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Anaplasma marginale, Theileria ovis and Theileria lestoquardi are intracellular pathogens that infect a wide variety of animals and cause enormous economic losses worldwide. The present investigation aims to report the occurrence and the phylogeny of these pathogens infecting sheep (N = 330) from Rajanpur District in Punjab (Pakistan) by using msp1b gene for A. marginale and 18 S rRNA for T. ovis and T. lestoquardi. Results revealed that prevalence rates of A. marginale, T. ovis and T. lestoquardi were 7.3%, 6.1% and 1.2%, respectively. Four (1.2%) and one (0.3%) sheep were found to be co-infected with two and three pathogens, respectively. Risk factor analysis revealed that rams were more infected with A. marginale compared to ewes (P = 0.015). In addition, it was observed that animals located in herds having dogs and those in small herds were found more infected, respectively, with T. ovis (P = 0.001) and T. lestoquardi (P = 0.009). Phylogenetic analysis revealed that Pakistani isolates of each detected pathogens clustered together and were closely related to other Pakistani isolates and those from worldwide countries such as Iran, Iraq, Turkey, Sudan, Tanzania, South Africa, and USA. This is the first molecular study reporting the natural infection of Pakistani sheep with these three pathogens. These data need to be taken into account in order to improve the productivity of the livestock sector which is one of main sources of income in the country., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Toxoplasma gondii (T. gondii) is a facultative heterogeneous parasite that belongs to Apicomplexa and can infect almost all warm-blooded animals, including ruminants, birds and humans. To date, no information is available about the molecular investigation of T. gondii in large ruminants from Pakistan. In the present study, prevalence, risk factors and genetic diversity of this parasite were evaluated by using PCR based on ITS-1 gene followed by sequencing of three selected positive PCR products. A total of 310 blood samples from cattle (N = 190) and buffaloes (N = 120) were collected from randomly selected farms located in Rajanpur district in Punjab (Pakistan). The overall infection rates of T. gondii were 12.2% (23/190) and 0% (0/120), respectively, in cattle and buffaloes. All studied epidemiological factors were not found associated with T. gondii infection in cattle. Sequence analysis of our T. gondii isolates infecting cattle revealed only one sequence considered as the most represented genetic variant (GV1) among T. gondii isolates around the world. Phylogenetic analysis revealed that ITS-1 partial sequences of our isolates clustered with those from T. gondii isolates infecting goats and birds from Pakistan and other isolates found in several animal species from different worldwide countries like China, Thailand, Poland, Canada, USA and Brazil. Our report indicates a natural infection with T. gondii of cattle for the first time in Pakistan by using molecular method. This study is important to the design of control strategy against this parasite in order to improve the output of livestock sector which is the main income source of the population in Pakistan., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: In this experimental study, we aimed to assess the acaricidal effects of Elettaria cardamomum L. essential oil (ECEO) against Hyalomma anatolicum tick in cattle from Saudi Arabia. Methods: Gas chromatography-mass spectrometry (GC-MS) was performed to identify the chemical composition of ECEO. The acaricidal, larvicidal, and repellent activity of ECEO against H. anatolicum was studied through the adult immersion test (AIT), the larval packet test (LPT), the vertical movement behavior of tick’s larvae technique, anti-acetylcholinesterase (AChE) activity, and oxidative enzyme activity. Results: By GC/MS, the most compounds were 1,8-cineole (34.3%), α-terpinyl acetate (23.3%), and α-pinene (17.7%), respectively. ECEO significantly (p < 0.001) increased the mortality rate as a dose-dependent response. After ECEO Treatment, number of eggs, egg weight, and hatchability significantly declined as a dose-dependent response. ECEO at concentrations of 5 µL/mL and above completely killed the larva. The LC50 and LC90 values for ECEO were 1.46 and 2.68 µL/mL, respectively. ECEO at concentrations of 10, 20, and 40 µL/mL showed 100% repellency activity up to 60, 120, and 360 min incubation, respectively. ECEO, especially at ½ LC50 and LC50, significantly inhibited GST and AChE activities of H. anatolicum larvae compared to the control group. Conclusions: We found promising adulticidal, larvicidal, and repellent effects of ECEO against H. anatolicum as a vector of theileriosis in Saudi Arabia. We also found that ECEO displayed these activities through inhibiting AChE and GST. Nevertheless, additional investigations are required to confirm the accurate mechanisms and the relevance of ECEO in practical application.

Canine babesiosis is a life-threatening haemoparasitic disease in dogs that is prevalent worldwide. In this study, the prevalence of Babesia vogeli (B. vogeli) was investigated in dogs from Egypt by using Polymerase Chain Reaction (PCR) assay, and associated risk factors were evaluated. In addition, phylogenetic position of B. vogeli Egyptian isolate was determined by sequencing. A total of 275 blood samples were taken from dogs located in four governorates belonging to the north of Egypt. Samples were examined by PCR targeting the B. vogeli 18S rRNA gene and this species was also confirmed by sequencing. Overall, the prevalence of B. vogeli was 5.1% among the studied dogs and the highest prevalence rate was found in the Giza governorate. Univariate logistic regression was used to evaluate each variable individually. The results revealed a significant association between the prevalence of B. vogeli infection and whether or not dogs were infested with ticks and the type of floor used in dog shelters. Additionally, tick infestation (OR 6.1, 95% CI 1.2-31.4), and living in shelters with soil floors (OR 3.8, 95% CI 0.8-17.8) were identified as potential risk factors for B. vogeli infection. Phylogenetic analysis was performed using B. vogeli 18S rRNA partial sequences with the hypervariable V4 region from GenBank. The Egyptian isolate was assigned to second sub-cluster with B. vogeli isolates from Japan, Venezuela and Paraguay within the B. vogeli/B. canis cluster. The present data will be useful to improve the understanding of canine babesiosis epidemiology and ways to control the disease in companion dogs., (© 2022. The Author(s).)

A novel phlebovirus, Punique virus (PUNV), was discovered and isolated in 2008 from sandflies from Northern Tunisia. PUNV is now classified as a unique member of the Punique phlebovirus species within the Phlebovirus genus in the Phenuiviridae family (order bunyavirales). In this study, we aimed to investigate the transmission dynamics of PUNV in Tunisia. Sandflies were collected during two consecutive years, 2009 and 2010, by CDC light traps. In 2009, a total of 873 sandflies were collected and identified to the species level. Phlebotomus perniciosus was the most abundant species. One pool of P. perniciosus females collected in autumn contained PUNV RNA, yielding an infection rate of 0.11%. The population densities of circulating sandfly species were assessed during May-November 2010 in Northern Tunisia by using sticky traps. Phlebotomus (Larroussius) perniciosus (71.74%) was the most abundant species, followed by Phlebotumus (Larroussius) longicuspis (17.47%), and Phlebotumus (Larroussius) perfiliewi (8.82%). The densities of dominant sandfly species were found to peak in early spring and again in the autumn. In 2010, species identification was not performed, and sandflies were only discriminated on the basis of sex and collection date. Out of 249 pools, three contained PUNV RNA. Each positive pool allowed virus isolation. The three pools of female sandflies containing PUNV RNA were collected in autumn with an infection rate of 0.05%. These findings provide further evidence that P. perniciosus is the main vector of PUNV in Tunisia, and this phlebovirus is endemic in Tunisia. Our findings provided strong evidence of intensive circulation of PUNV in sandflies and hosts through a viral infection buildup process between sandfly vectors and hosts starting at the beginning of the activity of sandflies in spring to reach a maximum during the second main peak in autumn.

