Coxiella burnetii and Borrelia spp. in peripheral blood of dromedary camels in Fars, Iran: molecular characterization, hematological parameters, and acute-phase protein alterations.

Background: Dromedary camels (Camelus dromedarius) are raised in extremely strict ecological conditions of deserts. Camels are vulnerable to many zoonotic infections. There are limited data on the occurrence of Q fever and borreliosis in camels, in Iran. Aims: The current study was focused on the occurrence of Coxiella burnetii and Borrelia spp. infection in the blood samples of Iranian camels using molecular assays. Effect of the presence of these infections on various hematological factors and some acute-phase proteins (Hp, a1AGP, SAA) were also investigated. Methods: Blood samples were collected from 113 clinically healthy camels to investigate the presence of the infections using nested PCR. Moreover, the sequence of positive samples was analyzed phylogenetically. Routine haematological tests were performed and the concentrations of acute-phase proteins were measured in serum using enzyme immunoassay. Results: PCR result showed that 6.19% (95% CI: 2.53-12.35%) (7/113) of camels were positive for C. burnetii. In addition, sequencing results of the corresponding gene of the outer membrane protein (com1) revealed two different genotypes of C. burnetii agent in camels from Southern Iran. In the PCR assay, Borrelia spp. DNA was not detected in the samples. No significant difference was observed in hematological parameters or acute-phase proteins between positive and negative Q fever camels except for mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW). Conclusion: Clinically healthy camels might be very important reservoirs of zoonotic pathogens. Q fever is not considered a notifiable disease in camels of Iran, and clinical cases may scarcely be recognized by the healthcare system. Due to a lack of adequate information, additional studies on the molecular epidemiology and clinical pathology aspects of C. burnetii infection in Iran are needed.

Immunological and bacteriological quality of fresh cow colostrum and passive immunity transfer in selected dairy farms in Fars, Iran.

Background: The quality of colostrum is affected by IgG level and microbial load. Aims: The quality of colostrum used in feeding dairy calves and passive immunity transfer in selected dairy farms in Fars province, Iran was investigated. Methods: A total of 75 colostrum and neonatal blood samples were collected from 11 herds. The immunological quality of colostrum was assessed using a Brix digital refractometer. The bacteriological quality was assessed by performing total plate count (TPC), total coliform count (TCC), spore-former count, fungi count, and species-specific PCR assay to detect some bacterial species. Results: The mean Brix of colostrum samples was 25.4% and 72% of the samples had a Brix score ≥22%. The mean serum Brix and the prevalence of failure of passive transfer (FPT) were 10% and 4%, respectively. The mean TPC, TCC, spore-former count, and fungi count were 3.6 × 105, 2.8 × 104, 3.2 × 104, and 1.1 × 104 CFU/ml, respectively. The results showed that 50, 5.9, and 4% of colostrum samples were positive for Staphylococcus spp., Salmonella spp. and Maycobacterium paratuberculosis, respectively. There was no evidence of contamination with Brucella spp., Corynebacterium bovis and Mycoplasma bovis. Conclusion: Considering all colostrum quality indicators comprehensively, only 37.3% of the studied samples met the industry standard. A large number of calves were at risk of receiving poor quality colostrum, especially in terms of microbial contamination. Further researches are needed to evaluate the colostrum management and the effect of bacterial contamination of colostrum on the health of neonate calves in this region.

Whole-genome and transcriptome analysis enhances precision cancer treatment options.

BACKGROUND: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies. PATIENTS AND METHODS: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA. DNA-based data, including mutations, copy number and mutation signatures, were combined with RNA-based data, including gene expression and fusions, to generate comprehensive WGTA profiles. A multidisciplinary molecular tumour board used WGTA profiles to identify and prioritize clinically actionable alterations and inform therapy. Patient responses to WGTA-informed therapies were collected. RESULTS: Clinically actionable targets were identified for 83% of patients, of which 37% of patients received WGTA-informed treatments. RNA expression data were particularly informative, contributing to 67% of WGTA-informed treatments; 25% of treatments were informed by RNA expression alone. Of a total 248 WGTA-informed treatments, 46% resulted in clinical benefit. RNA expression data were comparable to DNA-based mutation and copy number data in aligning to clinically beneficial treatments. Genome signatures also guided therapeutics including platinum, poly-ADP ribose polymerase inhibitors and immunotherapies. Patients accessed WGTA-informed treatments through clinical trials (19%), off-label use (35%) and as standard therapies (46%) including those which would not otherwise have been the next choice of therapy, demonstrating the utility of genomic information to direct use of chemotherapies as well as targeted therapies. CONCLUSIONS: Integrating RNA expression and genome data illuminated treatment options that resulted in 46% of treated patients experiencing positive clinical benefit, supporting the use of comprehensive WGTA profiling in clinical cancer care.

