Biochemical and genotyping analyses of camels (Camelus dromedaries) trypanosomiasis in North Africa.

Camels are considered an important food source in North Africa. Trypanosomiasis in camels is a life-threatening disease that causes severe economic losses in milk and meat production. Therefore, the objective of this study was to determine the trypanosome genotypes in the North African region. Trypanosome infection rates were determined by microscopic examination of blood smears and polymerase chain reaction (PCR). In addition, total antioxidant capacity (TAC), lipid peroxides (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were determined in erythrocyte lysate. Furthermore, 18S amplicon sequencing was used to barcode and characterizes the genetic diversity of trypanosome genotypes in camel blood. In addition to Trypanosoma, Babesia and Thelieria were also detected in the blood samples. PCR showed that the trypanosome infection rate was higher in Algerian samples (25.7%) than in Egyptian samples (7.2%). Parameters such as MDA, GSH, SOD and CAT had significantly increased in camels infected with trypanosomes compared to uninfected control animals, while TAC level was not significantly changed. The results of relative amplicon abundance showed that the range of trypanosome infection was higher in Egypt than in Algeria. Moreover, phylogenetic analysis showed that the Trypanosoma sequences of Egyptian and Algerian camels are related to Trypanosoma evansi. Unexpectedly, diversity within T. evansi was higher in Egyptian camels than in Algerian camels. We present here the first molecular report providing a picture of trypanosomiasis in camels, covering wide geographical areas in Egypt and Algeria.

Anti-inflammatory and Immunomodulatory Potency of Selenium-Enriched Probiotic Mutants in Mice with Induced Ulcerative Colitis.

Selenium-enriched Lactobacillus plantarum and Bifidobacterium longum mutants were used as a protector against Piroxicam-induced ulcerative colitis (UC). In this study, 32 BALB/c male mice were distributed to four groups: the control group, the Piroxicam group which was given 0.8 mg Piroxicam, SP and SB groups which were given 0.8 mg Piroxicam, and plus Lactobacillus plantarum and Bifidobacterium longum selenium-enriched mutants, respectively. Bodyweight; serum content of IgG, IgM, TNF-α, IL-2, IL-6, and IL-10; CBC; myeloperoxidase enzyme activity; histopathological examination of colon and spleen; and expression of TNF-α, IL-2, IL-6, and IL-10 genes in colon and spleen with qRT-PCR were determined. Bodyweight was found to reduce in the Piroxicam group and then recovery in the SB group. Serum content of IgG, IL-2, and IL-10 reduced in the Piroxicam group, whereas IgG, TNF-α, and IL-6 increased in the Piroxicam group in comparison to the other groups. Myeloperoxidase activity witnessed a significant increase in the Piroxicam group compared with the other groups. No significant differences were observed between all groups in measurements of red cells, hemoglobin, neutrophil, monocyte, eosinophil, and basophil in blood. Meanwhile, the white blood cells and platelets recorded the highest and lowest value, respectively, in the Piroxicam group. The colon of the Piroxicam group showed a noticeably massive infiltration of inflammatory cells in the lamina propria. These inflammations were mildly reduced in the SP group, while the reduction in the SB group was significant. In the Piroxicam group, splenic parenchyma saw an increase in the number of melanomacrophages, while hypertrophic plasma cells were observed in the SP group. The spleen of the SB group exhibits a nearly normal form. TNF-α and IL-6 genes had significantly upregulated in the colon of the Piroxicam group compared to the control group, while they were significantly downregulated in the SB group. In contrast, IL-2 and IL-10 genes had upregulated in the colon of the SB group compared to the control groups, while they had downregulated in the Piroxicam group. The expression of these genes had not recorded significant differences between all groups in the spleen. Therefore, this study recommends Bifidobacterium longum selenium-enriched mutants as anti-inflammatory and immunomodulatory supplements.

Evaluation of the effectiveness of two new strains of Lactobacillus on obesity-induced kidney diseases in BALB/c mice.