Hereditary hearing impairment (HI) is a common disease with the highest incidence among sensory defects. Several genes have been identified to affect stereocilia structure causing HI, including the unconventional myosin3A. Interestingly, we noticed that variants in MYO3A gene have been previously found to cause variable HI onset and severity. Using clinical exome sequencing, we identified a novel pathogenic variant p.(Lys50Arg) in the MYO3A kinase domain (MYO3A-KD). Previous in vitro studies supported its damaging effect as a 'kinase-dead' mutant. We further analyzed this variation through molecular dynamics which predicts that changes in flexibility of MYO3A structure would influence the protein-ATP binding properties. This Lys50Arg mutation segregated with congenital profound non-syndromic HI. To better investigate this variability, we collected previously identified MYO3A-KDs variants, p.(Tyr129Cys), p.(His142Gln) and p.(Pro189Thr), and built both wild type and mutant 3 D MYO3A-KD models to assess their impact on the protein structure and function. Our results suggest that KD mutations could either cause a congenital profound form of HI, when particularly affecting the kinase activity and preventing the auto-phosphorylation of the motor, or a late onset and progressive form, when partially or completely inactivating the MYO3A protein. In conclusion, we report a novel pathogenic variant affecting the ATP-binding site within the MYO3A-KD causing congenital profound HI. Through computational approaches we provide a deeper understanding on the correlation between the effects of MYO3A-KD mutations and the variable hearing phenotypes. To the best of our knowledge this is the first study to correlate mutations' genotypes with the variable phenotypes of DFNB30.Communicated by Ramaswamy H. Sarma.

The aim of this study was to enhance the rhizobacterium potential in horizontal subsurface flow constructed wetland (CW) system planted by Phragmites australis using specific and lytic phages. The bioinoculation of specific bacteriophage for target bacteria; Salmonella typhi , and the monitoring of bacterial inactivation under different conditions showed the effectiveness of this methodology to enhance bacteria reduction and consequentially ameliorate purification performance of this studied biological treatment system. The injection of the phage at a concentration equal to 10 3 UFP/mL within the rhizosphere of the inoculated filter (F) was allowed 1 U-Log 10 of improvement of bacterial inactivation compared to the control filter (T) nearly 1 logarithmic unit thus, a 90% improvement of bacteria reduction. When we increased the phage titer (10 5 UFP/mL), the bacterial reduction equal to 2.75 U-Log 10 (N/N 0 ) was registered that corresponds to a decrease of nearly 99.9%. According to the first-order model, the inactivation coefficient is equal to 2.29 min -1 (0.88 min -1 for the first experiment) and the bacterial reduction rate is 5 times higher than that determined for the control filter. This results show the positive impact of the phage in the bacterial inactivation and the improvement of water treatment of the biofilter C.

Species belonging to the genus Anaplasma (Rickettsiales) include bacteria of veterinary and public health importance. Beside the zoonotic Anaplasma phagocytophilum, A. platys, the etiological agent of canine cyclic thrombocytopenia, has been sporadically reported in clinically ill human patients. The ongoing emergence of novel strains related to this species in vertebrate hosts emphasises the need for genetic comparisons among strains identified in different regions of the world. In this paper we developed a PCR test suitable for amplification of the still undescribed gltA gene of Anaplasma strains related to A. platys from Mediterranean ruminants and applied on a panel of 248 samples. gltA sequencing allowed phylogenetic comparison with strains related to A. platys recently identified in China, and strains representative of the Anaplasmataceae family. Results suggest the designation of Candidatus A. turritanum, including Mediterranean A. platys - like strains, and Candidatus A. cinensis, including strains isolated in China. Data generated in this study are a solid reference for future epidemiological studies of novel unclassified strains related to A. platys and for their diagnosis and raise concern on their potential veterinary and public health implications encouraging investigating the suspected unexplored diversity within the genus Anaplasma in animals and human., (Copyright © 2021. Published by Elsevier GmbH.)

This review highlights the diagnostic methods used, the control strategies adopted, and the global epidemiological status of canine cyclic thrombocytopenia and granulocytic anaplasmosis at the animal-human interface. Canine anaplasmosis is an important worldwide disease, mainly caused by Anaplasma platys and A. phagocytophilum with zoonotic implications. A. platys chiefly infects platelets in canids, while A. phagocytophilum is the most common zoonotic pathogen infecting neutrophils of various vertebrate hosts. Diagnosis is based on the identification of clinical signs, the recognition of intracellular inclusions observed by microscopic observation of stained blood smear, and/or methods detecting antibodies or nucleic acids, although DNA sequencing is usually required to confirm the pathogenic strain. Serological cross-reactivity is the main problem in serodiagnosis. Prevalence varies from area to area depending on tick exposure. Tetracyclines are significant drugs for human and animal anaplasmosis. No universal vaccine is yet available that protects against diverse geographic strains. The control of canine anaplasmosis therefore relies on the detection of vectors/reservoirs, control of tick vectors, and prevention of iatrogenic/mechanical transmission. The control strategies for human anaplasmosis include reducing high-risk tick contact activities (such as gardening and hiking), careful blood transfusion, by passing immunosuppression, recognizing, and control of reservoirs/vectors.

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases. In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the "secondary" autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients., (Copyright © 2021. Published by Elsevier Masson SAS.)

Wild rodents are considered as potential carriers of several zoonotic vector-borne bacteria but their epidemiology is poorly understood in Tunisia. A total of 305 biological samples (100 spleens, 100 livers, 100 kidneys, and 5 pooled ectoparasites (Xenopsylla cheopis, Laelaps echidninus, Ornithonyssus sp., Hoplopleura sp. and eggs of the rat fleas)) were collected from 100 wild rodents from three Tunisian governorates. Molecular screening was performed to reveal infections with main vector-borne bacteria. Captured rodents belonged to three rodent genera and species including Rattus rattus (n = 51, 51%), Meriones shawi (n = 24, 24%) and Mus musculus (n = 25, 25%). Examined rodents were found to be heavily infested by the rat flea X. cheopis (n = 32, 47%) and the rat mite L. echidninus (n = 22, 32.3%). However, the rat mite Ornithonyssus sp. (n = 13, 19.1%) and the rat lice Hoplopleura sp. (n = 1, 1.5%) were rarely identified. Based on 16S rRNA and msp4 genes, infection with Anaplasmataceae bacteria was detected in six specimens of R. rattus and one M. shawi. Pathogenic A. phagocytophilum (n = 1), A. phagocytophilum-like 1 (Anaplasma sp. Japan) (n = 1), and A. ovis (n = 5) were identified. On the basis of ompB, ompA and gltA genes, infection with Rickettsia spp. was identified in three specimens of R. rattus and one of M. shawi. Five Rickettsia species of the spotted fever group, corresponding to R. monacensis, R. helvetica, R. massiliae, R. africae, and R. aeschlimannii, were detected in mixed infections. Bartonella henselae DNA was also found in two R. rattus, based on rpoB partial sequences. All revealed Anaplasma, Rickettsia and Bartonella bacteria were detected in spleen samples. Ehrlichia, Coxiella and Borrelia spp. were not identified in any of the tested samples. In Tunisia, this is the first report indicating infections with Anaplasma, Rickettsia and Bartonella spp. in wild rodents, particularly present alongside domestic livestock and human. This represents a serious risk of potential bacterial transmission. Thus, controlling rodent population in animal herds, residential areas and sensitizing local people to this risk seem absolutely necessary., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells. A. marginale DNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multiple A. marginale worldwide strains., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Belkahia, Ben Abdallah, Andolsi, Selmi, Zamiti, Kratou, Mhadhbi, Darghouth, Messadi and Ben Said.)

Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common inherited sensory impairment. It is particularly frequent in North African populations who have a high rate of consanguineous marriage. The c.242G>A homozygous variant in LRTOMT gene was previously established as pathogenic and is associated with NSHL in both humans and mice. The aim of this study is to determine the carrier frequency for the LRTOMT c.242G>A variant and also to estimate its age in addition to evaluating its diagnostic potential as a deafness biomarker among various populations and ethnicities in Northern African countries. A total of 179 Tunisian and 34 Libyan unrelated deafness patients were screened for this variant. The homozygous c.242G>A variant was found in 5.02% and 2.94% in Tunisian and Libyan families, respectively. Subsequent screening for this variant in 263 healthy controls of various ethnicities (136 Tunisian Berbers, 32 Andalusian and 95 Tunisian from undefined ethnic origin) revealed higher frequency for the heterozygous state among Tunisians of Berber origin only (19.11%). Genotyping 7 microsatellite markers nearby the variant location in ARNSHL patients who had the homozygous variant revealed the same haplotype suggesting a common founder origin for this variant. The age of this variant was estimated to be between 2025 and 3425 years (this corresponds to 3400 years when the variant rate was set at 10 -3 or 2600 years when the variant rate is set at 10 -2 ), spreading along with the Berber population who migrated to North Africa. In conclusion, the LRTOMT c.242G>A homozygous variant could be used as a useful deafness biomarker for North African ARNSHL patients meanwhile the heterozygous variant could be utilized in genealogical studies for tracing those of the Berber ethnic group., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Bovine anaplasmosis is an infectious disease with worldwide distribution. It spreads by various routes mainly through tick bites., Aims: This study aimed to investigate bovine related Anaplasma spp. in cattle from three northern governorates of Egypt by serological and molecular assays, to evaluate the associated risk factors and to analyze the phylogeny of revealed A. marginale isolates., Methods: During 2020, a total of 650 blood samples were collected from asymptomatic cattle in the governorates of Kafr El-Sheikh (n=240), Menofia (n=230), and Al-Gharbia (n=180). Sera samples were examined using the Anaplasma antibody test kit, cELISA v2. Blood genomic DNA of seropositive cattle was then examined by PCRs specific to A. marginale , A. centrale , and A. bovis . Selected positive samples were subjected to nucleotide sequencing. Risk factors (i.e. geographical area, breed, type of production, sex, age, herd size, season, husbandry system, tick infestation, and application of acaricides) were evaluated by logistic regression approach., Results: In total, 130 cattle (20%, 95% CI: 17.1-23.3) were recorded seropositive for Anaplasma species. Major risk factors associated with seropositivity were being crossbred, dairy cattle, aged more than 5 years, summer season, herd size of below 300, pasture grazing, tick infestation, and not being subjected to regular treatment with acaricides. By using species-specific PCR, only A. marginale was detected. Nucleotide sequencing showed the occurrence of two different msp4 genotypes., Conclusion: This study shows the high prevalence of A. marginale in cattle of Kafr El-Sheikh, Al-Gharbia, and Menofia. However, the connection between Anaplasma species and their tick vectors remains unknown in Egypt and merits further investigations. Since these infections primarily spread through ixodid tick bites, effective ectoparasite control strategies, regular examination of cattle and successful chemoprophylaxis are recommended., Competing Interests: The authors declare no conflict of interest.

Background: Variants in the HARS2 gene have been reported to be associated with nonsyndromic hearing loss (HL) and Perrault syndrome (PS), a rare recessive disorder marked by bilateral sensorineural HL and ovarian dysgenesis. Given the low number of pathogenic variants described in the HARS2 gene, no genotype/phenotype correlations have been established between variants in this gene and the clinical data. Materials and Methods: Whole blood was collected from four members of a Lebanese family with PS. An affected woman was evaluated for HL by clinical examination and audiological tests. Primary ovarian failure was analyzed according to age of primary or secondary amenorrhea, follicle stimulating hormone levels, and pelvic ultrasound. The existence of neurological symptoms and other associated conditions was checked. To identify the causative variant, we used a custom HaloPlex HS panel for next-generation sequencing of the coding sequences of six genes implicated in this syndrome. Results: We identified a novel homozygous HARS2 missense variant (c.260G>A; p.Arg87His), which is only the second homozygous variant in the HARS2 gene identified to date worldwide. This variant is predicted to be deleterious by multiple in silico analysis tools, moreover the Arg87 amino acid nearly is invariant among eight species. Based on molecular modeling analysis, this variation is predicted to disturb the proper folding of HARS2, which may reduce its aminoacylation efficiency. Clinical data are compared with the other cases recorded in the literature to help gain further knowledge with regard to the phenotype. Conclusion: Our results provide strong evidence corroborating the etiological association of this mutation with the HARS2-PS phenotype. HARS2 variants need to be searched for in patients with early-onset bilateral sensorineural HL and ovarian dysfunction in women so as to guarantee accurate endocrinological surveillance and management to minimize secondary complications.

Background: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species., Methodology: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples., Findings: Sequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex. A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples., Conclusion: The study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance., Competing Interests: The authors have declared that no competing interests exist.

We purpose in this study to develop a reliable and low-cost method for the detection of Viable but nonculturable (VBNC) bacteria. Indeed, after water disinfection, injured-VBNC bacteria can be underestimated using conventional assessment methods, causing false-negative results and, posing a significant and potential health risk. The VBNC bacterial survival strategy can hide the real microbial quality of treated water. To overcome this bacterial assessment limitation, we were used a specific and lytic phage to monitor the presence of active bacteria; Pseudomonas aeruginosa after photocatalytic treatment. Within 2 h of phage-target bacteria contact, the reduction of phage amplification rate ( A t ) can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of new infectious phages release despite their lose of cultivability in the usual media. The determination of phage reactivation coefficient ( R t ) after 2 and 8 h of phage-target cell contact time reveals the ability of phages to reactive their infectivity and their amplification in positive correlation with their host cells viability and activity. The increase in phage reactivation coefficient ( R t ) after an extension of the latent period was directly related to the positive interaction between infectious phages and potential active bacteria. The use of this method can improve the water disinfection process and avoid public health-hazardous especially related to the resuscitation of active-nonculturable bacteria mainly for pathogenic bacteria.

Tick-borne rickettsioses present a significant public health threat among emerging tick-borne diseases. In Tunisia, little is known about tick-borne Rickettsia pathogens. Therefore, the aim of this study was to investigate the presence of Rickettsia species in small ruminant ticks from Tunisia. Adult ticks ( n = 694) were collected from goats and sheep in northern Tunisia. Obtained ticks were identified as Rhipicephalus turanicus ( n = 434) and Rhipicephalus sanguineus sensu lato ( n = 260). Selected ticks ( n = 666) were screened for the presence of Rickettsia spp. by PCR targeting a partial sequence of the ompB gene followed by sequence analysis. Rickettsial DNA was detected in 122 (18.3%) tested tick samples. The infection rates in Rh. turanicus and Rh. sanguineus s.l. ticks were 23.4 and 9.5%, respectively. The overall prevalence of rickettsial DNA was markedly higher in ticks collected from goats (23.2%) compared to those infesting sheep (7.9%). The detection of rickettsial DNA was significantly higher in ticks from the governorate of Beja (39.0%) than those from the governorate of Bizerte (13.9%). Two additional genes, the outer membrane protein A gene ( ompA ) and the citrate synthase gene ( gltA ), were also targeted for further characterization of the detected Rickettsia species. Genotyping and phylogenetic analysis based on partial sequences ( n = 106) of the three different genes revealed that positive ticks are infected with different isolates of two Spotted Fever Group (SFG) Rickettsia , namely, Rickettsia massiliae and Rickettsia monacensis , closely related to those infecting camels and associated ticks from Tunisia, and humans and small ruminant ticks from neighboring countries like Italy, France, and Spain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Belkahia, Selmi, Zamiti, Daaloul-Jedidi, Messadi and Ben Said.)

