Possible Underlying Mechanisms for the Renoprotective Effect of Retinoic Acid-Pretreated Wharton's Jelly Mesenchymal Stem Cells against Renal Ischemia/Reperfusion Injury.

Objectives: The current work investigated the effect of Wharton jelly mesenchymal stem cells (WJ-MSCs) pretreated with all-trans-retinoic acid (ATRA) on renal ischemia in rats and the possible role of oxidative stress, apoptotic and Wnt/ß-Catenin signaling pathways, and inflammatory cytokines in their effects. Methods: The study included 90 male Sprague Dawley rats that were allocated to five groups (n = 18 rats): (I) Sham-operated group (right nephrectomy was performed); (II) Ischemia/reperfusion injury (IRI) group, a sham group with 45-min renal ischemia on the left kidney; (III) ATRA group, an ischemic group with an intravenous (i.v.) administration of ATRA 10 µM, 10 min post-surgery); (IV) WJ-MSCs group, an IRI group with an i.v. administration of 150 µL containing 7 × 106 WJ-MSCs, 10 min post-surgery; (V) WJ-MSCs + ATRA group, an IRI group with an i.v. administration of 150 µL of 7 × 106 WJ-MSCs pretreated with 10 µM ATRA. At the end of the experiments, serum creatinine, BUN micro-albuminuria (MAU), urinary protein, markers of redox state in the left kidney (MDA, CAT, SOD, and GSH), and the expression of Bax, IL-6, HIF-1α, Wnt7B, and ß-catenin genes at the level of mRNA as well as for immunohistochemistry for NFkB and ß-Catenin markers were analyzed. Results: The current study found that 45-min of renal ischemia resulted in significant impairment of kidney function (evidenced by the increase in serum creatinine, BUN, and urinary proteins) and deterioration of the kidney morphology, which was associated with a significant increase in redox state (evidenced by an increase in MDA and a decrease in GSH, SOD, and CAT), and a significant increase in inflammatory and apoptotic processes (evidenced by an increase in Bax and IL-6, NFkB, Wnt7B, ß-catenin and HIF-1α) in kidney tissues (p < 0.05). On the other hand, treatment with ATRA, WJ-MSCs, or a combination of both, caused significant improvement in kidney function and morphology, which was associated with significant attenuation of oxidative stress, apoptotic markers, and inflammatory cytokines (IL6 and NFkB) with the upregulation of HIF-1α and ß-catenin in kidney tissues (p < 0.05). Moreover, the renoprotective effect of WJ-MSCs pretreated with ATRA was more potent than WJ-MSCs alone. Conclusions: It is concluded that preconditioning of WJ-MSCs with ATRA may enhance their renoprotective effect. This effect could be due to the upregulation of the beta-catenin/Wnt pathway and attenuation of apoptosis, inflammation, and oxidative stress.

Biochemical and molecular effects of a commercial diuretic with herbal extract on experimentally induced urolithiasis in chickens.

This study evaluated the diuretic, antioxidant, anti-inflammatory, and immunological effects of a commercial diuretic (CD) (composed of ammonium chloride, potassium citrate, sodium chloride, ascorbic acid, biotin, halfa bar extract, and hexamine) on chickens with induced urolithiasis. A total of 100 one-day-old white Hy-Line chicks were fed a basal diet containing 20% crude protein (CP) and 1% Ca until they reached 48 days of age. Then, the birds were divided into five groups (G1-G5). G1 was fed a basal diet and kept as a negative control, G2 was fed a high protein (HP) diet containing 25% crude protein, G3 was fed high calcium (HC) diet containing 5% Ca, G4 was fed HP diet supplemented with CD, and G5 was fed HC diet supplemented with CD. The CD was supplemented with drinking water (at a dose of 0.5 ml/ liter) for 1 week. The experiment was held for 78 days. Clinical signs, postmortem lesions, and mortality rates were observed. Biochemical analytes, redox status biomarkers, and expression of interleukin-6 (IL-6) and interferon-gamma (IFN-γ) were measured. Tissue samples were taken for histopathological examination. No signs of CD toxicity were observed during the toxicity test prior to the experiment. Compared to all groups, birds in G2 and G3 showed impaired renal function and alterations in biochemical, redox status, lipid peroxidation, post-mortem, and histopathological lesions along with upregulation of IL-6 and IFN-γ in the kidney and spleen. In conclusion, commercial diuretic supplementation for one week improves renal function, redox status, immune and anti-inflammatory responses in chickens with induced urolithiasis.

