Summary
The novel coronavirus disease‐2019 (Covid‐19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability ...of advanced diagnostics and current need for cost‐effective methodology. The advent of the loop‐mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point‐of‐care test for the diagnosis of Covid‐19 in endemic areas, especially for low‐ and middle‐income countries.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Buparvaquone (BW 720C) is the major hydroxynaphtoquinone active against tropical theileriosis (Theileria annulata infection). Previous studies showed that buparvaquone, similarly to others ...hydroxynaphtoquinone, probably acts by binding to cytochrome b (cyt b) inhibiting the electron transport chain in the parasite. Several observations suggested that T. annulata is becoming resistant to buparvaquone in many endemic regions (Tunisia, Turkey and Iran), which may hinder the development of bovine livestock in these areas.
In the present study we sought to determine whether point mutations in T. annulata cytochrome b gene could be associated to buparvaquone resistance. A total of 28 clones were studied in this work, 19 of which were obtained from 3 resistant isolates (ST2/12, ST2/13 and ST2/19) collected at different time after treatment, from a field treatment failure and nine clones isolated from 4 sensitive stocks of T. annulata (Beja, Battan, Jed4 and Sousse). The cytochrome b gene was amplified and sequenced. We identified five point mutations at the protein sequences (114, 129, 253, 262 and 347) specific for the clones isolated from resistant stocks. Two of them affecting 68% (13/19) of resistant clones, are present in the drug-binding site Q02 region at the position 253 in three resistant clones and at the position 262 in 11 out of 19 resistant clones. These two mutations substitute a neutral and hydrophobic amino acids by polar and hydrophilic ones which could interfere with the drug binding capabilities. When we compared our sequences to the Iranian ones, the phylogenetic tree analyses show the presence of a geographical sub-structuring in the population of T. annulata.
Taken together, our results suggest that the cytochrome b gene may be used as a tool to discriminate between different T. annulata genotypes and also as a genetic marker to characterize resistant isolates of T. annulata.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry ...farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6′)-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6′)-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6′)-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally.
Bluetongue virus (BTV) is an arbovirus considered as a major threat for the global livestock economy. Since 1999, Tunisia has experienced several incursions of BTV, during which numerous cases of ...infection and mortality have been reported. However, the geographical origin and epidemiological characteristics of these incursions remained unclear. To understand the evolutionary history of BTV emergence in Tunisia, we extracted from Genbank the segment 6 sequences of 7 BTV strains isolated in Tunisia during the period 2000 to 2017 and blasted them to obtain a final dataset of 67 sequences. We subjected the dataset to a Bayesian phylogeography framework inferring geographical origin and serotype as phylodynamic models. Our results suggest that BTV-2 was first introduced in Tunisia in the 1960s and that since 1990s, the country has witnessed the emergence of other typical and atypical BTV serotypes notably BTV-1, BTV-3 and BTV-Y. The reported serotypes have a diverse geographical origin and have been transmitted to Tunisia from countries in the Mediterranean Basin. Interserotype reassortments have been identified among BTV-1, BTV-2 and BTV-Y. This study has provided new insights on the temporal and geographical origin of BTV in Tunisia, suggesting the contribution of animal trade and environment conditions in virus spread.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In this study, the genetic relationship and the population structure of western Mediterranean basin native sheep breeds are investigated, analyzing Maghrebian, Central Italian, and Venetian sheep ...with a highly informative microsatellite markers panel. The phylogeographical analysis, between breeds’ differentiation level (Wright’s fixation index), gene flow, ancestral relatedness measured by molecular coancestry, genetic distances, divergence times estimates and structure analyses, were revealed based on the assessment of 975 genotyped animals. The results unveiled the past introduction and migration history of sheep in the occidental Mediterranean basin since the early Neolithic. Our findings provided a scenario of three westward sheep migration phases fitting properly to the westward Neolithic expansion argued by zooarcheological, historical and human genetic studies.
We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, ...successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50fg/μl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This, is the first study that evaluates the L. infantum specific LAMP alongside other diagnostics tools for, CanL. Our results indicate a suitable sensitivity and specificity for the developed LAMP assay that could, has usefulness application on dogs and human L. infantum diagnosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The taxonomy of the Lesser Egyptian jerboa,
(
.)
(Dipodinae subfamily), was recently reevaluated, and the taxonomic status was defined by the presence of two cryptic species,
.
(Linnaeus 1758) and
.
...(Lichtenstein, 1823), with a higher genetic divergence in the sympatric North African populations than in other studied parapatric populations. Using phylogenetic analysis of the cytochrome b (
) gene from 46 specimens, we confirmed the new status in Tunisia; rodents were collected from two different biotopes belonging to the same locality at the ecological level (mountainous vs. Saharan) in the south of the country. The study of the eye lens weight of these specimens allowed the definition of a cutoff value (58.5 g), categorizing juveniles from adults. Moreover, this study confirmed the phylotaxonomic status of
.
in Tunisia, as recently illustrated, into two distinct species,
.
and
.
, and recorded for the first time the presence of two phylogroups among each of these rodent species. The lack of clear micro-geographical structure and biotope specificity between the two rodent species and their phylogroups was also highlighted.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Leishmaniases are caused by protozoan parasites of the genus Leishmania transmitted by females blood-feeding phlebotomine insects (Diptera: Psychodidae). In Tunisia, cutaneous and visceral ...leishmaniases are of public health concern. In Tunisia, 17 species of phlebotomine sand flies are described. Here we investigate natural infection in Tunisian mixed foci regions of leishmaniases. We trap female sandflies during the Leishmania transmission season in the country's central-eastern and northern parts. We investigate Leishmania infection using PCR-RFLP targeting the ITS1 ribosomal DNA, followed by enzymatic digestion with HaeIII; then, we identify sand flies using molecular methodologies. We confirm the presence of Phlebotomus papatasi and Phlebotomus perniciosus infected by L. major and L. infantum parasites in Tunisia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
This study shows, for the first time, natural Leishmania infection among Jaculus spp. in an endemic region of Tataouine, South Tunisia. To better characterize the transmission cycles in this complex ...focus of mixed transmission, Leishmania detection and species identification were performed by direct examination, internal transcribed spacer-1 (ITS1)-PCR-restriction fragment length polymorphism (RFLP), and sequencing of Jaculus (J.) jaculus (Linnaeus, 1758) and J. hirtipes (Lichtenstein, 1823) rodent species, which are frequently encountered in this area. Leishmania parasites were observed in 19 (41.3%) smears, while DNA parasites were detected in 28 (60.9%) Jaculus spp. spleens; among them, 12 (54.5%) were from 22 J. jaculus individuals and 16 (66.7%) were from 24 J. hirtipes individuals. Leishmania parasites were confirmed as Leishmania (L.) killicki (syn. L. tropica) in two J. hirtipes individuals (4.3%) and L. major (n = 24; 52.2%) in 10 J. jaculus and 14 J. hirtipes individuals. This finding represents the first evidence of natural infection with Leishmania parasites in rodents belonging to the Jaculus genus, providing the rationale to consider them as potential reservoir hosts of Old World Leishmania parasites in Tunisia and North Africa.
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