Pathogenic variants in Steroid 5 alpha reductase type 3 (SRD5A3) cause rare inherited congenital disorder of glycosylation known as SRD5A3-CDG (MIM# 612379). To date, 43 affected individuals have been reported. Despite the development of various dysmorphic features in significant number of patients, facial recognition entity has not yet been established for SRD5A3-CDG. Herein, we reported a novel SRD5A3 missense pathogenic variant c.460 T > C p.(Ser154Pro). The 3D structural modeling of the SRD5A3 protein revealed additional transmembrane α-helices and predicted that the p.(Ser154Pro) variant is located in a potential active site and is capable of reducing its catalytic efficiency. Based on phenotypes of our patients and all published SRD5A3-CDG cases, we identified the most common clinical features as well as some recurrent dysmorphic features such as arched eyebrows, wide eyes, shallow nasal bridge, short nose, and large mouth. Based on facial digital 2D images, we successfully designed and validated a SRD5A3-CDG computer based dysmorphic facial analysis, which achieved 92.5% accuracy. The current work integrates genotypic, 3D structural modeling and phenotypic characteristics of CDG-SRD5A3 cases with the successful development of computer tool for accurate facial recognition of CDG-SRD5A3 complex cases to assist in the diagnosis of this particular disorder globally., (© 2021 Wiley Periodicals LLC.)

In this study, we propose the development of a rapid and reliable method to control and to monitor microbial water quality. The methylene blue (MB) decolorization assay was based on the analysis of spectral profiles of dye in interaction with a different bacterial concentration. The determination of dye decolorization rate (DDR) shows a correlation between the MB reduction rate and the bacterial density. Moreover, the kinetic of the monomer and dimer equilibrium of MB in water mainly, the monitoring of bounded MB species in relationship with a knowed concentration of target bacteria, was allowed to establish a relationship between MB decolorization rate and bacterial density. Furthermore, this method was applied to evaluate the water quality after photocatalysis. Based on this method, the photocatalytic effects on bacterial density was highlighted by the decrease in DDR after photocatalytic treatment with fractioned times (0 to 5 h); this increase was followed by a decrease of bounded MB species and, an increase in free MB forms miming the reduction of bacterial density due to the biocide effects of photocatalysis process. However, the analysis of spectra profiles shows a weak but a continuous decrease in bounded MB dimer and monomer forms in the treated water samples exempt of culturable bacteria. Moreover, the MB spectra profiles were tended toward a negative control spectrum without superposition. Thus, the possibility of the presence of viable but non-culturable bacteria was expected; therefore, to optimize this tertiary water treatment process, an extending on proceeding time was recommended to avoid the bacterial resuscitation after photocatalysis.

Introduction: Hearing impairment (HI) is characterized by complex genetic heterogeneity. The evolution of next generation sequencing, including targeted enrichment panels, has revolutionized HI diagnosis., Objectives: In this study, we investigated genetic causes in 22 individuals with non- GJB2 HI., Methods: We customized a Haloplex HS kit to include 30 genes known to be associated with autosomal recessive nonsyndromic HI (ARNSHI) and Usher syndrome in North Africa., Results: In accordance with the ACMG/AMP guidelines, we report 11 pathogenic variants; as follows; five novel variants including three missense ( ESRRB- Tyr295Cys, MYO15A -Phe2089Leu and MYO7A -Tyr560Cys) and two nonsense (USH1C- Gln122Ter and CIB2- Arg104Ter) mutations; two previously reported mutations (OTOF- Glu57Ter and PNPT1 -Glu475Gly), but first time identified among Tunisian families; and four other identified mutations namely WHRN- Gly808AspfsX11, SLC22A4 -Cys113Tyr and two MYO7A compound heterozygous splice site variants that were previously described in Tunisia. Pathogenic variants in WHRN and CIB2 genes, in patients with convincing phenotype ruling out retinitis pigmentosa, provide strong evidence supporting their association with ARNSHI. Moreover, we shed lights on the pathogenic implication of mutations in PNPT1 gene in auditory function providing new evidence for its association with ARNSHI. Lack of segregation of a previously identified causal mutation OTOA -Val603Phe further supports its classification as variant of unknown significance. Our study reports absence of otoacoustic emission in subjects using bilateral hearing aids for several years indicating the importance of screening genetic alteration in OTOF gene for proper management of those patients., Conclusion: In conclusion, our findings do not only expand the spectrum of HI mutations in Tunisian patients, but also improve our knowledge about clinical relevance of HI causing genes and variants., Competing Interests: The authors declared that there is no conflict of interest., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)

Bartonellosis is one of the clinically underdiagnosed emerging bacterial diseases among domestic livestock, particularly in camels. Until now, the natural infection of camels with Bartonella species was not investigated in Tunisia. In the attempt of filling this gap in knowledge, a total of 412 dromedary camels (Camelus dromedarius) as well as 300 associated ticks (Hyalomma dromedarii (160; 53.4%), H. impeltatum (131; 43.6%) and H. excavatum (9; 3%) were screened for the presence of Bartonella spp. by PCR followed by a sequencing step through the amplification of the rpoB gene. Positive samples were then tested and further characterized by the combined use of the ftsZ and gltA genes. Fifteen camels (3.6%) were found to be positive to Bartonella spp. However, there was no evidence of Bartonella DNA in any of the analyzed ticks. Risk factors' analysis shows that camels derived from arid and semi-arid bioclimatic areas were more infected than those originated from desert area. Molecular characterization and phylogenetic analysis revealed the occurrence of novel B. henselae genotypes closely related to those isolated from humans, cats, and lions. By combining the characteristics of each single gene with those of concatenated sequences, we report here the first molecular detection of B. henselae in the dromedary camel suggesting a possible involvement of camelids as hosts or reservoirs in the transmission cycle of this emerging bacterium in arid and saharan areas., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anaplasma ovis, the causative agent of ovine anaplasmosis in tropical and subtropical countries, is a tick-borne obligatory intraerythrocytic bacterium of sheep, goats and wild ruminants. In Tunisia, data about the molecular phylogeny and the genetic diversity of A. ovis isolates are limited to the analysis of msp4 and groEL genes. The aim of this study was to genetic characterize 40 A. ovis isolates infecting 28 goats, 10 sheep, one camel and one Rhipicephalus turanicus tick located in different geographic regions of Tunisia on the basis of 3 partial genes (gltA, groEL and msp1a). Sequence analysis revealed 6 and 17 different genotypes in the partial gltA and groEL genes, respectively. Phylogenetic analysis revealed, as expected for the groEL gene, that sequences from small ruminants and their infesting ticks clustered separately from those isolated from camels. The analysis of amino-acid Msp1a sequences identified 18 novel genotypes of Msp1a repeats from 20 A. ovis isolates. These Msp1a repeats were highly variable with 33-47 amino-acids, and the number of repeats is one for 19 isolates infecting 18 goats and one R. turanicus tick, and 4 for a single isolate found in one sheep. Phylogenetic trees based on Msp1a partial sequences revealed that the N-terminal region of Msp1a protein appear to be relatively more informative phylogeographically compared to other markers especially according to countries. The presented data give a more detailed knowledge regarding the molecular phylogeny and the genetic diversity of A. ovis isolates occurring in different animal species and their associated ticks in Tunisia., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Rift Valley fever (RVF) is a mosquito-borne zoonosis that severely impacts livelihoods, national and international economies, and human health. Few studies have investigated the prevalence of this infection in Tunisian livestock. The present report aimed to update the epidemiological status and identify the risk factors associated with this RVF virus infection in the one-humped dromedary camel from arid areas. A total of 470 sera of apparently healthy camels (Camelus dromedarius) were collected from six governorates from southern and central Tunisia. Samples were tested by a competitive Enzyme Linked Immunosorbent Assay (ELISA). An overall, 162 camels (34%, 95%CI: 0.1-0.4) were seropositive to RVF virus antigen. Logistic regression model revealed three potential risk factors associated with the infection. A meaningful high seropositivity was observed among aged camels (>10 years-old) (40%) (P=0.001; OR=3.367). Besides, camels raised in small flocks particularly intended for meat production showed a high level of seropositivity (37%) (P=0.013; OR=13.173). Animals having close contact with other ruminants showed high seroprevalence (37%) (P=0.022; OR=10.919). This report indicated that Tunisian one-humped dromedaries were exposed to this virus and may contribute to its dissemination among farmers and other livestock. Furthers studies are urgently required to isolate and characterize this virus, evaluate the potential risk of human infection particularly in farmers, veterinarians and slaughterhouse workers and finally to program a serious strategy for RVF control., Competing Interests: Declaration of Competing Interest The authors declared that they have no potential conflicts of interest with respect to the research, authorship, and publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

