Spatial scale influences the distribution of viral diversity in the eukaryotic virome of the mosquito Culex pipiens.

Our knowledge of the diversity of eukaryotic viruses has recently undergone a massive expansion. This diversity could influence host physiology through yet unknown phenomena of potential interest to the fields of health and food production. However, the assembly processes of this diversity remain elusive in the eukaryotic viromes of terrestrial animals. This situation hinders hypothesis-driven tests of virome influence on host physiology. Here, we compare taxonomic diversity between different spatial scales in the eukaryotic virome of the mosquito Culex pipiens. This mosquito is a vector of human pathogens worldwide. The experimental design involved sampling in five countries in Africa and Europe around the Mediterranean Sea and large mosquito numbers to ensure a thorough exploration of virus diversity. A group of viruses was found in all countries. This core group represented a relatively large and diverse fraction of the virome. However, certain core viruses were not shared by all host individuals in a given country, and their infection rates fluctuated between countries and years. Moreover, the distribution of coinfections in individual mosquitoes suggested random co-occurrence of those core viruses. Our results also suggested differences in viromes depending on geography, with viromes tending to cluster depending on the continent. Thus, our results unveil that the overlap in taxonomic diversity can decrease with spatial scale in the eukaryotic virome of C. pipiens. Furthermore, our results show that integrating contrasted spatial scales allows us to identify assembly patterns in the mosquito virome. Such patterns can guide future studies of virome influence on mosquito physiology.

Molecular prevalence and associated infection risk factors of tick-borne protozoan and rickettsial blood pathogens in small ruminants.

BACKGROUND: Tick-borne blood pathogens cause highly pathogenic diseases, which are associated with substantial economic losses in ruminants. Despite this, epidemiological research on these pathogens remains neglected in many countries. This study initiated a regional epidemiological survey that included the detection of molecular prevalence, associated risk factors, and gene sequencing, combined with phylogenetic analysis, targeting the two main tick-borne blood protozoan and rickettsial pathogens of Babesia, Theileria, and Anaplasma that infect small ruminants. One hundred blood samples were collected from 76 sheep and 24 goats. RESULTS: Microscopic examination of Giemsa-stained blood films revealed that 73% of the samples were infected with at least one species of the three blood pathogenic organisms. Molecular diagnosis based on the 18 S rRNA for Babesia and Theileria species and the major surface protein 4 (msp4) for Anaplasma species, revealed that 43% of the small ruminants were infected with at least one of these pathogens. The animal's sex was the most significant associated risk factor, with 49.4% of female animals infected compared with only 4% of male animals (P < 0.05). The open breeding system recorded the highest infection rate for tick-borne blood pathogens. Homology-based and phylogenetic analyses indicated that the specific isolate species were Babesia ovis (B. ovis), Theileria ovis (T. ovis), and Anaplasma ovis (A. ovis), with sequences showing significant identities with isolates from sheep, goats, and other animal species, and geographically diverse countries in Africa, Asia, and Europe, in addition to Egypt. CONCLUSION: This was the first molecular evidence of B. ovis, T. ovis, and A. ovis infections in sheep and goat populations in the North Coast region of Egypt. More extensive studies are required to develop an epidemiological map of blood pathogenic organisms, while more effective control strategies are required to reduce the burden of tick-borne pathogens on small ruminants.

Prevalence of intestinal parasitic infections and their associated risk factors among preschool and school children in Egypt.