Alteration of fatty acid profile of milk in Holstein cows fed Bacillus coagulans as probiotic: a field study.

BACKGROUND: Probiotics may improve milk quality and the general health status of animals. AIMS: The effects of dietary Bacillus coagulans PRM101 on milk components, milk fatty acids (FA), and some health indicators of dairy cows were investigated. METHODS: The probiotic was added to the feed of 12 Holstein cows (2 g/cow: 2 × 1011 CFU/cow) for 63 days compared to a control group fed on the basal ration (n=11). Milk and blood samples were taken on days 0, 21, 42, and 63. RESULTS: The yields of milk and energy corrected milk (ECM; computed from milk weight and its fat and protein content) decreased linearly and similarly (P=0.60) in both groups. The treatment cows, however, showed quadratic increases in the weights of milk (P=0.03) and ECM (P=0.04) at d42 of the study. Energy corrected milk (d42, P<0.05) and crude protein content of milk (d42, P<0.05; d63, P<0.1) were higher in the cows receiving the probiotic. The proportions of heptadecanoic (C17:0; P=0.002) and linoleic (C18:2; P=0.077) acids in milk fat (g/100 g fat) were higher in the treatment cows on d63. Milk total antioxidant capacity (TAC), malondialdehyde (MDA), and similarly, amyloid A (AA) and haptoglobin (Hp) of milk and blood were not affected. Total antioxidant capacity and MDA were negatively correlated in the control group (r=-0.669, P=0.005). Heptadecanoic acid correlated negatively with milk MDA (r=-0.611, P=0.035) and positively (r=0.591, P=0.043) with serum Hp in the treatment cows. CONCLUSION: Dietary B. coagulans PRM101 may improve the proportions of C17:0 and C18:2 FA in milk. Some improvements in milk protein and the health status of the cows may also be anticipated.

The effect of carboxymethyl cellulose coating incorporated with clove oil nanoemulsion on quality of shrimp (Litopenaeus vannamei) during refrigerated storage.

BACKGROUND: The quality of shrimp, as high perishable seafood, can readily be effected by microbial, chemical, and physical alterations during storage. AIMS: In this study, carboxymethyl cellulose (CMC) coating either alone or incorporated with variable levels of clove oil nanoemulsion (CNE) (10, 20, and 30 mg/ml) was developed to sustain the quality of shrimp during refrigerated storage. METHODS: Changes in microbial, chemical, textural, color (L*, a*, b*), and sensory parameters of the studied groups evaluated through distinct experimental approaches. RESULTS: Based on our results, the pattern of increase in the aerobic plate count (APC) and psychrotrophic bacterial count (PBC) in different groups were reported as the following order: CMC + CNE < CMC < control. Also, CMC and CNE integration decreased the upward trend of total volatile basic nitrogen (TVB-N), and thiobarbituric acid reactive substances (TBARS) compared to that of the control group during storage at 4°C for 10 days (P<0.05). During the storage period, the decrease of the L* value in the control group was greater than the others; whereas, the lowest a* and b* values were observed in this group (P<0.05). Besides, the textural and sensory properties of coated shrimp were significantly more acceptable (P<0.05). CONCLUSION: Integration of CNE in CMC coating promisingly improves the quality of shrimp during refrigerated storage.