TEDIS, an information system dedicated to patients affected with neuro-developmental disorders including autism, focuses on patient data generated during in-depth clinical assessment in nine expert centers in Ile-de-France region. Long term partnership involving methodologists and domain experts is necessary to support quality data production and analyses and to guarantee quality data and information governance in a domain characterized by frequent evolutions in clinical assessment instruments and in diagnostic criteria and classification.

Rickettsia species are adapted to a wide range of specific animal hosts. Camels (Camelus dromedarius) have been identified as a carrier of various zoonotic pathogens and became a focus of growing public health interest. This study reported the occurrence of rickettsial infection in camels and infesting ticks from five Tunisian governorates. Based on ompB PCR, eight out of 293 camels (2.7%) were found to be infected with Rickettsia spp. Furthermore, 13 tick specimens of Hyalomma impeltatum (10.4%) and 9 of H. dromedarii (8.0%) harboured DNA of Rickettsia bacteria with an overall prevalence rate of 9.2% (22/237). Molecular prevalence of Rickettsia infection varied significantly according to tick infestation for camels and among tick genders. Five rickettsial species, showing a potential public health interest, were revealed by sequencing. Based on ompB partial sequences, five species were identified corresponding to R. aeschlimannii, R. monacensis, R. helvetica and R. massiliae in camels and to R. africae, R. aeschlimannii, R. monacensis and R. helvetica in ticks. Based on ompA typing, three species were revealed corresponding to R. africae and R. monacensis in camels and to R. africae, R. aeschlimannii and R. monacensis in ticks. This is the first report consolidating the hypothesis that camels may serve as potential hosts for Rickettsia spp. and Hyalomma spp. ticks as possible vectors in arid and Saharan areas of Tunisia. The present data highlight the importance of preventive measures and survey that must be implemented in camel herds in order to limit the spread of these vector-borne bacteria to animals and humans., (© 2019 Blackwell Verlag GmbH.)

The genus Anaplasma currently comprises 6 bacterial species mostly pathogenic to animals and/or human, including the zoonotic species Anaplasma phagocytophilum, the causative agent of tick-borne fever (TBF) of ruminants, and of granulocytic anaplasmosis of horses, dogs and human. Recently, novel potentially non-pathogenic strains related to A. phagocytophilum have been identified in Japan, China, and Tunisia. This paper reports the identification, molecular typing, and evolutionary history of novel Anaplasma strains (A. phagocytophilum-like 1 and 2), related to but distinct from A. phagocytophilum in Mediterranean area of Europe and Africa. PCR-RFLP and phylogenetic analyses based on 16S rRNA provided evidence for the circulation of A. phagocytophilum-like 1 strains in Europe. Phylogeny based on groEL gene showed the inclusion of Sardinian and Tunisian A. phagocytophilum-like 1 strains in a unique clade distinct from, but related to that of Japanese strains. Results suggest that genetic diversity within the genus Anaplasma is much greater than expected and provide information useful for the development of specific and effective diagnostic and prophylactic tools., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest associated with the current study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To date, no information is available regarding the infection of camels (Camelus dromedarius) by Anaplasma ovis in North African region. Several animal species can be infected by A. ovis which further complicates its natural infection cycle. In this paper, we investigated the occurrence and the genetic diversity of A. ovis in camels and ticks collected from them in Tunisia and the risk factor analysis. Camel blood samples (n = 412) and tick (n = 300) samples, identified as Hyalomma dromedarii (n = 149, 49.6%), H. impeltatum (n = 142, 47.3%) and H. excavatum (n = 9, 3%), were analyzed by conventional PCR followed by the sequencing of msp4 and groEL genes. A. ovis DNA was identified in five camels (1.2%), but not in infesting ticks (0%). The microscopic examination revealed the specific infection of camel erythrocytes by Anaplasma inclusions. The msp4 and groEL typing confirmed the natural infection of camels by A. ovis and revealed two different msp4 genotypes earlier detected in Tunisian small ruminants and their infested ticks, and five different and novel groEL genetic variants forming a separately sub-cluster within A. ovis cluster. The occurrence of different A. ovis strains specific to camels associated with a low prevalence of this Anaplasma species in camels may enrich knowledge regarding the distribution and the transmission cycle of this bacterium in arid and Saharan areas of Tunisia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys. Understanding the phylogenetic relations among these species is fundamental to perform an accurate identification and an informative intra-specific analysis. Heat shock groESL operon is frequently employed in phylogenetic analysis of Anaplasma species and, for the most cases, the use of partial sequences of this operon is randomly done without knowing the most appropriate regions to be used either in species attribution or in intra-specific diversity analysis. In this study, on the basis of all fully and nearly complete groESL sequences available in the GenBank, we firstly selected a minimum partial length sequence which allows species delineation and gives a similar topology to that found by analyzing the complete sequence. By using other in silico analyses, we obtained two minimal partial sequences that are the most interesting to describe intra-specific diversity within A. ovis and A. centrale. Our results raise concern on the use of randomly selected partial sequences of groESL operon employed for the detection and the characterization of Anaplasma species and provide additional background about minimum length groESL operon required for Anaplasma species attribution and strains diversity analysis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tick-borne bacteria are considered to be emerging in camels, but data about their occurrence in Tunisian dromedaries and their infesting ticks remain scarce. In this study, 412 camel blood samples and 327 partially engorged ticks were collected and tested for the presence of Coxiella burnetii and/or related strains, and Rickettsiales bacteria. Coxiella burnetii was detected in 9 Hyalomma impeltatum and 3 H. dromedarii with an overall prevalence rate of 3.6% (12/327). Candidatus Midichloria mitochondrii DNA was identified in 16 H. impeltatum and 10 H. dromedarii with an overall prevalence rate of 8% (26/327). Six ticks (2%) were found to be co-infected with these two bacteria. No positive DNA camel blood sample was observed for both bacteria. Genotyping and phylogenetic analysis of obtained C. burnetii partial sequences based on the IS1111 and htpB genes revealed 99-100% similarity to the pathogenic C. burnetii strains isolated from humans, ruminants and ticks, and that were genetically distant to those isolated from all endosymbiotic related strains (Coxiella-like bacteria). The analysis of the rickettsial partial sequences of the 16S rRNA gene showed 100% similarity to Ca. M. mitochondrii strains infecting Ixodid ticks and dogs. This is the first time that C. burnetii and Ca. M. mitochondrii have been detected in ticks from Tunisia, which raises the possibility of the involvement of Hyalomma tick species in the active diffusion of these bacteria among camels, other domestic animals and humans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Livestock constitute habitual hosts and carriers for several infectious pathogens which may represent a serious public health concern affecting the readiness of military forces and lead to wide economic losses. The present report aimed to investigate the prevalence of some haemopathogens infecting military livestock, particularly, dromedaries, sheep and horses using Giemsa-stained blood smears. A total of 300 animals (100 from each species) were selected, clinically examined and sampled. Trypanosoma spp. (22.0%), Anaplasma spp. (17.0%) and Babesia spp. (1.0%) were identified in camels' blood. Six dromedaries were found to be co-infected by Trypanosoma and Anaplasma organisms (6.0%). Camels of female gender, infested by ticks and showing clinical signs were statistically more infected by Trypanosoma spp., compared to those of male gender, free of ticks and apparently healthy (P= 0.027, 0.000 and 0.004, respectively). Babesia spp. infection (1.0%) was identified, for the first time in Tunisia, in one adult female camel that presented abortion and anemia. Anaplasma spp. was the only haemopathogen identified in examined sheep (6.0%) and horses (17.0%). Horses infested by Hippobosca equina flies and sheep infested by Rhipicephalus turanicus ticks were more infected by Anaplasma spp. than other non-infested animals (P=0.046 and 0.042, respectively). Hyalomma dromedarii, H. impeltatum and H. excavatum were the most prevalent diagnosed ticks removed from camels with an intensity of infestation of 1.2 ticks per animal. However, in sheep, only R. turanicus was identified. H. equina and Tabanus spp. were the potential hematophagous flies found in dromedaries and horses herds. This useful data must be taken into consideration during animal treatment and vectors' control programs in Tunisian military farms which help to limit the diffusion of vector-borne diseases, keep our livestock healthy and reduce economic losses.

