Influence of environmental and seasonal factors on microbial contamination levels in clam production areas in southern Tunisia: Escherichia coli, Salmonella spp., hepatitis A virus and norovirus. / Influence des facteurs environnementaux et saisonniers sur le niveau de contamination des zones de production des palourdes par Escherichia coli, Salmonella spp., le virus de l'hépatite A et les norovirus dans la région de Sfax (Sud tunisien).

To study the influence of environmental and biological factors on levels of contamination by Escherichia coli, Salmonella spp., hepatitis A virus (HAV) and norovirus in clam production areas, an epidemiological study was conducted on 791 samples of live clams (Ruditapes decussatus), 539 of which were sent for bacteriological analysis and 252 for detection of norovirus and HAV. These samples were collected in different production areas in the Sfax region of southern Tunisia over four consecutive years, from March 2013 to December 2016. The prevalence of positive samples was 36% for E. coli, 11% for Salmonella spp., 19% for norovirus and 3% for HAV. There was a significant correlation between contamination by E. coli and by Salmonella spp., as well as between contamination by noroviruses and by HAV and between contamination by noroviruses and by Salmonella spp. Temperature, the presence of migratory birds and tourism are the main factors affecting microbial contamination levels in bivalve molluscs.

Pour étudier l'influence des facteurs environnementaux et biologiques sur le niveau de contamination des zones de production des palourdes par Escherichia coli, Salmonella spp., le virus de l'hépatite A (VHA) et les norovirus, une enquête épidémiologique a été réalisée en utilisant 791 échantillons de palourdes vivantes (Ruditapes decussatus), dont 539 destinés à des analyses bactériologiques et 252 destinés à la détection des norovirus et du VHA. Ces échantillons ont été collectés dans différentes zones de production de la région de Sfax (Sud de la Tunisie) durant quatre années consécutives, du mois de mars 2013 au mois de décembre 2016. La prévalence d'échantillons positifs était respectivement de 36 % pour E. coli, de 11 % pour Salmonella spp., de 19 % pour les norovirus et de 3 % pour le VHA. La corrélation était fortement significative entre la contamination par E. coli et celle par Salmonella spp., ainsi qu'entre la contamination par les norovirus et celle par le VHA et entre la contamination par les norovirus et celle par Salmonella spp. La température, la présence d'oiseaux migrateurs et l'activité touristique sont les principaux facteurs associés au niveau de contamination microbienne des mollusques bivalves.

Los autores describen una investigación epidemiológica encaminada a estudiar la influencia de los factores ambientales y biológicos sobre el nivel de contaminación de zonas de producción de almejas por Escherichia coli, salmonelas, virus de la hepatitis A (VHA) y norovirus. Para ello se llevó a cabo una investigación epidemiológica: en el curso de cuatro años consecutivos (de marzo de 2013 a diciembre de 2016), se obtuvieron en distintas zonas productivas de la región de Sfax (sur de Túnez) 791 muestras de almejas vivas (Ruditapes decussatus), de las que 539 fueron sometidas a análisis bacteriológicos y 252 sirvieron para detectar norovirus y el VHA. La prevalencia de muestras positivas resultó respectivamente del 36% para E. coli, del 11% para Salmonella spp., del 19% para los norovirus y del 3% para el VHA. Había una correlación muy significativa entre la contaminación por E. coli y la contaminación por salmonelas, y también entre la presencia de los norovirus y la del VHA y entre la de los norovirus y la de salmonelas. La temperatura, la presencia de aves migratorias y la actividad turística son los principales factores asociados al nivel de contaminación microbiana de los moluscos bivalvos.

A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors.