BACKGROUND: Kidney disease (KD) is a public health problem worldwide and is an important factor in peripheral vascular disease, arrhythmias, heart failure, acute myocardial infarction, stroke, and angina. Obesity has been indicated as an effective cause of kidney diseases. So, this study aims to use two new strains of Lactobacillus to reduce the metabolic disorders and kidney insufficiency associated with obesity. METHODS: Fifty BALB/c male mice were divided into five groups (control, obesity, obesity pro1, obesity pro2, and obesity mix). The bodyweight, cholesterol profile, urea, and creatinine levels in urine and serum were all measured. Histopathological analysis and expression of Opn, Vim, Ngal, Kim-1, and αKlotho genes for kidney tissues were performed. RESULTS: The results indicated that body weight, cholesterol profile, urea, and creatinine levels in serum and urine had the lowest significance (P ˂ 0.05) in the obesity mix group and the highest significance in the obesity group. HDL had the highest significance (P ˂ 0.05) in the obesity mix group and the lowest significance (P ˂ 0.05) in the obesity group. Expression of Opn, Vim, Ngal, and Kim-1 genes was the most upregulated in the obesity group compared with the other groups, and there were nonsignificant differences (P > 0.05) between the obesity pro1 and obesity mix groups and the control group. Expression of αKlotho gene was significantly reduced (P ˂ 0.05) in the obesity group compared with the control group. CONCLUSION: This study demonstrated that the combination of pro1 and pro2 strains could reduce kidney inflammation and necrosis.

Inhibition of induced-hepatic cancer in vivo through IQGAP1-shRNA gene therapy and modulation of TRAIL-induced apoptosis pathway.

Background: Liver cancer is the deadliest malignancy among common tumors. It is the top cause of cancer-related deaths in Egypt, and it is characterized by increasing occurrence among the population. The objective of this study was to determine the outcome of pre-treatment of IQGAP1-shRNA on induced mouse hepatocellular carcinoma model and evaluate the potency of this IQGAP1-shRNA plasmid to recover hepatic cancer as a new tool of cancer therapy. Therefore, we will use RNA interference (RNAi) technology to silence IQGAP1 oncogene to completely recover the chemically induced models for hepatic cancer by designing short RNAi specific for IQGAP1 gene in HCC cells in vivo and construct new vectors suitable for this purpose. We assigned mice into three groups: the first negative control group (NC) was injected with saline, the second control group was injected with shRNA (shNC), the third positive control group was injected with diethylnitrosamine (DENAA), and the fourth group was treated with the IQGAP1-shRNA prior to its exposure to DENA. Results: Our results revealed that the treated group with IQGAP1-shRNA with DENA developed very few cases of hepatic cancer when compared with the positive control group. The positive control group exhibited significant increases in the liver function level as well as a decrease in serum albumin levels when compared to both the treated and the negative control groups. The altered levels of the serum α-fetoprotein as well as of the tumor necrosis factor-alpha, and interleukin-4 in DENA-treated mice were significantly ameliorated by IQGAP1-shRNA administration. Flow cytometer analyses have indicated that the silencing of IQGAP1 cannot significantly modulate DENA-induced apoptosis in the circulating blood cells. Moreover, the elevated mRNA expression levels of IQGAP1, IQGAP3, KRas, HRas, interleukin-8, nuclear factor kappa B, caspase-3, caspase-9 and Bcl-2, were significantly decreased by the IQGAP1-shRNA treatment. However, the IQGAP2, DR4, DR5, p53 and BAX genes were found to be significantly up-regulated post-therapy. In agreement with these findings, IQGAP1-shRNA was able to modulate the DENA-induced histological changes in the mice liver which were represented by severe necrosis and hydropic degenerative changes. Conclusion: Our study revealed that IQGAP1-shRNA was able to preserve hepatocyte integrity and the liver histological architecture through the regulation of the expression of IQGAPs, Ras, TRAILs and IL-8 receptors, as well as of pro-apoptotic and anti-apoptotic genes. Therefore, the silencing of IQGAP1 could be part of a promising therapeutic strategy against hepatic cancer.

New Approach about the Signaling Crosstalk between IQGAPs/ NF- κB/IL-8 and PDCD5/p53/TRAIL Pathways that Modulate Malignant Transformation in Hepatocellular Carcinoma.

OBJECTIVE: We aimed to investigate the signalling crosstalk of TNF-related apoptosis-inducing ligand, TRAIL death receptors, tumour protein p53, and programmed cell death (PDCD5) with IQGAPs. Also, we targeted the crosstalk between IQGAPs genes with decoy receptors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukins -8 (IL-8) and its receptor genes in a designed model of hepatocellular carcinoma induced in male Balb/c mice. METHODS: The presence of HCC was confirmed by histological and morphological alterations. In parallel to the incidence of hepatic cancer, we found lung, heart, and kidney cancer after treatment with DEN. RESULTS: Our results show that the expression of mRNA of IQGAP1, TRAIL decoy receptors, NF-κB, and IL-8 genes was elevated in hepatocellular carcinoma, as compared to normal liver tissue, while their expression was further up-regulated by increasing the dose of diethylnitrosamine. The expression of IQGAP2, TRAIL death receptors, p53, and PDCD5 was significantly down-regulated in HCC (p˂0.05). For confirmation of gene expression, protein levels of both IQGAP1 and P53 were measured by western blot analysis, which showed that diethylnitrosamine enhanced protein expression of IQGAP1 and diminished that of p53. CONCLUSION: IQGAPs have a direct signaling relationship with p53, IL-8, and TRAIL family. This interaction is recognized as a key signalling pathway for hepatocellular carcinogenesis.

