expressions in the serum of Holstein cows, and influences bacterial flora in the intestines of Japanese Black calves. The findings may be helpful for further study on the efficacies of oral administration of β-(1 → 3),(1 → 6)-D-glucans on domestic animals.

CLONING OF DENGUE VIRUS TYPE 3 (INDONESIAN STRAIN D3-1703) NON STRUCTURAL-1 GENE INTO pYES2/CT VECTOR

Dengue is an infectious disease caused by dengue virus. Dengue endemic region includes America, Western Pacific, Africa, East Mediterranian, and South East Asia including Indonesia. An early diagnostic system specific for Indonesia is needed to control dengue in Indonesia. In this research, cloning of Non Structural 1 (NS1) gene from dengue virus type 3 (Indonesian strain D3-1703) into pYES2/CT vector was performed. In the long run, NS1 recombinant protein will be expressed in Saccharomyces cerevisiae for diagnostic materials. Polymerase Chain Reaction (PCR) amplification of NS1 gene fragments were done with optimal annealing temperature at 55 oC. NS1 gene fragment and pYES2/CT were cut by BamH I and Not I enzymes. The digested pYES2/CT was dephosphosrylated using Calf Intestine Alkaline Phospatase enzyme. Ligation with the vector:insert ratio of 1:12 and 1:20 resulted in 6 and 5 recombinant colony candidates respectively. Restriction enzyme and PCR verifications showed that 5 recombinant plasmids contained NS1 gene. Sequencing of the first 600 bp of one recombinant plasmid was performed. The blastn analysis showed that it had a 99% identity with dengue virus type 3 strain FW06. Finally, it was shown that NS1 clone within pYES2/CT was in the correct Open Reading Frame and ready to be expressed in S. cerevisiae.

Diversity of Salmonella spp. serovars isolated from the intestines of water buffalo calves with gastroenteritis

BackgroundSalmonellosis in water buffalo (Bubalus bubalis) calves is a widespread disease characterized by severe gastrointestinal lesions, profuse diarrhea and severe dehydration, occasionally exhibiting a systemic course. Several Salmonella serovars seem to be able to infect water buffalo, but Salmonella isolates collected from this animal species have been poorly characterized. In the present study, the prevalence of Salmonella spp. in water buffalo calves affected by lethal gastroenteritis was assessed, and a polyphasic characterization of isolated strains of S. Typhimurium was performed.ResultsThe microbiological analysis of the intestinal contents obtained from 248 water buffalo calves affected by lethal gastroenteritis exhibited a significant prevalence of Salmonella spp. (25%), characterized by different serovars, most frequently Typhimurium (21%), Muenster (11%), and Give (11%). The 13 S. Typhimurium isolates were all associated with enterocolitis characterized by severe damage of the intestine, and only sporadically isolated with another possible causative agent responsible for gastroenteritis, such as Cryptosporidium spp., Rotavirus or Clostridium perfringens. Other Salmonella isolates were mostly isolated from minor intestinal lesions, and often (78% of cases) isolated with other microorganisms, mainly toxinogenic Escherichia coli (35%), Cryptosporidium spp. (20%) and Rotavirus (10%). The S. Typhimurium strains were characterized by phage typing and further genotyped by polymerase chain reaction (PCR) detection of 24 virulence genes. The isolates exhibited nine different phage types and 10 different genetic profiles. Three monophasic S. Typhimurium (B:4,12:i:-) isolates were also found and characterized, displaying three different phage types and three different virulotypes. The molecular characterization was extended to the 7 S. Muenster and 7 S. Give isolates collected, indicating the existence of different virulotypes also within these serovars. Three representative strains of S. Typhimurium were tested in vivo in a mouse model of mixed infection. The most pathogenic strain was characterized by a high number of virulence factors and the presence of the locus agfA, coding for a thin aggregative fimbria.ConclusionsThese results provide evidence that Salmonella is frequently associated with gastroenteritis in water buffalo calves, particularly S. Typhimurium. Moreover, the variety in the number and distribution of different virulence markers among the collected S. Typhimurium strains suggests that within this serovar there are different pathotypes potentially responsible for different clinical syndromes.