The Vibrio splendidus clade has previously been associated with epidemic outbreaks of various aquatic animals, as in the case of the cupped oyster, Crassostrea gigas. To investigate whether involved strains could present a clonal origin and to identify possible alternative background carriage animals or zooplankton, a large epidemiological survey was conducted on isolates of the splendidus clade. For this purpose, Vibrio strains were isolated from various samples including oysters, mussels, sediments, zooplankton, and sea water on the basis of a North/South gradient of the European sea water zone (Ireland, The Netherlands, France, Italy, and Spain). A total of 435 isolates were successfully associated to the V. splendidus clade using real time polymerase chain reaction with 16S specific primers and probes. A multiple-locus variable-number tandem-repeat analysis (VNTR) was conducted on all isolates based on a multiplex PCR-VNTR with a set of primer pairs designed from the V. tasmaniensis LGP32 genome. Preliminary validation of the primers on a set of collection strains from the V. splendidus clade confirmed that the former V. splendidus-related LGP32 and relative strains were related to V. tasmaniensis rather than to the type strain V. splendidus LMG 4042. The VNTR analysis was then successfully conducted on 335 isolates which led to the characterization of 87 different profiles. Our results showed that (1) the high diversity of VNTR did not enlighten significant correlation between a specific pattern and the origin of collected samples. However, populations isolated from animal samples tend to differ from those of the background environment; (2) oyster mortality events could not be linked to the clonal proliferation of a particular VNTR type. However, few different patterns seemed successively associated with samples collected during peaks of oyster's mortality. (3) Finally, no correlation could be seen between specific VNTR patterns and sequence phylogeny of the virulence factors vsm and ompU that were detected among strains isolated during as well as outside mortality events. These results, combined with incongruence observed between the ompU and vsm phylogenetic trees, suggested both large diffusion of strains and massive lateral gene transfer within the V. splendidus clade.

Embryonic exposure to dimethoate and/or deltamethrin impairs sexual development and programs reproductive success in adult male offspring mice.

Pesticides can be toxic to desirable plants and animals, including humans. The aim of this study was to investigate the reproductive effects of low doses of pesticides on male offspring of exposed pregnant mice. Three groups of five female mice were treated daily by oral gavage with dimethoate (5 mg kg(-1) per day), deltamethrin (5 mg kg(-1) per day) and their mixture at 5 mg kg(-1) per day from day 3 to day 21 of pregnancy. Fertility, sexual behaviour and a number of reproductive endpoints, such as organ weights, sperm evaluations and testicular histology, were examined on four adult male offspring of exposed pregnant mice. When compared with control, a dose of deltamethrin 5 mg kg j(-1) causes a decrease in the absolute and relative weight of the testes of exposed mice and it affects their fertility by reducing the density, mobility and vitality of sperm and increasing the number of abnormal forms of these cells (P ≤ 0.01). The same results were obtained in mice exposed to a dose of 5 mg kg j(-1) combination of dimethoate and deltamethrin. This study demonstrated that deltamethrin and combination of dimethoate and deltamethrin caused a decrease in the absolute and relative weight of the testes, which affected the sperm parameters of male offspring of exposed mice to a low dose of these pesticides during pregnancy.

MHC class I and class II genes in Tunisian patients with reactive and undifferentiated arthritis.

OBJECTIVE: To study HLA class I and class II association in Tunisian patients with reactive (ReA) and undifferentiated arthritis (UA). METHODS: The study included 17 patients with ReA defined according to the European Spondylarthropathy Study Group criteria for spondylarthropathy (SpA), 11 patients classified as having undifferentiated arthritis and 100 unrelated healthy controls. HLA class I antigens were typed serologically and HLA class II alleles were genotyped molecularly by the polymerase chain reaction with sequence-specific primers technique. RESULTS: There was a major difference between HLA alleles in ReA and UA patients when compared separately with controls. Increased frequencies of HLA-B27 (p=7.76 10-12, OR=59.30), HLA-B51 (p=0.015, OR=4.91) and HLA-DRB1*04 (p=0.033, OR=2.90) alleles were found in patients with ReA but not in patients with UA. HLA-B27 was not expressed totally in our cohort of UA patients. A significant increase of HLA-B15 (p=0.002, OR=18.40) and a moderate increase of HLA-B7 (p=0.043, OR=5.15) was found in patients with UA, but not in patients with ReA. In the B27 negative patients, HLA-DRB1*04 association with ReA was found independently of B27. CONCLUSION: Our data confirmed a significant association of HLA-B27 with ReA in the Tunisian population. Our results also suggested that some of the additional HLA antigens were associated with ReA including HLA-B51 and HLA-DRB1*04 alleles. UA seemed to have a genetic background different from ReA in Tunisian patients.