Protective and therapeutic effects of two novel strains of Lactobacilli on diabetes-associated disorders induced by a high level of fructose.

Diabetes is a metabolic disorder described as insufficient secretion of insulin in the pancreas or the inability of the existing insulin to function properly. It poses a greater risk on human health as it is considered the base of several diseases. Thus, this study was designed to evaluate two novel strains of Lactobacillus in handling pancreas disorders. 50 BALB/c male mice were divided into five groups; (a) feeding on normal diet only as control group, (b) given 21% fructose in drinking water as diabetes group, (c) feeding on Lactobacillus rhamnosus strain Pro2 (MT505335.1) plus 21% fructose as LR group, (d) feeding on Lactobacillus plantarum strain Pro1 (MT505334.1) plus 21% fructose as LP group and (e) mixture of two strains plus 21% fructose as Mix group. The serum content of glucose, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) was determined. Pancreases histopathology was examined. Expression of GH, IGF1, and GLP-1 genes was measured in the liver and pancreas by RT-qPCR. Serum content of glucose, ALT, and AST significantly increased in diabetes group, and significantly reduced in (LP) and (Mix) groups compared with control. Pathological changes occurred in the exocrine and endocrine components of the diabetes group pancreas. Besides, islet cells are almost entirely disturbed and acinar cells degenerated. However, in (LP) and (Mix) groups, the pathological changes significantly decreased and became related to the control group. Expression of GH, IGF1, and GLP-1 genes was significantly downregulated in the liver and pancreas of mice given fructose compared with control. Expression of these genes was either significantly upregulated in groups (LP and Mix) or identical to the control group. This study shows that the strain Pro1 (MT505334.1) or a combination of two strains is useful in reducing diabetic risk.

A study on mutation points of GDF9 gene and their association with prolificacy in Egyptian small ruminants.

BACKGROUND: Genetic variants of the GDF9 gene were considered to be the potent gene markers for improving fecundity traits in Egyptian sheep and goats. Also, these favorable gene variants could be applied in the breeding program by gene-assisted selection (GAS), aiming towards the potential amelioration of reproduction and production in such small ruminants. The present investigation was designed to evaluate the genetic variants of the GDF9 gene on fecundity traits including the mean number of lambing "MNL" and mean number of twin production "MNTP" of Egyptian sheep and goats. RESULTS: This experiment involved 113 mothers, 83 of sheep and 30 of goats, at first, second, third, and fourth parity, and also 26 young females, 12 of sheep and 14 of goats at age of sexual maturation. T-ARMS-PCR analysis was performed on five mutation points (G1, G4, G6, G7, and G8). In sheep, the heterozygous mothers of G4 had significant elevation (P ≤ 0.05) of MNL and MNTP than wild-type homozygous ewes. However, the heterozygous mothers of G1 and G6 gave a reduction of MNL and MNTP as compared to mothers with wild-type genotypes. The ewes of G7 had heterozygous genotype (AG), and the ewes of G8 had wild type (CC). In goat, G4 and G7 were polymorphic, and G1, G6, and G8 were monomorphic type. Based on these findings, it must be selected the young sheep females of heterozygous in G4, and the young goat females of heterozygous in G4 and G7 for participating in a successful breeding program, because they will have potential high fecundity traits. CONCLUSION: The present results confirmed that the genetic variants of the GDF9 gene were considered to be the major gene markers for enhancement of the prolificacy in Egyptian sheep and goats and could be applied in a successful breeding program by gene-assisted selection (GAS) in small ruminants.

Study the Effect of Microinjection of Zn, Fe, and Cu Loaded in Montmorillonite on Development Activities in Fertilized Chicken Eggs.