Comprehensive comparison between 222 CTLA-4 haploinsufficiency and 212 LRBA deficiency patients: a systematic review.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) haploinsufficiency (CHAI) and lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency (LATAIE) are newly identified inborn errors of immunity with shared molecular pathomechanisms and clinical manifestations. In this review, we aimed to provide differential comparisons regarding demographic, clinical, immunological and molecular characteristics between these two similar conditions. A literature search was conducted in PubMed, Web of Science and Scopus databases and included studies were systematically evaluated. Overall, 434 (222 CHAI and 212 LATAIE) patients were found in 101 eligible studies. The CHAI patients were mainly reported from North America and western Europe, while LATAIE patients were predominantly from Asian countries. In CHAI, positive familial history (P < 0·001) and in LATAIE, consanguineous parents (P < 0·001) were more common. In CHAI patients the rates of granulomas (P < 0·001), malignancies (P = 0·001), atopy (P = 0·001), cutaneous disorders (P < 0·001) and neurological (P = 0·002) disorders were higher, while LATAIE patients were more commonly complicated with life-threatening infections (P = 0·002), pneumonia (P = 0·006), ear, nose and throat disorders (P < 0·001), organomegaly (P = 0·023), autoimmune enteropathy (P = 0·038) and growth failure (P < 0·001). Normal lymphocyte subsets and immunoglobulins except low serum levels of CD9+ B cells (14·0 versus 38·4%, P < 0·001), natural killer (NK) cells (21 versus 41·1%, P < 0·001), immunoglobulin (Ig)G (46·9 versus 41·1%, P = 0·291) and IgA (54·5 versus 44·7%, P = 0·076) were found in the majority of CHAI and LATAIE patients, respectively. The most frequent biological immunosuppressive agents prescribed for CHAI and LATAIE patients were rituximab and abatacept, respectively. Further investigations into the best conditioning and treatment regimens pre- and post-transplantation are required to improve the survival rate of transplanted CHAI and LATAIE patients.

Interleukin-8 gene expression and apoptosis induced by Salmonella Typhimurium in the presence of Bacillus probiotics in the epithelial cell.

AIMS: This study aimed to evaluate the effects of three Bacillus probiotics on Salmonella Typhimurium, and interleukin-8 (IL-8) gene expression in the co-culture of the Bacillus and the pathogen in vitro. METHODS AND RESULTS: Bacillus subtilis, Bacillus indicus and Bacillus coagulans were initially turned to spore and heat-inactivated forms. The cellular damages of the probiotics on the HT-29 cells were investigated individually and in combination with S. Typhimurium using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorescence assays. To extract cell free supernatants (CFS) of the probiotics, they were cultured in selective media. The inhibitory activity of CFSs were then assayed against the pathogen. The gene expression of IL-8 of the HT-29 cells was evaluated by real-time PCR in all the groups. The results showed that the CFSs of three probiotics could inhibit the growth of S. Typhimurium by more than 50%. Inhibitory effects of B. indicus and B. subtilis CFSs were related to the production of pepsin-sensitive compounds, except B. coagulans in which the high inhibitory effect was due to organic acids. The spores of the three probiotics and the heat-inactivated forms of B. subtilis and B. coagulans could reduce the cytotoxicity of S. Typhimurium. The cell viability also increased applying both forms probiotics against the pathogen. In all co-culture groups, the IL-8 gene expression induced by S. Typhimurium was reduced. CONCLUSIONS: The three Bacillus probiotics can be considered as proper candidates for the prevention and treatment of S. Typhimurium food poisoning. SIGNIFICANCE AND IMPACT OF THE STUDY: Applying probiotics as live bacteria is universally noted in foods. This study tried to discover the effects of Bacillus probiotics in the form of spore or even heat-killed bacteria against S. Typhimurium and evaluate ratio of IL-8 gene expression in cell culture. The most effective Bacillus probiotic will be recommended. This approach will help to use probiotics as nonvegetative cells in foods to fight gastrointestinal pathogens.

A web-based daily care training to improve the quality of life of mothers of children with cerebral palsy: A randomized controlled trial.

BACKGROUND: Mothers of moderately to severely affected children with cerebral palsy (CP) have to spend a long time to take care of their children. This time-consuming responsibility affects their physical and psychosocial health. Therefore, mothers as caregivers are required to receive special training to take care of their children. AIMS: The aim of this study was to evaluate the effectiveness of a developed web-based intervention for daily care training of children with CP on their mothers' quality of life (QOL), anxiety, depression, stress, and their musculoskeletal pain. METHODS AND PROCEDURES: This study was a single blind randomized controlled trial. 91 mothers of children with CP with Gross Motor Function Classification System (GMFCS) levels III, IѴ, and Ѵ, who aged from 4 to 12 years were assigned to the intervention and control groups using block randomization. Mothers in the control group received their routine face to face occupational therapy intervention and mothers in the intervention group received 12 weeks web-based intervention. QOL, depression, anxiety, stress, and pain were measured before and after the intervention in both groups. OUTCOMES AND RESULTS: The results of analysis of covariance showed that after controlling the mean score of pretest of pain, the mean score of post-tests in the intervention and control groups was significantly different (P < 0.05). The mean scores of physical health and total QOL scores of post-tests in the intervention group were significantly higher than the control group with controlling pretest scores. CONCLUSIONS AND IMPLICATIONS: Designed web-based intervention affects the caregivers' QOL and pain significantly. This intervention can be used to provide daily care training for mothers of children with CP.