In this study, we have monitored the potential activity of a foodborne and waterborne pathogenic bacterium, Salmonella typhi, under starvation conditions. The interaction between lytic phage and starved-VBNC pathogenic bacteria was studied to establish reliable methods for the detection of active cells before resuscitation. The analysis of phage kinetic parameters has demonstrated the flexibility of lytic with the quantity and mainly the quality of host cells. After 2 h of phage-starved-VBNC bacteria interaction, the reduction of phage amplification rate can reveal the ability of specific-lytic phage to recognize and to attach to their host cells with a probability of burst and release of infectious phages by active bacteria. After an extension of the latent period, the boost of the phage amplification rate was directly related to the positive interaction between potential intracellular 'engaged' phages and potential active bacteria. Furthermore, the modeling of the Salmonella-specific phage growth cycle in relationship with starved host cells can highlight the impact of the viability and the activity state of the host cells on the phage's growth cycle.

Background/aims: Mandibular angle fractures fixated with plate osteosynthesis techniques have to withstand the effects of muscle attachments. Individual variations in the craniofacial morphology may alter the biomechanical resistance of the bone-plate construct. The aim of the present study was to determine the influence of variations in the mandibular plane angle (MPa) on the biomechanical stability of sheep mandibular angle fractures (MAFs)., Materials and Methods: Sixty sheep hemi-mandibles were used. The mandibles were positioned on a test jig that simulated low (15°, group L), normal (25°, group N), and high (35°, group H) MPa. Unfavorable MAFs were created with thin diamond cutting disks. One four-hole, 9.0-mm-spacing, standard titanium miniplate of 2.0 mm thickness and 5.0-mm-long screws were inserted at the superior border of the alveolar bone in monoplanar orientation. Specimens were then subjected to vertical loads between 10 N and 150 N in a universal testing machine. The displacement values at each 10 N force increment and the load magnitude at which 3.0 mm displacement limit was reached were recorded., Results: Starting from 40 N, the displacement values at each 10 N increment in the H group were significantly higher than those of the L and N groups until 150 N (P < 0.05). The force magnitude required to reach 3.0 mm of displacement in the H group was significantly lower than that for the L and N groups (P < 0.05 for each)., Conclusions: The one-miniplate monoplanar fixation technique used in sheep MAF with high MPa is more likely to offer lower biomechanical resistance to the vertical forces applied over the molar region than do the normal and low MPa., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In Tunisia, most of Anaplasma species and unclassified strains have been detected in several animals, but data on the occurrence of Anaplasma spp. in ticks are still lacking. In this study, we report the molecular evidence, genetic characterization and phylogeny of Anaplasma spp. in ticks collected from small ruminants. A total of 395 ticks (178 males and 179 females) were collected from sheep (n = 215) and goats (n = 180). Tick species were identified as 232 Rhipicephalus turanicus, 99 Rhipicephalus sanguineus sensu lato, 34 Rhipicephalus bursa and 30 Rhipicephalus annulatus. Overall infection rate of Anaplasma spp. was 5.6% (20/357 analyzed ticks). All positive ticks were collected from goats and found to be infected by A. ovis. R. turanicus is the most infected tick species by A. ovis (7.9%) followed by R. sanguineus s.l. (2.5%) with an absence of infection in R. bursa and R. annulatus. A. ovis prevalence rate varied significantly according to bioclimatic areas and geographic regions. GroEL typing and phylogenetic analysis revealed that these analyzed ticks are infected with various and novel strains of A. ovis. The use of PCR-RFLP method complemented with sequencing and phylogenetic analysis based on 16S rRNA gene confirm that one R. turanicus tick, positive to A. ovis, is co-infected with A. phagocytophilum-like 2 (0.3%). Specific A. phagocytophilum, A. phagocytophilum-like 1, A. marginale, A. centrale, A. bovis, and A. platys and related strains were not detected in any of the tested ticks. Present data expand knowledge about tick-borne bacteria present in ticks and further clarify the transmission cycles of these bacteria and their different elements in Tunisia., (Copyright © 2018. Published by Elsevier B.V.)

The main goal of the present study was to enhance the rhizobacterium potential in a horizontal subsurface flow constructed wetland system planted with Phragmites australis, through environmentally friendly biological approaches. The bioinoculation of antagonist bacteria has been used to promote higher rhizosphere competence and improve pathogenic bacteria removal from wastewater. The experiment was performed both with single and sequential bioinoculation. The results showed that strain PFH 1 played an active role in pathogenic bacteria removal, remarkably improving inactivation kinetics of the pathogenic tested bacterium Salmonella typhi in the plant rhizosphere. The single bioinoculation of selected bacteria into the rhizosphere of P. australis improved the kinetics of S. typhi inactivation by approximately 1 U-Log 10 (N/N 0 ) (N is the number of viable cultured bacteria at time t, N 0 is the number of viable and cultivable bacteria at time t 0 ) compared to the control. By a series of multi-bioinoculations, the enhancement of pathogenic bacteria reduction compared to the inhibition rate in the pilot-scale control was of 2 U-Log 10 (N/N 0 ). These findings suggested that this strain represents a promising candidate to enhance water purification in constructed wetlands.

