Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt.

BACKGROUND: Tick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene. RESULTS: We collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries. CONCLUSION: This study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

A transmission electron microscopy investigation suggests that telocytes, skeletal muscles, myoblasts, and stem cells in common carp (Cyprinus carpio) respond to salinity challenges.

BACKGROUND: Telocytes are modified interstitial cells that communicate with other types of cells, including stem cells. Stemness properties render them more susceptible to environmental conditions. The current morphological investigation examined the reactions of telocytes to salt stress in relation to stem cells and myoblasts. The common carp are subjected to salinity levels of 0.2, 6, and 10 ppt. The gill samples were preserved and prepared for TEM. RESULTS: The present study observed that telocytes undergo morphological change and exhibit enhanced secretory activities in response to changes in salinity. TEM can identify typical telocytes. This research gives evidence for the communication of telocytes with stem cells, myoblasts, and skeletal muscles. Telocytes surround stem cells. Telopodes made planar contact with the cell membrane of the stem cell. Telocytes and their telopodes surrounded the skeletal myoblast. These findings show that telocytes may act as nurse cells for skeletal stem cells and myoblasts, which undergo fibrillogenesis. Not only telocytes undergo morphological alternations, but also skeletal muscles become hypertrophied, which receive telocyte secretory vesicles in intercellular compartments. CONCLUSION: In conclusion, the activation of telocytes is what causes stress adaptation. They might act as important players in intercellular communication between cells. It is also possible that reciprocal interaction occurs between telocytes and other cells to adapt to changing environmental conditions.

Polystyrene nanoplastic and engine oil synergistically intensify toxicity in Nile tilapia, Oreochromis niloticus : Polystyrene nanoplastic and engine oil toxicity in Nile tilapia.

Polystyrene nanoplastic (PS-NPs) and Engine oil (EO) pose multiple ecotoxic effects with increasing threat to fish ecosystems. The current study investigated the toxicity of 15 days exposure to PS-NPs and / or EO to explore their combined synergistic effects on Nile tilapia, Oreochromis niloticus (O. niloticus). Hematobiochemical parameters, proinflammatory cytokines, and oxidative stress biomarkers as well as histological alterations were evaluated. The experimental design contained 120 acclimated Nile tilapia distributed into four groups, control, PS-NPs (5 mg/L), EO (1%) and their combination (PS-NPs + EO). After 15-days of exposure, blood and tissue samples were collected from all fish experimental groups. Results indicated that Nile tilapia exposed to PS-NPs and / or EO revealed a significant decrease in almost all the measured hematological parameters in comparison to the control, whereas WBCs and lymphocyte counts were significantly increased in the combined group only. Results clarified that the combined PS-NPs + EO group showed the maximum decrease in RBCs, Hb, MCH and MCHC, and showed the maximum significant rise in interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in comparison to all other exposed groups. Meanwhile, total antioxidant capacity (TAC) showed a significant (p < 0.05) decline only in the combination group, whereas reduced glutathione (GSH) showed a significant decline in all exposed groups in comparison to the control. Both malondialdehyde (MDA) and aspartate aminotransferase (AST) showed a significant elevation only in the combination group. Uric acid showed the maximum elevation in the combination group than all other groups, whereas creatinine showed significant elevation in the EO and combination group when compared to the control. Furthermore, the present experiment proved that exposure to these toxicants either individually or in combination is accompanied by pronounced histomorpholgical damage characterized by severe necrosis and hemorrhage of the vital organs of Nile tilapia, additionally extensively inflammatory conditions with leucocytes infiltration. We concluded that combination exposure to both PS-NPs and EO caused severe anemia, extreme inflammatory response, oxidative stress, and lipid peroxidation effects, thus they can synergize with each other to intensify toxicity in fish.

Resistance of Nile tilapia fed with Padina boergesenii extract to Pseudomonas putida infection.

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1ß), beta-defensin (ß-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection.

Expression profiles of four Nile Tilapia innate immune genes during early stages of Aeromonas veronii infection.

OBJECTIVE: During Egypt's hot summer season, Aeromonas veronii infection causes catastrophic mortality on Nile Tilapia Oreochromis niloticus farms. Egypt is ranked first in aquaculture production in Africa, sixth in aquaculture production worldwide, and third in global tilapia production. This study aimed to investigate, at the molecular level, the early innate immune responses of Nile Tilapia to experimental A. veronii infection. METHODS: The relative gene expression, co-expression clustering, and correlation of four selected immune genes were studied by quantitative real-time polymerase chain reaction in four organs (spleen, liver, gills, and intestine) for up to 72 h after a waterborne A. veronii challenge. The four genes studied were nucleotide-binding oligomerization domain 1 (NOD1), lipopolysaccharide-binding protein (LBP), natural killer-lysin (NKL), and interleukin-1 beta (IL-1ß). RESULT: The four genes showed significant transcriptional upregulation in response to infection. At 72 h postchallenge, the highest NOD1 and IL-1ß expression levels were recorded in the spleen, whereas the highest LBP and NKL expression levels were found in the gills. Pairwise distances of the data points and the hierarchical relationship showed that NOD1 clustered with IL-1ß, whereas LBP clustered with NKL; both genes within each cluster showed a significant positive expression correlation. Tissue clustering indicated that the responses of only the gill and intestine exhibited a significant positive correlation. CONCLUSION: The results suggest that NOD1, LBP, NKL, and IL-1ß genes play pivotal roles in the early innate immune response of Nile Tilapia to A. veronii infection, and the postinfection expression profile trends of these genes imply tissue-/organ-specific responses and synchronized co-regulation.