Molecular detection and identification of Babesia bovis and Trypanosoma spp. in one-humped camel (Camelus dromedarius) breeds in Egypt.

BACKGROUND AND AIM: Camels are a unique source of milk and meat, which helps recover from several diseases that affect humans worldwide. In Egypt, one of the great obstacles for this industry is tick-borne diseases. This study aimed to characterize blood parasite infections, such as Babesia (B.) bovis and Trypanosoma (T.) spp. in one-humped camel (Camelus dromedarius) (n=142) breeds in Halayeb and Shalateen, Egypt, through phylogenetic analysis. MATERIALS AND METHODS: The prevalence of B. bovis and Trypanosoma spp. was identified in camels using polymerase chain reaction (PCR) assays targeting the Rhoptry-Associated Protein-1 and internal transcribed spacer 1 genes, respectively. A nested PCR technique was conducted to detect B. bovis. At the same time, KIN multispecies PCR assay was employed to diagnose and classify trypanosome DNA in camels. RESULTS: B. bovis was detected in 4/142 camels with an infection rate of 2.81%. Sequencing and phylogenetic analyses revealed that the strain of B. bovis isolated from this population was closely related to strains isolated from Argentine, the United States, and Brazil. Moreover, Trypanosoma evansi was detected in 8/142 camels with an infection rate of 5.63%. Sequencing and phylogenetic analyses revealed that this isolated strain T. evansi was closely related to Trypanosoma theileri detected from cattle in Brazil. CONCLUSION: The obtained data indicated the existence of B. bovis and T. evansi in camels from two provinces of Egypt. The obtained findings have economic significance and reflect the importance of implementing effective prevention and control methods across Egypt to reduce the incidence of B. bovis and T. evansi in camels.

Fucoidan ameliorates acute and sub-chronic in vivo toxicity of the fungicide cholorothalonil in Oreochromis niloticus (Nile tilapia).

Fucoidans are sulfated glycans from marine algae that have both anti-cancer and anti-microbial properties. Chlorothalonil is a fungicide and insecticide commonly used in agriculture. Chlorothalonil is relatively toxic to fish and can potentially affect the aquaculture practices. In this study, we determined whether fucoidan administration would offer any protection from acute and subchronic toxicity of chlorothalonil on Nile tilapia. First, we tested the effect of chlorothalonil (20 to 140 µg/L, water-applied) on Nile tilapia in an acute exposure (six days). Survival analysis was performed, together with assessment of histopathology, oxidative stress (i.e., antioxidant status, hydrogen peroxide levels, malondialdehyde and nitric oxide levels) and immunohistochemistry to measure indicators of hepatic damage (i.e., caspase 3, p53, mini-chromosome maintenance proteins (MCM), and glutathione peroxidase). Chlorothalonil induced mild to severe histopathological alterations that were dose-dependent in various tissues of Nile tilapia. Chlorothalonil also induced oxidative stress as indicated by elevated biochemical markers. The highest recorded mortalities were associated with p53 expression. Additional feeding experiments were conducted with fucoidan (8 g/kg diet), following acute (40 µg/L for seven days) and sub-chronic (20 µg/L for six weeks) chlorothalonil application in Nile tilapia. Many of these same biochemical biomarkers of stress, oxidative damage response, and tissue pathology (evidence for hepatic neoplasm) were ameliorated by fucoidan, suggesting a protective effect of the compound. Agrochemicals are ubiquitous on a global scale, and the use of fucoidan as a feed additive may be beneficial for protecting aquatic animal health and aquaculture species from the impacts of chemical run-off.