Intestinal parasitic infections (IPIs) are among the major public health problems globally, particularly in developing countries like Egypt. This study aimed to evaluate prevalence and risk factors associated with IPIs among preschool and school children in Egypt. A cross-sectional study was conducted on 996 randomly selected preschool and school-aged children in Gharbia governorate during January to April 2018. Stool specimens were examined for the presence of the parasite by direct smear and the formol-ether concentration methods. The overall prevalence of IPIs was 46.2%. Entamoeba histolytica and Ascaris lumbricoides were the most predominant parasites (12.7% per each). This is followed by Enterobius vermicularis (8.6%), Giardia lamblia (7.1%), Cryptosporidium parvum (1.5%), Heterophyes heterophyes (1.4%), Hymenolepis nana (0.7%), Hookworms (0.6%), Fasciola hepatica (0.5%) and Dipylidium caninum (0.4%). Infected children with no symptoms (26.8%) were significantly (P < 0.001) more frequent than those with medical complaint (19.4%). Socio-demographic predictors of IPIs were preschool age (OR = 4.9; P < 0.001; 95%CI 3.3-7.3), living in rural dwellings (OR = 1.96; P < 0.001; 95%CI 1.5-2.5), and belonging to a low-income family (OR = 4.7; P < 0.001; 95%CI 2.3-9.3). The absence of safe drinking water, lack of hand washing (after soil contact, or before meals, or after toilet usage), and eating unwashed vegetables were risk factors for IPIs in the study region (OR = 1.3-6.9, P < 0.001 -P = 0.05). Higher odds for exposure to potential zoonotic parasites were evident in children with pets in their homes for G. lambia and D. caninum (OR = 2.1-8.3; P = 0.02 -P = 0.04), children having household reared ruminants for C. parvum (OR = 10.4; P < 0.001), and children that play with stray animals for E. histolytica and Hookworm (OR = 1.8-6.3; P = 0.04 -P = 0.05)compared to other children with no animal contact. The present study highlights the importance of periodic screening and treatment of IPIs in children, deworming companion animals, and public education for effective prevention of IPIs in children in Egypt.

Prevalence of tick-borne haemoparasites and their perceived co-occurrences with viral outbreaks of FMD and LSD and their associated factors.

Species of Theileria, Babesia, and Anaplasma are Tick-borne pathogens (TBPs) that are prevalent throughout the world, particularly in the tropical and subtropical regions. Associated diseases of Theileriosis, Babesiosis, and Anaplasmosis, respectively, represents a major threat to livestock production in many countries. TBPs have a high prevalence in different geographical locations in Egypt. Foot and mouth disease (FMD) and Lumpy skin disease (LSD) are considered endemic bovine viral diseases in Egypt. Our clinical observations during the epidemics of LSD and FMD viruses showed higher prevalence rates for the TBPs. To investigate this correlation, a total of 670 samples from cattle and buffalo were collected during the summers of 2017 and 2018 distributed throughout ranches and smallholders in two geographical locations in Egypt. Two farms with a recent clinical outbreak of LSD with a total of 270 animals, while the other location included three farms with a recent FMD outbreak with a combined 400 cattle. Examined animals were classified mainly according to age, gender, species, breed (native versus crossbred), and the presence of ticks. Whole blood samples were collected for TBPs and viral (LSD and FMD) examinations, while tissue specimens were collected for detection of FMD and LSD viruses by real-time PCR. Our results confirmed significantly higher prevalence rates for the TBPs in LSD-positive than LSD-negative animals, while no significant difference could be detected for the prevalence rate of the TBPs in the FMD positive and negative groups. The prevalence of Babesia and Theileria was significantly (P < 0.05) higher in cross-breeds than native cattle. Infections with Anaplasma and co-infections with Babesia-Anaplasma and Theileria-Anaplasma were significantly higher in native than cross-breeds cattle. The intensity of parasitic infection (parasitemia) has a significant difference in the positive groups for the two viruses compared to the negative groups. These results collectively confirming the enhancing role of LSD on the prevalence rate of the haemoprotozoal infections leading to more serious outcomes to the livestock infections, and therefore the control of haemoprotozoal infections should be implemented as a part of viral epidemics control.

Livoneca redmanii (Isopoda, Cymothoidae) in meagre Argyrosomus regius: parasitological and molecular diagnosis and proposed control measure.