Characterizing Two New Henneguya Species in the Respiratory Organs of African Sharptooth Catfish.

Henneguya species are myxozoans, a suborder of Cnidaria, which can affect the gills and extrarespiratory organs of the African sharptooth catfish, Clarias gariepinus. This research describes natural infection-induced histological alterations caused by the Henneguya species present. The Henneguya species were also identified molecularly using DNA sequenced from infected tissue cysts, and phylogenetically analyzed. Clinical investigations revealed cyst-like nodules on the fish gill filaments and extrarespiratory organs. Within a milky fluid inside the cysts were several Henneguya-like spores. Henneguya sp. infested 27.5% of the fish, with the highest prevalence in the gills compared to the extrarespiratory organs. The Henneguya species parasitized the gill and the dendritic tissues, resulting in histopathological characteristics. The plasmodia's developmental stages resulted in destructive damage which manifested as marked necrosis, which was replaced by a focal aggregation of inflammatory cells. Amplification of the 18S ribosomal DNA from the fish parasites was followed by sequencing, which confirmed their identities as new species Henneguya qenabranchiae n. sp. and Henneguya qenasuprabranchiae n. sp. with 99.53 and 99.64% identities, respectively, to Henneguya sp. 1 HS-2015. The two C. gariepinus myxozoans shared some characteristics based on morphologic and phylogenetic analysis as previously published, where it was proposed that they were a sister lineage to Henneguya species in Egypt, and it is now proposed that they are new species.

Henneguya species infecting the gastrointestinal tract of Clarias gariepinus from the Nile River.

As part of a study on parasitic infection in the African sharptooth catfish Clarias gariepinus, we found cysts of varying sizes in the stomach and intestine that contained myxospores with morphological features resembling those of the genus Henneguya. The present investigation was carried out with data on spore morphology and histopathology. Additionally, the myxozoan was identified using a molecular-based approach with 18S small subunit rDNA sequences. Based on the morphological characterization and tissue specificity of Myxozoa, 2 species of Henneguya were identified in the catfish stomach and intestine. Several histopathological changes were observed in the intestine which may affect fish performance and survival. The phylogenetic position of nucleotide sequences of the Henneguya species identified here were clustered with other fish-infecting Henneguya species. These sequences were deposited in GenBank. It appears that they potentially represent 2 species, denominated Henneguya sp. 1 and Henneguya sp. 2 according to the samples originating from the stomach and intestine, respectively. Although future investigations are needed for detailed morphological and molecular descriptions, this study documents the likely occurrence of infection with Henneguya noted for the first time, to our knowledge, in the digestive system of C. gariepinus in Egypt.

Migrating and Nerve Fibers-Associated Eosinophilic Granule Cells in the Olfactory Organ of Catfish (Clarias garpienius): Light and Electron Microscopic Evidence.

Piscine mast cells or eosinophilic granule cells (EGCs) of fish are equivalent to the mammalian mast cells. Recently, a better understanding of EGCs functions is allowed because of the growing interest in fish models. Herein, we present a trial to furnish data regarding the distribution of the EGCs in the fish olfactory organ, an issue that has not been reported so far. Regarding their distribution, two kinds of EGCs had been identified. An intra-epithelial one was detected in the olfactory epithelium lining of the olfactory lamellae. The stromal one was identified in the connective tissue core of the olfactory lamellae and in the lamina propria underlying the olfactory epithelium. Some were detected in the capillary lumen. The cytoplasm of the EGCs reacted strongly with the MMP-9 antibody. Stimulating a migration perspective for the olfactory EGCs which was confirmed by their location in the blood capillaries. Several EGCs could be verified in close relation, some underneath the epineurium, with the nerve fiber. Mutually, this verifies the existence of intra-epithelial and stromal migrating EGCs in the catfish olfactory organ and their inclusion in the olfactory immune response. Additionally, this provides evidence for an immune­nervous interaction to influence both the immune reactions and the nervous scheme in catfish.

Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt.

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

Morphological changes in intraepithelial and stromal telocytes in Cyprinus carpio in response to salinity stress.