Biochemical and molecular investigation of oxidative stress associated with urolithiasis induced by increased dietary calcium or protein in chickens.

This study was carried out to evaluate the effects of induced urolithiasis by high dietary calcium (Ca) or protein levels on biochemical analyte levels, redox status, selected inflammatory cytokines and histopathology in chickens. A total of 90 one-day-old white Hy-Line chicks were fed basal control diets containing 20% crude protein (CP) and 1% Ca until they reached 44 days of age. After that, the birds were divided into three groups (30 birds per group). All management factors (light, temperature, ventilation, stock density and diet) were identical among the three groups throughout the study except for the dietary Ca and protein percentages. Group I was fed a control diet containing 20% CP and 1% Ca, group II was fed a high-Ca diet containing 5% Ca, and group III was fed a high-protein diet containing 25% CP. Our findings clearly demonstrated that dietary imbalance (caused by high-Ca or high-CP levels) per se in chickens was physiologically harmful, as it was accompanied by post-mortem lesions; biochemical, redox status and histopathological alterations; and upregulation of inflammatory cytokines (interleukin (IL)-1ß and IL-6). In particular, the birds fed the high-Ca diet clearly exhibited the most obvious alterations in most of the endpoints. In conclusion, this study constitutes the first extensive investigation of the effects of high-Ca or high-protein diets induced urolithiasis on growth performance, redox status, inflammatory cytokine levels and pathological characterization in chickens.

The effect of fucoidan or potassium permanganate on growth performance, intestinal pathology, and antioxidant status in Nile tilapia (Oreochromis niloticus).

Fucoidans are marine algal sulfated glycans that are widely used as dietary additives in aquaculture. These glycans are recognized as beneficial supplements for their antimicrobial, anti-inflammatory, anticancer, and antiviral properties. Potassium permanganate is another commonly used chemical that is used in aquaculture to treat infections in fish. Despite their widespread use, there are few data available regarding the potential sublethal toxicity associated with fucoidan and potassium permanganate treatments of fish. In this study, we investigated the effect of each compound on the growth, intestinal health, and antioxidant status of Nile tilapia (Oreochromis niloticus). Both compounds affected the growth of experimental fish compared with untreated fish. However, while growth parameters were positively associated with the dose of fucoidan administered, growth was negatively associated with the dose of potassium permanganate in Nile tilapia. Fucoidan treatment was observed to improve the intestinal health of fish based upon increases in intestinal villous area, intestinal villous length and width, and the intraepithelial lymphocyte number and decreases in the total intestinal bacterial count compared with untreated fish. Conversely, potassium permanganate induced intestinal epithelium proliferation and villous branching, a histopathological response typically observed with chemical irritants. Both fucoidan and potassium permanganate decreased levels of oxidative and nitrosative stress markers and enhanced the antioxidant status in multiple organs. Taken together, fucoidan dietary application improved the growth, intestinal health, and antioxidant status in Nile tilapia, supporting the use of this compound as a promising feed additive for aquaculture production. Conversely, potassium permanganate baths have negative effects on fish growth at higher doses and appeared to act as a gastrointestinal irritant in tilapia. This study improves knowledge regarding the biochemical and histological responses in Nile tilapia to two widely used aquaculture-related treatments.

Antibacterial and antioxidant activity of clove oil against Streptococcus iniae infection in Nile tilapia (Oreochromis niloticus) and its effect on hepatic hepcidin expression.