Isopodiosis in cultured meagre Argyrosomus regius was investigated at 3 farms in the northern lakes of Egypt throughout 2018, based upon prevalence rate, parasitological examination, and molecular identification by PCR targeting the large ribosomal subunit 16S of the rRNA gene. Further, the susceptibility of A. regius to isopod infection was experimentally evaluated under hyposalination of 25, 15, and 8 ppt for 1 wk. The isolated isopod stages were morphologically identified as Livoneca redmanii Leach, 1818 with prevalence rates of 77.05 and 77.9% in Al-Madiyyah and Sidi Krir, respectively. The highest prevalence and salinity of 78.85% and 30-34 ppt were reported in El Matareya. DNA sequencing and molecular analysis confirmed the identification of L. redmanii. A. regius experimentally infected with L. redmanii under a hyposalination protocol at 15 and 8 ppt showed marked reduction in mortality (20 and 50%, respectively), and infection prevalence (40 and 63.33%, respectively). The serum lysozyme concentration and nitric oxide of treated fish at 15 and 8 ppt were significantly increased compared to those held at 25 and 33 ppt, whereas serum osmolality levels were higher at 25 and 33 ppt. To our knowledge, this is the first molecular characterization of L. redmanii in cultured A. regius in Egypt.

Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk.

Toxocara vitulorum is an Ascarid nematode infecting the small intestine of buffalo and cattle particularly neonate calves, with the postnatal route through milk is the main infection source. However, little is known about shedding rates and the optimum detection methods of T. vitulorum larvae in the milk of the infected bovine hosts. In this study, we aimed to evaluate the use of two methods, microscopy and PCR, and their detection limits both under the experimental and natural infection situations. In doing this, T. vitulorum eggs extracted from naturally occurring adult female worms were successfully subjected to experimental embryonation, and larvae were implemented in experimental infection of milk in ascending infection doses of 0, 1, 5, 10, 20, 50 larvae/2-ml milk samples. With the except of negative control, microscopy-based examination detected larvae in all samples, albeit with means, ranges, and the total number of larvae were detected in exponential rates relative to larvae densities in milk samples. PCR technique corresponded well to microscopy in detecting genomic DNA of T. vitulorum larvae in all milk samples down to a single larva/sample. On the other hand, and by applying the same methodology approach on 50 naturally-occurring bovine colostrum/milk samples, 13 (26%) and 20 (40%) samples were tested positive for T. vitulorum infection by microscopy and the PCR-based detection, respectively. Of these, 11 out of 26 buffalo samples (42.30%) and 2 out of 24 cow samples (8.33%) were tested positive by microscopy, while 16 (61.54%) and 3 (12.50%) of buffalo and cow samples were tested positive by PCR, respectively. By applying the Agreement Coefficient, substantial agreement (0.77) between molecular and microscopy detection was detected from all tested samples. In conclusion, larvae of T. vitulorum were unequivocally detected by microscopy and molecular methods in milk samples both under the experimental and natural field situations. Nevertheless, slightly higher rates by PCR than microscopy were obtained when detecting naturally-infected milk samples. To the best of our knowledge, this is the first in situ detection of larvae of T. vitulorum in the milk of the naturally infected animals.

Assessment of the inhibitory effects of disinfectants on the embryonation of Ascaridia columbae eggs.

This study was set out with the aim of assessing the effects of the most commonly available commercial disinfectants on embryogenesis of A. columbae eggs. In addition to the distilled water treatment as a control group, four disinfectants were tested that included formalin, povidone iodide, TH4, and Virkon-S, in three independent experiments. In the first experiment, an overnight incubation with the working concentration of disinfectants resulted in significant inhibition of 80%, 85%, and 98% of embryonic development at day 9 post-treatment with formalin, povidone iodide, and TH4, respectively. This inhibition was continued through days 12 and 15 with the three tested disinfectants. Virkon-S did not affect embryogenesis with larval development comparable to that of the control group. In the second more dissected experiment, contact times of 10, 20, 30, and 60 minutes were set out for each disinfectant with embryogenesis inhibition results echoed that of the first experiment, with all disinfectants but Virkon-S inhibited larval development in significant proportions of eggs. Again, Virkon-S was very neutral in its effect on embryogenesis. When pigeon fecal matters were mixed with eggs and were subjected to disinfectants, discrepancies to results of the first two experiments were observed with only formalin inhibited embryogenesis in considerable proportion of eggs. Thus, and with the exception of Virkon-S, disinfectants tested at levels similar to those applicable in poultry in-houses and farms exhibited potent ovicidal activities on free eggs. To our knowledge, this is the first study involving the application of the commonly used in-poultry houses disinfectants to inactivate or delay the embryogenesis of bird Ascarids. The future perspective will potentially involve the in-field applications of the efficient disinfectants to eliminate or reduce the dissemination of infections with bird Ascarids in the chicken, turkey, pigeons, and other poultry houses.