Telocytes establish connections and communicate with various types of cells and structures. Few experimental studies have been performed on telocytes. In this study, we investigated the effect of salinity stress on telocytes in relation to osmoregulatory, immune, and stem cells. After exposing the common carp to 0.2 (control), 6, 10, or 14 ppt salinity, we extracted and fixed gill samples in glutaraldehyde, processed and embedded the samples in resin, and prepared semi-thin and ultrathin sections. Two types of telocytes were identified: intraepithelial and stromal telocytes. Intraepithelial telocytes were found to form part of the cellular lining of the lymphatic space and shed secretory vesicles into this space. Stromal telocytes were observed to shed their secretory vesicles into the secondary circulatory vessels. Both intraepithelial and stromal telocytes were enlarged and exhibited increased secretory activities as salinity increased. They exerted their effects via direct contact and paracrine signaling. The following changes were observed in samples from fish exposed to high salinity levels: chloride cells underwent hypertrophy, and their mitochondria became cigar-shaped; pavement cells were enlarged, and their micro-ridges became thin and elongated; stromal telocytes established contact with stem cells and skeletal myoblasts; skeletal muscle cells underwent hypertrophy; and macrophages and rodlet cells increased in number. In conclusion, our findings indicate that intraepithelial and stromal telocytes respond to salinity stress by activating cellular signaling and that they play major roles in osmoregulation, immunity, and regeneration.

Lactoferrin improves the immune response and resistance of silver carp, a hematological, light (histochemical and immunohistochemical), fluorescent, and scanning electron microscopic study.

Lactoferrin is a multifunctional protein that has roles in iron metabolism, cell generation and differentiation, and antibacterial, antiviral, and antiparasitic activity. The study aim was to evaluate the effect of dietary lactoferrin powder on the innate immune response and disease resistance of silver carp (Hypophthalmichthys molitrix) infected with Vibrio vulnificus. The study also investigates immune cells in different organs including gills, intestine, spleen, liver, and pancreas using light, scan electron microcopy. Two diet regimes, a basic diet and a 600-mg/kg bovine lactoferrin-supplemented diet were used. The fish were fed for 30 successive days. We included 270 apparently healthy silver carp (H. Molitrix) with an average body weight of 10 ± 2 g/fish. Addition of lactoferrin to the diet led to an increase in the activity of non-specific immune parameters; lymphocytes, and in the monocytes percentage, and in total protein, serum globulin, and albumin, with a corresponding increase in phagocytic activities and indices. The histological analysis revealed increase immune cells such as lymphocytes in intestine, rodlet cells in liver, gills, and pancreas, and the macrophages in liver, pancreas as well as spleen. The lactoferrin-supplemented diet significantly increased survival and disease resistance following V. vulnificus challenge, which seemed to correspond to an enhancement of non-specific immune functions in H. molitrix. RESEARCH HIGHLIGHTS: The study evaluates the effect of dietary lactoferrin powder on the innate immune response and disease resistance of silver carp (Hypophthalmichthys molitrix) infected with Vibrio vulnificus. The study also investigates immune cells in different organs including gills, intestine, spleen, liver, and pancreas using light, scan electron microcopy. Lactoferrin increases the activity of nonspecific immune parameters; lymphocytes, and in the monocytes percentage, and in total protein, serum globulin, and albumin, with a corresponding increase in phagocytic activities and indices. The histological analysis revealed increase immune cells such as lymphocytes in intestine, rodlet cells in liver, gills, and pancreas, and the macrophages in liver, pancreas as well as spleen.

Detection of aflatoxin-producing fungi isolated from Nile tilapia and fish feed.

Contamination of fish by fungi and their mycotoxins poses major health concerns to human and animals. Therefore, our study was aimed to investigate Aspergillus flavus (A. flavus) infections and the levels of aflatoxins in Nile tilapia, Oreochromis niloticus (O. niloticus), and fish feed. Samples from O. niloticus and fish feed (n=25 for each) were randomly collected from private fish farms at Qena province, Egypt, during the winter season. Different Aspergillus spp. were detected in 60 % and 64 % of O. niloticus and fish feed, respectively. HPLC-based analysis revealed aflatoxin-producing activity in 75 % and 83 % of A. flavus isolates from fish and fish feed, respectively. While 96 % of O. niloticus muscles and fish feed samples were contaminated with aflatoxins, the detected levels were below the permissible limits, i.e. 20 µg/kg. Moreover, experimental infection with toxicogenic A. flavus isolates was conducted to evaluate their pathogenicity in O. niloticus. Expectedly, experimental infections of O. niloticus with A. flavus were associated with several clinical symptoms reported in naturally infected fish, e.g. yellow coloration with skin ulceration, hemorrhagic ulcerative patches on gills and skin, corneal opacity, fin rot and abdominal distention. Furthermore, aflatoxicogenic A. flavus isolates from fish were sensitive to herbal clove oil. Even though the measured levels of aflatoxin were below permissible limits, effort should be placed on further reduction of exposure to genotoxic and carcinogenic mycotoxins.