This study was designed to evaluate the modulating effect dietary clove essential oil (CL) has on the antioxidant and immunological status of Nile tilapia following Streptococcus iniae (Si) infection. Fish were placed on either control or (1.5 and 3%) CL-supplemented diets for 4 weeks. After sampling, the remaining fish in the control group were divided into 2 groups: an unchallenged (negative control) and an Si-challenged positive control. On the other hand, the remaining fish in CL-supplemented groups were challenged with Si, and mortality was checked for two weeks before the final sampling. Serum immunological parameters, tissue antioxidants, and oxidative stress markers were determined. Moreover, hepatic hepcidin expression was also measured in different groups. The obtained results showed improvements in blood phagocytic, bactericidal, lysozyme, and respiratory burst activities in CL-supplemented fish before and after the Si challenge. Si-challenge caused a remarkable increase in tissue malondialdehyde (MDA) levels that was inhibited by CL supplementation. The activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in tissues were significantly elevated in a dose-dependent manner in CL-supplemented groups in both pre- and post-challenge experiments; renal SOD did not show any differences. Hepatic nitric oxide (NO) level was significantly decreased in CL-supplemented fish in a dose-dependent manner. In the post-challenge experiment, nitrosative stress was apparent in the liver and kidney; however, CL supplementation was sufficient to reverse it. Interestingly, a remarkable induction of the hepatic hepcidin expression was observed in all CL-supplemented groups in the pre-challenge experiment and Si-challenged fish, underscoring the role of CL as an antibacterial through inducing hepatic hepcidin expression to combat S. iniae infection. CL-supplementation was associated with lower mortality rates after Si-challenge, which was more pronounced in CL-3% supplemented fish. In conclusion, our results demonstrate that CL has a potent antioxidant role via increasing antioxidant enzymes' activities and antagonizing lipid peroxidation. Moreover, CL has an immune-stimulant effect by inducing the hepatic hepcidin expression and immunological markers in response to S. iniae infection.

Effect of omega-3 rich diet on the response of Japanese quails (Coturnix coturnix japonica) infected with Newcastle disease virus or avian influenza virus H9N2.

This study was performed to evaluate the effects of omega-3 supplementation on growth performance, clinical signs, post-mortem lesions, haemagglutination inhibition (HI) antibody titres, gene expression and histopathology in quails (Coturnix coturnix japonica) infected with Newcastle disease virus (NDV) and avian influenza virus (AIV) H9N2. One hundred, 40-day-old male quails were divided into 5 groups: G1, fed a control basal diet; G2A, infected with NDV; G2B, infected with H9N2; G3A, infected with NDV and given omega-3, and G3B, infected with H9N2 and given omega-3. The dietary omega-3 supplementation was continued for 4 weeks: two weeks before infection and two weeks after intranasal infection with virulent NDV and AIV H9N2. Our results revealed significant differences (P < 0.05) in growth performance, HI antibody titres, clinical signs, post-mortem lesions, mortality, viral shedding rates, immunological parameters, and histopathological lesions between the treated (G3A and G3B) and untreated (G2A and G2B) groups. In conclusion, dietary omega-3 supplementation for 4 weeks can improve growth performance and alleviate the deleterious immunological and pathological effects of NDV and AIV H9N2 infection in quails.

Immunological and pathological effects of vitamin E with Fetomune Plus® on chickens experimentally infected with avian influenza virus H9N2.