This study aims to reduce embryonic mortality, increase body weight, and improve immune system in chicken. A total of 240 eggs were assigned to three treatments (n = 60) and injected with cooper (Cu), zinc (Zn), and iron (Fe) loaded by montmorillonite (Mnt), and one untreated group (n = 60). Some hormones and enzymes related with growth were measured in terms of serum, and expression of some genes related to growth, immune, and programmed cell deaths that were determined in the liver and spleen of chicken by RT-qPCR. The embryonic death on the fifth and seventh days after injecting eggs with Fe-Mnt was less obvious than in other groups. The heaviest body weight was recorded for Fe-Mnt and Cu-Mnt treatment. Fe-Mnt treatment had higher serum GSH, SOD, GH, and Myostatin contents and lower MDA than those in the other treatments. Cu-Mnt treatment included the highest contents of CAT enzyme and IGF-1 hormone in serum. The highest expression of IGF-1, GH, BCL6, and SYK genes in liver tissue were recorded by Zn-Mnt, IGFBP2, FGF8, and IFNW1 genes by Cu-Mnt, and TC1RG1 and IFNW1 genes by Fe-Mnt in spleen tissue. In conclusion, Fe-Mnt was the best treatment for reducing embryonic mortality, and increasing body weight of chickens and expression of growth and immune genes, followed by Cu-Mnt treatment.

Metabolic Acidosis in Postsurgical Patient on Canagliflozin and Metformin: A Case Report.

Canagliflozin is a novel drug for diabetes mellitus with the mechanisms of inducing glucosuria through inhibition of the sodium-glucose cotransporter 2 in the kidney independent of insulin activity. We are reporting euglycemic ketoacidosis with severe life-threatening metabolic acidosis. The 2 patients described had type 2 diabetes mellitus and were in a state of relative starvation after abdominal surgery. The first patient had been given an oral diet but was restricted with regard to calorie and sugar intake. The second patient had been nil per os since the operation.

Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilum to nine antimicrobials.

A multi-laboratory broth microdilution method trial was performed to standardize the specialized test conditions required for the fish pathogens Flavobacterium columnare and F. psychrophilum. Nine laboratories tested the quality control (QC) strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 against 10 antimicrobials (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline, and trimethoprim/sulfamethoxazole) in diluted (4 g l-1) cation-adjusted Mueller-Hinton broth incubated at 28 and 18°C for 44-48 and 92-96 h, respectively. QC ranges were set for 9 of the 10 antimicrobials. Most of the minimal inhibitory concentration (MIC) distributions (16 of 18, 9 drugs at both temperatures) for A. salmonicida ATCC 33658 were centered on a single median MIC ± 1 two-fold drug dilution resulting in a QC range that spanned 3 dilutions. More of the E. coli ATCC 25922 MIC distributions (7 of 16) were centered between 2 MIC dilutions requiring a QC range that spanned 4 dilutions. A QC range could not be determined for E. coli ATCC 25922 against 2 antimicrobials at the low temperature. These data and their associated QC ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI M49-A Guideline. This method represents the first standardized reference method for testing fish pathogenic Flavobacterium spp.

Improved method for determining antibiotic susceptibility of Flavobacterium columnare isolates by broth microdilution.

A simple and reproducible microdilution method was developed to test the susceptibility of the bacterium Flavobacterium columnare to antibiotics in vitro. The testing was conducted at 28 degrees C for 44-48 h at two dilutions of Mueller-Hinton broth (DMHB) using a standardized inoculum, a reference isolate of Escherichia coli ATCC25922 as a quality control organism, positive and negative control wells, and standardized custom-made microtiter plates. The E. coli ATCC25922 and F. columnare ATCC23463 (the species type strain) had significantly better growth in DMHB at 1:5 (4 g/L) than at 1:7 (3 g/L). The E. coli ATCC25922 was found to be acceptable as a reference isolate and produced minimum inhibitory concentration (MIC) values similar to those in the range published by the Clinical and Laboratory Standards Institute derived using standard Mueller-Hinton broth. The new method was used to determine the MIC of 23 F. columnare isolates (representing the three genotypes of the species) to enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, ormetoprim/sulfadimethoxine, and oxolinic acid.

Simultaneous multiresidue determination of tetracyclines and fluoroquinolones in catfish muscle using high performance liquid chromatography with fluorescence detection.