Q fever, caused by Coxiella burnetii, is a zoonotic disease responsible of abortion in ruminants. Few studies have investigated the prevalence of this infection in camels (Camelus dromedarius). The present report aimed to highlight the epidemiological status and identify the risk factors associated with C. burnetii infection in one-humped dromedary that is the most productive livestock species in arid areas. A total of 534 sera of healthy camels were collected in eight governorates from southern and central Tunisia. Samples were tested by an indirect Enzyme linked Immunosorbent Assay (ELISA). Results were analyzed using the Chi-square test and logistic regression. Overall, 237 camels (44%, 95%CI: 0.40-0.49) were seropositive to C. burnetii. Statistical analysis pointed out four potential risk factors associated with infection. A meaningful high seropositivity was observed in female camels with a previous history of abortion (70%) (OR = 4.186, 95%CI: 2.05-8.51). Seroprevalence was higher in aged camels (>10 years-old) (48%) (OR = 2.91, 95%CI: 1.37-6.17). Besides, camels, intended for meat production from small herds showed a high level of infection (52%) (OR = 2.43, 95%CI: 1.3-4.5). Coxiellosis evolved in dromedary herds throughout the year, however infection was significantly important in autumn (60%) (OR = 4.13, 95%CI: 1.86-9.17) and winter (56%) (OR = 5.52, 95%CI: 2.50-12.16). Bioclimatic stage, gender, tick infestation and contact with other ruminants were not risk factors in camel's infection by C. burnetii. Our reports confirm that Tunisian one-humped dromedaries had been exposed to this bacterium and could contribute to its dissemination among farmers and other livestock animals. Furthers studies are required to evaluate the prevalence of Q fever among people professionally exposed like farmers, veterinarians and slaughterhouse workers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

ZnO nanorods (NRs) with an average length and diameter of 186 and 20 nm, respectively, were prepared through a mild solvothermal route and used as photocatalysts either as dispersed powder or immobilized on glass slides. The ZnO NRs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Dispersed ZnO NRs and, to a lesser extent, immobilized ZnO NRs were demonstrated to exhibit high photocatalytic activity under simulated sunlight of low intensity (5.5 mW/cm²) both for the degradation of the Orange II dye and for Escherichia coli bacterial decontamination (2.5-fold survival decrease after 180 min irradiation for immobilized NRs). SEM, atomic force microscopy (AFM), fluorescence spectroscopy, and epifluorescence microscopy demonstrate that cell surface damages are responsible of bacterial inactivation. The immobilized ZnO NRs could be reused up to five times for bacterial decontamination at comparable efficiency and therefore have great potential for real environmental applications.

Anaplasma marginale, which is responsible for bovine anaplasmosis in tropical and subtropical regions, is a tick-borne obligatory intraerythrocytic bacterium of cattle and wild ruminants. In Tunisia, information about the genetic diversity and the phylogeny of A. marginale strains are limited to the msp4 gene analysis. The purpose of this study is to investigate A. marginale isolates infecting 16 cattle located in different bioclimatic areas of northern Tunisia with single gene analysis and multilocus sequence typing methods on the basis of seven partial genes (dnaA, ftsZ, groEL, lipA, secY, recA and sucB). The single gene analysis confirmed the presence of different and novel heterogenic A. marginale strains infecting cattle from the north of Tunisia. The concatenated sequence analysis showed a phylogeographical resolution at the global level and that most of the Tunisian sequence types (STs) formed a separate cluster from a South African isolate and from all New World isolates and strains. By combining the characteristics of each single locus with those of the multi-loci scheme, these results provide a more detailed understanding on the diversity and the evolution of Tunisian A. marginale strains., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The genus Anaplasma belonging to the Anaplasmataceae family (order Rickettsiales) comprises obligate intracellular Gram-negative bacteria of veterinary and public health importance. Six species and five types of strains genetically related are currently assigned to the genus Anaplasma including Anaplasma marginale, A. centrale, A. bovis, A. phagocytophilum, A. ovis and A. platys as classified species, and "A. capra", A. odocolei sp. nov., A. phagocytophilum-like 1 (Anaplasma sp.-Japan), A. phagocytophilum-like 2 (Anaplasma sp.-China) and A. platys-like (also named Candidatus Anaplasma camelii) as unclassified strains. Most of these Anaplasma species and strains have been molecularly identified in several animal and/or tick species in the north of Africa. The aim of this review is to summarize the current knowledge about molecular epidemiology, associated risk factors and genetic diversity of Anaplasma species and related strains infecting animals and/or their incriminated tick vectors in North Africa. All these data should be considered when establishing of common management and control programs for anaplasmosis infecting humans and different animal species in North African countries., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The aim of this work was the genetic characterization of cefotaxime-resistant enterobacteria from animals (53 samples), the surface water of rivers (17 samples), and wastewater treatment plants (43 samples) in Tunisia. A total of 48 (42.4%) cefotaxime-resistant isolates were recovered. An extended spectrum beta-lactamase (ESBL) phenotype with a positive double-disk synergy test (DDST) was exhibited by 34 (70.8%) and 14 (29.1%) isolates from water and animal origins, respectively. Isolates from water were identified as: Escherichia coli (n = 17), Hafnia spp. (n = 13), Citrobacter spp. (n = 1), Enterobacter cloacae (n = 1), Klebsiella pneumoniae (n = 1), and K. oxytoca (n = 1). Animal isolates were identified as: E. coli (n = 11), E. cloacae (n = 1), Hafnia spp. (n = 1), and K. pneumoniae (n = 1). PCR investigation of blaCTX-M, blaTEM, and blaSHV genes showed that amongst the 48 isolates with a positive DDST, 41 (87.5%) carried the blaCTX-M gene, 1 isolate harbored the blaSHV gene, and 1 isolate coharbored blaCTX-M with blaSHV genes. The class 1 and 2 integrons were detected in 27 (56.2%) and 1 (2%) isolates, respectively. Our study showed a significant occurrence of ESBL-producing enterobacteria in animals and aquatic environments with a predominance of blaCTX-M genes., (© 2019 S. Karger AG, Basel.)

The present study aimed to estimate the molecular prevalence of Theileria annulata and Trypanosoma evansi infection in cattle in Northern Tunisia. A total number of 96 cattle from five farms were evaluated. T. annulata and T. evansi prevalences were 61% [56/66] and 10% [7/13], respectively, at a confidence interval (CI) of 95%, while co-infection was present in 6% [4/8] of the tested animals at a CI of 95%. There was a significant correlation between age and the prevalence of T. annulata infection, whereas, there was no significant association shown with the age of cattle and T. evansi infection. Sequence and phylogenetic analyses showed that the T. annulata Tams1 gene and T. evansi ITS1 rDNA gene were highly conserved with 97.1-100% and 98.3-100% sequence identity, respectively.