Avian influenza virus (AIV) H9N2 infection causes economic losses on poultry farms, and immunostimulants are essential for improving chicken immunity. This study evaluated the immunological and pathological effects of vitamin E with Fetomune Plus® (a commercial product based on a yeast extract and vitamins) on chickens experimentally infected with AIV H9N2. Three groups of white Hy-Line chicks were included. The G1 group was kept as an uninfected untreated control, the G2 group was intranasally infected with the AIV H9N2 strain (0.5 ml of 106 50% egg infectious dose (EID50)), and the G3 group was infected and treated with vitamin E (200 mg/kg of diet) and Fetomune Plus® (1 ml/liter of drinking water) for four weeks. The gene expression of interferon-gamma (IFN-γ), interleukin (IL)-6, and IL-2 was determined at 3, 5 and 7 days post-infection (PI). Virus shedding titers and rates and haemagglutination inhibition (HI) antibody titers were detected. Clinical signs, mortalities and post-mortem lesions were recorded. The birds were weighed, and relative organ weights were calculated. Tissue specimens were taken for histopathological examination and immunohistochemistry (IHC). The expression of IFN-γ in the duodenum revealed a significant increase in G2 compared to G3 at 3 days PI, while the duodenal and splenic expression of IL-6 was significantly increased in G2 compared to G3 at 5 days PI. IL-2 was overexpressed in the duodenum in G3 compared to G2 at 3 and 5 days PI. A significant decrease (P ≤ 0.05) in the virus shedding titer and an increase in the HI titers were detected in G3 compared to G2. The clinical signs and the mortality rate were clearly appeared in G2 than in G3. By IHC, lower H9N2 staining intensity was observed in the examined organs from G3 than in those from G2. In conclusion, as a first report, vitamin E with Fetomune Plus® supplementation for four weeks could improve the immunological and pathological effects of H9N2 infection on chickens.

Molecular identification of Trichodina compacta Van As and Basson, 1989 (Ciliophora: Peritrichia) from cultured Oreochromis niloticus in Egypt and its impact on immune responses and tissue pathology.

Trichodinids are peritrichous ciliated protozoa that affect both wild and cultured fishes. Several Trichodina species have low host specificity and are morphologically distinct, facilitating their identification based primarily on the presence of adhesive discs and the number of attached denticles. A trichodinid species named Trichodina compacta was first reported by Van As and Basson (1989) (Protozoa: Ciliophora: Peritrichia). However, in trichodinid infestations, morphological characteristics are insufficient for identifying the infesting species. Therefore, molecular and phylogenetic analyses are considered to be promising and useful tools for identifying the infesting species. This study aimed to achieve the molecular identification of a trichodinid infestation in Nile tilapia and to construct the phylogenetic relationships between the identified species and other peritrichous parasites. Moreover, we also aimed to study the pathological and immunological impacts of trichodinids on fry tissue to improve our understanding of the immune responses of teleost fish to trichodinae parasitic infestations and develop a better control method. Here, we used molecular techniques to identify the isolated trichodina species as T. compacta and demonstrated that Trichodina infestation in Nile tilapia is associated with remarkable immunogenic and inflammatory responses (increased il-1ß expression and decreased il-8 and tgf-ß expression). These findings improve our understanding of the responses of teleost fish to trichodinid parasite infestation and will be helpful for the development of novel control strategies that reverse the inflammatory and immunogenic alterations that occur in infested fish.

Molecular characterization of the vitamin D receptor (VDR) gene in Holstein cows.

Vitamin D plays a vital role in calcium homeostasis, growth, and immunoregulation. Because little is known about the vitamin D receptor (VDR) gene in cattle, the aim of the present investigation was to present the molecular characterization of exons 5 and 6 of the VDR gene in Holstein cows. DNA extraction, genomic sequencing, phylogenetic analysis, synteny mapping and single nucleotide gene polymorphism analysis of the VDR gene were performed to assess blood samples collected from 50 clinically healthy Holstein cows. The results revealed the presence of a 450-base pair (bp) nucleotide sequence that resembled exons 5 and 6 with intron 5 enclosed between these exons. Sequence alignment and phylogenetic analysis revealed a close relationship between the sequenced VDR region and that found in Hereford cattle. A close association between this region and the corresponding region in small ruminants was also documented. Moreover, a single nucleotide polymorphism (SNP) that caused the replacement of a glutamate with an arginine in the deduced amino acid sequence was detected at position 7 of exon 5. In conclusion, Holstein and Hereford cattle differ with respect to exon 5 of the VDR gene. Phylogenetic analysis of the VDR gene based on nucleotide sequence produced different results from prior analyses based on amino acid sequence.