Efficient methods are needed for analysis of veterinary drug residues in food. A number of methods are available for single analytes. Multiresidue methods are now increasingly available. It is still rare, however, to find methods not involving mass spectrometry which allow for analysis of more than one class of drug residue. An efficient multiresidue method for the simultaneous determination of fluoroquinolones (FQs) and tetracyclines (TCs) in catfish muscle has now been developed. This method involves an extraction of the analytes with a mixture of acetonitrile and citrate buffer containing magnesium chloride. After centrifugation and evaporation of the supernatants, the residues are determined using high performance liquid chromatography with fluorescence detection. With this method, five fluoroquinolones and three tetracyclines were determined in fortified catfish muscle at levels of 20, 50, and 100 ng g(-1). Average recoveries for ciprofloxacin (CIP), sarafloxacin (SAR), danofloxacin (DANO), enrofloxacin (ENRO), difloxacin (DIF), oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) were in the range of 60-92% with good relative standard deviations. The limits of quantitation ranged from 0.15 to 1.5 ng g(-1). Utilization of the method to successfully analyze catfish muscle samples incurred with enrofloxacin and with oxytetracycline is described.

Laboratory efficacy of florfenicol against Streptococcus iniae infection in sunshine bass.

An experimental feeding trial was performed to evaluate the efficacy of florfenicol (FFC) in controlling Streptococcus iniae infection in sunshine bass (female white bass Morone chrysops x male striped bass M. saxatilis). Five dosage levels of FFC in medicated feed were administered daily: 0, 5, 10, 15, and 30 mg of active ingredient/kg of fish body weight. Treatment was started within 22-24 h postchallenge by waterborne exposure to virulent S. iniae. The FFC medication was continued for 10 consecutive days, followed by a 25-d posttreatment observation. At the conclusion of the experiment, FFC treatment significantly increased the survival of S. iniae-challenged sunshine bass from 4.2% in the nonmedicated (positive control) group to 69.2% in the 5-mg/kg dosage group, 86.7% in the 10-mg/kg group, and 94.2% in the 15- and 30-mg/kg groups. Survival was significantly higher in the 15- and 30-mg/kg treatment groups than in the 5-mg/kg treatment group; differences among the 10-mg/kg and higher dosage groups were not significant. Survival curve analysis using a log-rank test indicated no significant difference between curves for the 10- and 15-mg/kg groups but a significant difference between curves for the 5- and 10-mg/kg groups. At the end of the experiment, no carriers were detected in any challenged group receiving an FFC-medicated diet, but the bacterium was recovered from the nonmedicated challenged survivors of the infection. The results of the experiment suggest that the optimum therapeutic daily dose of FFC is between 10 and 15 mg/kg body weight for 10 d.

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer.

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.

Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC43622 strain to Flavobacterium johnsoniae.

Species-specific polymerase chain reaction (PCR) primers have been designed to identify the causative agent of columnaris disease, Flavobacterium columnare. The 16S rRNA gene sequences of F. columnare (eight sequences representing the different genotypes of the species) and related species (18 sequences) were aligned and compared to choose specific regions that are unique to F. columnare and do not have significant intraspecies variability. The species-specific regions in the 16S rRNA gene were used to design a pair of species-specific PCR primers, ColF and ColR. The PCR technique produced a specific amplicon of about 675 base pairs (bp) in 27 isolates of F. columnare and there was no amplification in the closely related species. The specificity of the amplified product was confirmed by digesting with HhaI. The PCR primers did not produce a 675 bp product with F. columnare ATCC43622 strain. This ATCC43622 strain was characterized by biochemical and ribotyping methods and renamed Flavobacterium johnsoniae. The American Type Culture Collection has confirmed these findings and made the change.

Europium-sensitized luminescence determination of oxytetracycline in catfish muscle.

An europium-sensitized time-resolved luminescence (TRL) method was developed to determine oxytetracycline (OTC) in cultivated catfish muscle. Extraction of OTC from fish muscle was performed with pH 4.0 ethylenediaminetetraacetic acid (EDTA)-McIlvaine buffer and clean up with hydrophilic-lipophilic balanced copolymer solid phase extraction (SPE) cartridges. The eluate was used without further concentration for TRL measurement in pH 9.0 micellar tris(hydroxylmethyl)aminomethane (TRIS) buffer. Cetyltrimethylammonium chloride (CTACl) was used as surfactant and EDTA as a co-ligand. The excitation and emission wavelengths were set at 388 and 615nm, respectively. The linear dynamic range was 0-1000ngg(-1) (R(2)=0.9995). The recovery was 92-112% in the fortification range of 50-200ngg(-1) and the limits of detection (LOD) ranged from 3 to 7ngg(-1). Incurred catfish samples were used to demonstrate the performance of the method around 100ngg(-1), the European Union maximum residue level.