Diagnostic value of an enzyme-linked immunosorbent assay using the recombinant CT694 species-specific protein of Chlamydia trachomatis.

AIM: To study the performance of the CT694 protein in relation to the microimmunofluorescence (MIF) and the pELISA tests for the serodiagnosis of Chlamydia trachomatis infections. METHODS AND RESULTS: The CT694 protein was produced as recombinant protein and was used as antigen in ELISA test for the detection of C. trachomatis IgG antibodies. The performance of the developed ELISA test was compared to the MIF test at two cut-off values of 16 and 64, and to the specific pELISA test using a panel of 342 sera. These sera were from children MIF C. trachomatis and Chlamydophila pneumoniae negative, patients MIF C. pneumoniae positive, patients MIF C. trachomatis positive, patients suspected to have chlamydial infections diagnosed by the Cobas Amplicor test, healthy blood donors and prostitutes. Our results indicate that the developed ELISA test has performed better compared with the MIF and the pELISA tests. The highest performance was obtained when comparing the developed ELISA test in relation to the pELISA, yielding an overall sensitivity and specificity of 85% and 87% respectively. CONCLUSIONS: The CT694 ELISA showed the best performance when compared to the species-specific pELISA test and may be used for the serodiagnosis of C. trachomatis infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The CT694 ELISA test responds to the criteria of both sensitivity and specificity according to the MIF and pELISA tests and may be used for serodiagnosis of C. trachomatis infections.

Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples.

The purpose of this study was threefold: to compare semen and first void urine (FVU) specimens from asymptomatic infertile men for the detection of Chlamydia trachomatis, genital ureaplasma, and genital mycoplasma infections using in-house inhibitor-controlled polymerase chain reaction (PCR)-microtiter plate hybridization assay; to determine the prevalence of those organisms in infertile men in Tunisia; and to study the relationship between these bacteria and male infertility. Paired urine and semen specimens from 104 patients were examined by in-house PCR for the presence of DNA of Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and genital mycoplasmas (Mycoplasma hominis and Mycoplasma genitalium). Semen analysis was assessed according to the guidelines of the World Health Organization. Nominal scale variables, the Mann-Whitney test, and the Kruskal-Wallis nonparametric analysis of variance test were used for statistical analysis. There was a very high concordance (>95%) and a very good agreement (kappa > 0.9) between the detection of Chlamydia trachomatis, genital ureaplasmas, and Mycoplasma hominis in semen and corresponding FVU specimens. Our findings also show a high concordance (81.1%) and a good agreement (kappa = 0.79) between the detection of Mycoplasma genitalium in both specimens. C trachomatis, genital mycoplasmas, and genital ureaplasmas were found to be widespread among infertile male patients in Tunisia, as shown by their respective prevalences of 43.3%, 18.3%, and 14.4%. The mean values of seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte count were not significantly related either to the detection of C trachomatis DNA or to that of genital ureaplasma or mycoplasma DNA in semen specimens. Using our in-house PCR, both semen and FVU were found to be sensitive diagnostic specimens for the detection of C trachomatis, ureaplasmas, and mycoplasmas. The FVU, a less invasive and self-collected specimen, can serve as a marker for the presence of these organisms in the genital tract and can be used as a reliable way of detecting asymptomatic carriers of infection.

Screening for bacterial pathogens in semen samples from infertile men with and without leukocytospermia.

We aimed to study the correlation between leukocyte counts in semen and bacterial pathogens in seminal samples of infertile men, and to establish the minimum leukocyte count associated with significant bacteriospermia. A total of 116 patients who underwent evaluation of fertility were investigated using routine semen analysis according to the guidelines of the WHO and bacterial pathogens analysis by culture and in-house PCR assay. The overall prevalence of bacteriospermia in semen samples was 56.9% independent of the presence of leukocytes. The most common bacterial species detected were Chlamydia trachomatis (41.4%), Ureaplasma urealyticum (15.5%) and Mycoplasma hominis (10.3%). The receiver operating characteristic curve analysis demonstrated that the sensitivity/specificity for detecting bacteria at a cut off level of >or=1 x 10(6) leukocytes per ml (which is the WHO defined level for leukocytospermia) was 20.3%/81.5%. The highest sensitivity/specificity ratio was found in semen samples with a cut-off level of >or=0.275 x 10(6) leukocytes per ml, which is best shown with the odds ratio of 2.47. A significant correlation was found between bacteriospermia and leukocytospermia at the cut-off level of >or=0.275 x 10(6) leukocytes per ml of semen samples (P = 0.032). We proposed that this is a possible new cut-off level to predict the presence of bacteria in semen of infertile men.