To date, there have been no reports on seasonal variations of Anaplasma spp. in South Mediterranean small ruminants. In this longitudinal field study, single and mixed Anaplasma spp. infections in small ruminants from five different governorates belonging to three bioclimatic zones from the North of Tunisia were evaluated according to seasons. A total of 1685 blood small ruminant samples were collected in spring (355 sheep and 241 goats), summer (249 sheep and 202 goats), autumn (236 sheep and 186 goats) and winter (132 sheep and 84 goats). Molecular survey of A. ovis and A. bovis showed that average prevalence rates were 35.6% (minimum 30.7% in spring and maximum 43.6% in autumn) and 7.4% (minimum 0.9% in spring and maximum 18.1% in summer), respectively, in sheep, and 46% (minimum 21.7% in summer and maximum 65.5% in winter) and 10.1% (minimum 2.2% in autumn and maximum 23.8% in summer), respectively, in goats. A. phagocytophilum was not detected in all investigated animals. The infection profiles of A. ovis and A. bovis show that anaplasmosis caused by A. ovis is endemic in small ruminants from all investigated bioclimatic areas during the four seasons but conversely, A. bovis infection is highly intensified only in the summer. A. ovis and A. bovis infections were validated by sequencing. The comparison of the 16S rRNA sequences of A. bovis variants showed 100% identity between Tunisian variants isolated from goats, sheep and cattle. The analysis of A. ovis msp4 sequences revealed two different genetic variants previously described in Italy. This is the first survey outlining seasonal dynamics of Anaplasma spp. infections in Tunisian small ruminants. This situation should to be taken into account if anaplasmosis control programs in these domesticated animals are envisaged., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In cattle, anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, and A. bovis. To date, no information concerning the seasonal dynamics of single and/or mixed infections by different Anaplasma species in bovines are available in Tunisia. In this work, a total of 1035 blood bovine samples were collected in spring (n=367), summer (n=248), autumn (n=244) and winter (n=176) from five different governorates belonging to three bioclimatic zones from the North of Tunisia. Molecular survey of A. marginale, A. centrale and A. bovis in cattle showed that average prevalence rates were 4.7% (minimum 4.1% in autumn and maximum 5.6% in summer), 7% (minimum 3.9% in winter and maximum 10.7% in autumn) and 4.9% (minimum 2.7% in spring and maximum 7.3% in summer), respectively. A. phagocytophilum was not detected in all investigated cattle. Seasonal variations of Anaplasma spp. infection and co-infection rates in overall and/or according to each bioclimatic area were recorded. Molecular characterization of A. marginale msp4 gene indicated a high sequence homology of revealed strains with A. marginale sequences from African countries. Alignment of 16S rRNA A. centrale sequences showed that Tunisian strains were identical to the vaccine strain from several sub-Saharan African and European countries. The comparison of the 16S rRNA sequences of A. bovis variants showed a perfect homology between Tunisian variants isolated from cattle, goats and sheep. These present data are essential to estimate the risk of bovine anaplasmosis in order to develop integrated control policies against multi-species pathogen communities, infecting humans and different animal species, in the country., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The objective was to characterize Staphylococcus aureus isolated from two wastewater treatment plants (WWTPs) located in Tunis City (Tunisia), during the period 2014-2015. Genetic lineages, antibiotic resistance mechanisms and virulence factors were determined for the recovered isolates. S. aureus isolates were recovered from 12 of the 62 wastewater samples tested (19.35%), and one isolate/sample was characterized, all of them being methicillin-susceptible (MSSA). Six spa types (t587, t674, t224, t127, t701 and t1534) were found among the 12 isolates, and the spa-t587, associated with the new sequence type ST3245, was the most predominant one (7 isolates). The remaining isolates were assigned to five clonal complexes (CC5, CC97, CC1, CC6 and CC522) according to the sequence-type determined and/or the spa-type detected. S. aureus isolates were ascribed to agrI (n = 3), agrII (n = 7) and agrIII (n = 1); however, one isolate was non-typeable. S. aureus showed resistance to (number of isolates): penicillin (12), erythromycin (7), tetracycline (one) and clindamycin (one). Among the virulence factors investigated, only one isolate harboured the tst gene, encoding the TSST-1 (toxic shock syndrome toxin 1). Despite the low number of studied isolates, the present study reports the occurrence of both human- and animal-associated S. aureus clonal complexes in WWTPs in Tunisia.

Molecular diagnosis of Anaplasma platys and related strains (A. platys-like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a versatile bacterium, which can infect or colonize a variety of host species. The objective of this study was to characterize S. aureus isolates recovered from nasal swabs of 167 healthy ewes sampled from 12 farms in different areas of Tunisia during the period of 2014-2015. Genetic lineages, virulence factors and antibiotic resistance mechanisms were determined for recovered isolates. S. aureus was detected in 45 out of 167 tested samples (26.9%). All isolates were methicillin-susceptible (MSSA) and the majority of them were susceptible to tested antibiotics with few exceptions (% of resistance): penicillin (8.8), ciprofloxacin (4.4), and tobramycin or tetracycline (2.2, each). Twelve different spa types were detected (t15098, t15099, t1773, t3576, t1534, t5428, t3750, t5970 t254, t2883, t127 and t933), two of them were new (t15098 and t15099). S. aureus isolates were ascribed to agrI (n=23), agrII (n=1) and agrIII (n=20), and one was non-typeable. According to the sequence-type (ST) determined and/or the spa-type detected, the 45S. aureus isolates were assigned to six clonal complexes, with CC522 (44.4%) and CC130 (37.7%) being the most common lineages. Twenty-one (46.6%) and two (4.2%) isolates harbored the tst and eta genes encoding TSST-1 and ETA, respectively. In conclusion, nares of healthy ewes could be a reservoir of MSSA CC522 and CC130, lineages associated with TSST-1 and ETA that might represent a risk to human health., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Accurate diagnosis of animal and zoonotic diseases, such as granulocytic anaplasmosis, is crucial to estimate risk during control programs. In this study, 16S rRNA nested PCR and RFLP assay were combined to investigate the presence of Anaplasma phagocytophilum and genetically related strains (namely A. phagocytophilum-like 1 and 2) in 936 Tunisian ruminants. By using this method, A. phagocytophilum was not detected in any of the tested animals, while A. phagocytophilum-like 1 and A. phagocytophilum-like 2 were detected at variable prevalence rates in sheep, goats and cattle at coinfection rates respectively of 3.9, 2.5 and 0.5%. Sequence analysis validated RFLP data, and confirmed the co-occurrence of two potentially novel species closely related to A. phagocytophilum in Tunisian ruminants. Phylogeny indicated the presence of genetic variants shared by different ruminant species for each type of A. phagocytophilum-like strains. Results raise concern on the use and interpretation of indirect and direct tests traditionally employed for detecting pathogenic A. phagocytophilum strains in ruminants and in other vertebrates' species, and provide additional background to improve classification of bacterial species closely related to A. phagocytophilum, and to reconstruct their evolutionary history., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Autism spectrum disorders (ASD) are complex neuro-developmental disorders affecting children in early age. Diagnosis relies on multidisciplinary investigations, in psychiatry, neurology, genetics, electrophysiology, neuro-imagery, audiology, and ophthalmology. To support clinicians, researchers, and public health decision makers, we developed an information system dedicated to ASD, called TEDIS. It was designed to manage systematic, exhaustive and continuous multi-centric patient data collection via secured internet connections. TEDIS will be deployed in nine ASD expert assessment centers in Ile-DeFrance district. We present security policy and infrastructure developed in context of TEDIS to protect patient privacy and clinical information. TEDIS security policy was organized around governance, ethical and organisational chart-agreement, patients consents, controlled user access, patients' privacy protection, constrained patients' data access. Security infrastructure was enriched by further technical solutions to reinforce ASD patients' privacy protection. Solutions were tested on local secured intranet environment and showed fluid functionality with consistent, transparent and safe encrypting-decrypting results.