The effect of exercise on reelin level in the hippocampus of diabetic rats.

AIM: Diabetes is one of the most common diseases which can attenuate brain function by destroying hippocampus neurons, while reelin is a largely secreted extracellular matrix glycoprotein in the hippocampus causing synaptic plasticity, promoting postsynaptic structures and maturing neurons. The aim of this study was to assess the effect of exercise, as an external factor for neurogenesis in the brain, on reelin levels and memory improvement in diabetic rats. METHOD: Thirty rats were randomly allocated into three groups; healthy sedentary, diabetic sedentary and diabetic exercise-trained. The experimental group was treadmill-exercised at speed 22 m/min for 1 hour, 5 days per week. Finally, spatial memory of rats was tested and reelin levels were measured. RESULTS: The results showed that short-term exercise improved spatial memory in diabetic rats but had no effect on reelin levels in the hippocampus of diabetic rats. CONCLUSION: Diabetes reduced the spatial memory without altering the reelin levels while exercise improved spatial memory without altering the reelin levels (Fig. 4, Ref. 33).

Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen.

The Asian citrus psyllid (Diaphorina citri) is the insect vector responsible for the worldwide spread of 'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host-microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

The use of inulin as fat replacer and its effect on texture and sensory properties of emulsion type sausages.

The present study aimed to investigate the possibility of reducing energy content in emulsion type sausages by replacing fat with inulin. In the manufactured product, the fat content was reduced to 6%-18% and replaced by inulin and water. The quality of the resulting product was determined by chemical and texture profile analyses (TPA), color measurement and sensory evaluation. The results showed that replacing fat with inulin led to a significant energy content reduction of up to 64% (with 6% inulin and 12% water). In addition, color measurement, sensory evaluation and TPA were comparable to the traditional product in the inulin treated samples. The overall acceptability of all experimental groups was adequate; therefore, inulin is suggested as a good replacement for fat in emulsion type sausages.

The effects of probiotic, prebiotic and synbiotic diets containing Bacillus coagulans and inulin on rat intestinal microbiota.

An in vivo experiment was conducted to study the effects of probiotic Bacillus coagulans spores, with and without prebiotic, inulin, on gastrointestinal (GI) microbiota of healthy rats and its potentiality to survive in the GI tract. Forty-eight male Wistar rats were randomly divided into four groups (n=12) and fed as follows: standard diet (control), standard diet supplied with 5% w/w long chain inulin (prebiotic), standard diet with 10(9)/day spores of B. coagulans by orogastric gavage (probiotic), and standard diet with 5% w/w long chain inulin and 10(9) spores/day of B. coagulans by orogastric gavage (synbiotic). Rats were fed the diets for 30 days. At day 10, 20 and 30 of experiment, 24 h post administration, four rats from each group were randomly selected and after faecal collection were sacrificed. Small intestine, cecum, and colon were excised from each rat and used for microbial analysis. Administration of synbiotic and probiotic diets led to a significant (P<0.05) increment in lactic acid bacteria (LAB), total aerobic and total anaerobic population compared the prebiotic and control diets. A significant decrease in Enterobacteriaceae counts of various segments of GI tract (except small intestine) in synbiotic, probiotic and prebiotic fed groups were also seen. The obvious decline in spores count through passing GI tract and high surviving spore counts in faecal samples showed that spores are not a normal resident of GI microbiota and affect intestinal microbiota by temporary proliferation. In conclusion, the present study clearly showed probiotic B. coagulans was efficient in beneficially modulating GI microbiota and considering transitional characteristics of B. coagulans, daily consumption of probiotic products is necessary for any long-term effect.

Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz.

The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended.

An epidemiological survey on the determination of Taenia saginata cysticercosis in Iran, using a PCR assay.