Molecular prevalence of Chlamydia and Chlamydia-like bacteria in Tunisian domestic ruminant farms and their influencing risk factors.

Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.

Endocrine disrupting potential and reproductive dysfunction in male mice exposed to deltamethrin.

Pesticide exposure may affect semen quality and male fertility in humans. The aim of the present work was to elucidate the adverse effects of deltamethrin (Delta), a synthetic pyrethroid, on exposed male mice and their offspring. Adult male Albino/Swiss mice received deltamethrin (5 mg/kg) daily for 35 days and mated with untreated females to produce offspring. Classical measurements of ejaculate and sperm quality and testicular histopathological changes were assessed. Deltamethrin treatment affects sperm quality and quantity in the ejaculated semen of mice that had also markedly impaired libido as measured by indices of mating and fertility and number of pregnant females housed with male mice exposed to this pesticide. Exposure mice to deltamethrin significantly decreased their testosterone and inhibin B levels and affected reproductive performance. Testes of exposed mice showed marked histopathological alterations as compared to the control group. The mice exposed to 5 mg/kg body weight/day of deltamethrin showed severe alterations of the seminiferous tubules, sloughing of the germ cells, the vacuolization of germ cell cytoplasm, and the disruption of spermatogenic cells compared to the control group. Altered pregnancy outcomes were directly attributed to damage of sperm of male mice exposed to deltamethrin compared to the control group. We concluded that exposure to deltamethrin affected the reproductive system of male mice explored by altered total sperm density, motility, and morphology in mice spermatozoa.

Usefulness of enzyme linked immunosorbent assays species specific in the detection of Chlamydia trachomatis and Chlamydophila pneumoniae IgG antibodies in patients with genital infections or respiratory tract infections.

Chlamydia trachomatis (Ct) and Chlamydophila pneumoniae (Cpn) are obligate intracellular bacteria causing genital tract infections (GTI) and respiratory tract infections (RTI), respectively. Antigenic cross-reactivity between the two species may complicate serologic diagnosis. In this study, we compared the performance of two ELISA tests in relation to microimmunofluorescence (MIF) for the detection of Ct and Cpn IgG antibodies. We also explored the degree of cross-reactivity by ELISA and MIF. Among 278 positive sera for Cpn and/or Ct IgG antibodies in the MIF, 153 were from patients with GTI and 125 were from patients with RTI. These sera were tested by our in house MIF test and by two commercial ELISA: SeroCP and SeroCT for the detection of anti-Cpn IgG antibodies and anti-Ct IgG antibodies, respectively. In sera from patients with RTI, correlation between MIF and SeroCP was 92%. The specificity of this test was 38.5%. In fact, among the 140 sera from patients with GTI and that cross-reacted in MIF, only six were confirmed by the two ELISA tests as having IgG antibodies to Ct. The correlation between MIF and SeroCT was 80%. The specificity of this test was 100%. Indeed, among the 65 sera from patients with RTI with cross-reactions in MIF, 30 sera showed a negative SeroCT test. SeroCT was highly specific and could diminish considerably the extent of cross-reactions. Whilst, SeroCP test was not specific enough to distinguish between the presence of IgG antibodies and Cpn or Ct.

Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature.

AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells. Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test. However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer. hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained. During the temperature-induced resuscitation, constant total cell counts were observed. Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase. We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells. CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer. hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer. hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn.

Chlamydia trachomatis and male infertility in Tunisia.