Amyloodiniosis in cultured Dicentrarchus labrax: parasitological and molecular diagnosis, and an improved treatment protocol.

Amyloodinium ocellatum, the causative agent of amyloodiniosis (marine velvet, velvet disease), affects marine and brackish fish in various warm and temperate habitats. We recorded disease outbreaks with high morbidity and mortality rates in marine-cultured European seabass Dicentrarchus labrax fry at 2 locations in northwest Egypt. The sudden outbreak, high morbidity and mortality rates, and skin lesions with a velvety appearance in affected fish all indicated A. ocellatum infection. This was further confirmed by microscopic findings of the parasitic stage (trophonts) in skin and gill smears. While ecological factors including water temperature and salinity were all amenable to parasite establishment and propagation, mortality rates differed between the 2 farms, with rates of mortality well correlated with prevalence and intensity of A. ocellatum infections. Characterization by PCR targeting rDNA gene fragments and subsequent DNA sequencing and phylogenetic analysis further confirmed the molecular identity of the A. ocellatum isolate, which was genetically similar to isolates from other geographical locations. Finally, an improved treatment method using dual hyposalination and copper sulfate exposure to increase the efficiency and decrease the toxicity of copper sulfate was tested. The gradual reduction in water salinity coupled with copper sulfate treatment was more efficient at controlling the disease than only applying copper sulfate. To our knowledge, this is the first parasitological and molecular characterization of A. ocellatum in marine cultures in Egypt. The high molecular identity and close phylogenetic relationship further confirmed the monophyletic nature of A. ocellatum isolates.

Knockdown of APC/C-associated genes and its effect on viability and cell cycle of protozoan parasite of Trypanosoma brucei.

In the eukaryotic pathogen of Trypanosoma brucei, the anaphase promoting complex or cyclosome (APC/C) is composed of ten subunit proteins which are conserved in kinetoplastid protozoan parasites. During the course of APC/C characterization by PTP tagging and mass spectrometry, some other proteins were found to be associated in substoichiometric ratio to APC/C. These proteins could not be assigned as APC/C core components as they are below the threshold imposed by mass spectrometry identification and therefore they are termed non-core APC/C-associated proteins. Here in this study, functional roles of these proteins were investigated through reverse genetics approach. mRNAs of protein-encoding genes were individually knocked down by RNA interference and the resulting phenotypes were assayed through functional assays such as growth curve, cell cycle progression by flow cytometry, and DNA profiles by DAPI staining and microscopy examination. Based on the presented data, these proteins are playing essential functions in the cell biology of T. brucei; and more specifically, in regulating its cell cycle progression. Thus, the non-core APC/C-associated proteins appear to play important roles in complementing APC/C specialized function in the cell cycle of T. brucei.

A minimal anaphase promoting complex/cyclosome (APC/C) in Trypanosoma brucei.

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C's. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.

The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei.

The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.

Functional characterization of cohesin SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei.

Minichromosomes in the nuclear genome of Trypanosoma brucei exhibit unusual patterns of mitotic segregation. To address whether differences in their mode of segregation in relation to large chromosomes are reflected at a molecular level, we characterized two different proteins that have highly conserved functions in eukaryotic chromosomes segregation: the SMC3 protein, a component of the chromatid cohesion apparatus, and the protease separase that resolves the cohesin complex at the onset of anaphase and has, in other organisms, additional functions during mitosis. Using in situ hybridization we show that RNA interference-mediated depletion of SMC3 has no visible effect on the segregation of the minichromosomal population but interferes with the faithful mitotic separation of large chromosomes. In contrast, separase depletion causes missegregation of both mini- and large chromosomes. We also show that SMC3 persists as a soluble protein throughout the cell cycle and only associates with chromatin between G1 and metaphase. Separase is present in the cell during the entire cell cycle, but is excluded from the nucleus until the metaphase-anaphase transition, thereby providing a potential control mechanism to prevent the untimely cleavage of the cohesin complex.