OBJECTIVE: Chlamydia trachomatis is a common sexually transmitted micro-organism. The impact of chlamydial infection on semen parameters and male fertility is controversial. Our purpose was to determine the prevalence of C. trachomatis in the male partners of infertile couples in Tunisia and to assess the relationship between chlamydial infection markers and male infertility. METHODS: Chlamydial DNA in urethral and in semen specimens was determined by using the Cobas Amplicor polymerase chain reaction (PCR) assay and chlamydial immunoglobulin G (IgG) antibodies were measured by micro-immunofluorescence in serum samples in 92 male partners, with or without pathological standard semen parameters, according to the guidelines of the World Health Organization (sperm count, progressive sperm motility, sperm morphology and sperm viability). In parallel, chlamydial infection markers in endocervical material were determined by PCR and chlamydial IgG antibodies were measured by micro-immunofluorescence in serum samples from the female partners of the patients. RESULTS: C. trachomatis was found in 35.9% (33/92) of the male partners of the infertile couples and in 38% (35/92) of their female partners. There was a significant correlation between the detection of C. trachomatis in both partners (p = 0.004). C. trachomatis DNA was detected in 18.5% (17/92) of urethral specimens and in 16.3% (15/92) of semen specimens. Chlamydial IgG antibodies were present in 9.8% (9/92) of the serum samples. A standard semen analysis showed that 88% (81/92) were pathological. Sperm viability, progressive sperm motility, morphology and sperm concentration were abnormal in 73.8%, 70.2%, 34.5% and 13%, respectively, of the 92 evaluated semen specimens. Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with or without chlamydial infection markers showed that only the presence of C. trachomatis DNA in semen samples can affect sperm motility. Parameters of the standard semen analysis were not significantly related either to the detection of chlamydial DNA in urethral samples or to the presence of serum chlamydial antibodies. CONCLUSION: Our results show that C. trachomatis seems to be widespread among the male partners of infertile couples in Tunisia and show that this organism can affect sperm motility and, thus, can play an important role in the etiology of male infertility.

Culture-negative endocarditis due to Chlamydia pneumoniae.

We report on the case of a 54-year-old woman diagnosed as having culture-negative endocarditis (clinical and histopathologic evidence compatible with a recent episode of endocarditis). The responsibility of Chlamydia pneumoniae in this episode of endocarditis was suggested by a serological study and was then confirmed by the positive results of PCR and in situ hybridization tests with aortic and mitral valves tissues. To our knowledge, this is the first case of endocarditis due to C. pneumoniae confirmed by molecular biology-based techniques.

Detection of Chlamydia trachomatis in semen and urethral specimens from male members of infertile couples in Tunisia.

The sequelae to infection with Chlamydia trachomatis in women are an established cause of tubal infertility. However, little is known about chlamydial infection and male infertility. The main objective of this study was to evaluate the presence of asymptomatic C. trachomatis infections in urethral and semen specimens from the male members of infertile couples by means of four different methods: the direct fluorescence antibodies assay, cell culture, the Roche Cobas Amplicor polymerase chain reaction, and the presence of chlamydial local IgA antibodies by the recombinant antibody-enzyme-linked immunosorbent assay. One or more chlamydial infection markers were detected in 42 (45.7%) of the 92 examined urethral and semen specimens from the male partners of infertile couples. C. trachomatis was detected in 23.9% (22/92) of urethral specimens and in 35.9% (33/92) of semen specimens. Although there was a significant correlation between the detection of one or more chlamydial infection markers in urethral and semen specimens (p = 0.01), no significant correlation was found between the detection of C. trachomatis DNA in these samples. Furthermore, no significant association was found between the presence of chlamydial local IgA antibodies and the detection of C. trachomatis. The discrepancies in positive results found between some techniques for the detection of C. trachomatis in urethral and semen specimens might be explained by variations in the sensitivities and specificities of the tests carried out and the use of specimens from different anatomical locations. Our findings suggest that C. trachomatis seems to be widespread among the male partners of infertile couples in Tunisia. The detection of C. trachomatis in urethral or semen specimens can serve as a marker for the presence of this organism in the genital tract, which is not necessarily the cause of male infertility. The study of the correlation between the detection of chlamydial infection markers and the parameters of male fertility seems to be necessary in order to determine the direct link between chlamydial infection and male infertility and to choose the most efficient technique and most suitable specimen with which to diagnose C. trachomatis-associated male infertility.