Bovine cysticercosis caused by Taenia saginata is a zoonotic disease affirming routine inspection measures for the postmortem detection of cysticerci (cysts) in beef destined for human consumption. Detection is based on gross examination of traditional carcase predilection sites; although there is evidence to suggest that examination of other sites may offer improvements in sensitivity. In the current study, a biomolecular-based assay was employed to confirm and differentiate T saginata cysticercosis from other comparable parasitic infection in cattle carcases. Out of 7371 cattle carcases routinely inspected, 72 (0.97 per cent) were initially detected, from which 57 (79.16 per cent), 11(15.27 per cent) and 4 (5.55 per cent) were recorded in masseter muscle, heart and diaphragm, respectively. The PCR assay was also conducted to confirm different stages of the cysts, being able to detect the cyst, and to discriminate its various degenerative stages with other parasitic structures. The technique was proposed as a reliable tool to differentiate the cysticerci and, thus, could be used in further epidemiological studies as there was no difference in view of negative PCR results in lesions found by routine inspection.

Endurance exercise training and diferuloyl methane supplement: changes in neurotrophic factor and oxidative stress induced by lead in rat brain.

Lead is a highly neurotoxic agent that particularly affects the developing central nervous system. In the current study we investigated the neuroprotective effects of exercise training and/or diferuloyl methane (DM) supplement, which is known as curcumin, on lead acetate-induced neurotoxicity in the rat hippocampus. Sixty rats were randomly divided into six groups: 1) lead acetate, 2) DM supplement, 3) endurance training, 4) training+ DM supplement, 5) sham and 6) base. The rats in the training groups performed treadmill running consisting of 15 to 22 m · min(-1) for 25 to 64 min, 5 times a week for 8 weeks. All groups except sham received lead acetate (20 mg · kg(-1)), whereas the sham group received DM solvent. In addition, the DM and training + DM groups received DM solution (30 mg · kg(-1)) intraperitoneally. Chronic administration of lead acetate resulted in a significant increase in the malondialdehyde (MDA) in plasma, but not in the hippocampus. In addition, it led to significantly decreased brain-derived neurotrophic factor (BDNF) in the hippocampus and total antioxidant capacity (TAC) levels, as compared to the sham group. Treadmill running, DM supplementation, or both resulted in a significant decrease in MDA levels and significantly increased BDNF and TAC levels, as compared to the lead acetate group. These results provide a rationale for an inhibitory role of DM supplement and regular exercise in the attenuation of lead-induced neurotoxicity.

Expression of antigenic determinants of the haemagglutinin large subunit of novel influenza virus in insect cells.

The global outbreak of novel A/H1N1 spread in human population worldwide has revealed an emergency need for producing a vaccine against this virus. Current influenza vaccines encounter problems with safety issues and weak response in high-risk population. It has been established that haemagglutinin is the most important viral antigen to which antibody responses are directed, and recombinant subunit vaccines, haemagglutinin of influenza A and B viruses, have been considered in order to facilitate vaccine production. In the present study, we have focused on construction of a recombinant baculovirus encoding the large subunit of novel influenza virus A/H1N1 haemagglutinin. The full genome of haemagglutinin was cloned into pGEM-TEasy vector and sequenced. The large subunit of the haemagglutinin gene was amplified by PCR using specific primers and cloned into pFast- BacHTc donor plasmid, which was then confirmed by restriction enzyme analysis and sequencing and transformed into E. coli DH10Bac competent cells. The bacmid DNA was transfected into insect cells to produce recombinant baculovirus. Expression of recombinant haemagglutinin in insect cells was determined by SDS-PAGE and immunoblotting. It has been shown that the recombinant haemagglutinin (rHA) obtained from the baculovirus insect cell expression system has suitable immunogenicity in human and can be considered as a candidate flu vac- cine. Here we produced large amounts of the HA1 protein of novel influenza A/H1N1 (Iranian isolate) in insect cells. The immunogenicity and efficacy of the recombinant HA1 will be evaluated as a vaccine candidate and compared to the recombinant HA1 produced in a prokaryotic system.

Apoptosis of ejaculated human sperm is induced by co-incubation with Chlamydia trachomatis lipopolysaccharide.

BACKGROUND: Previous work has shown that co-incubation of human sperm with Chlamydia trachomatis serovars E and LGV leads to premature sperm death and that this is due primarily to chlamydial lipopolysaccharide (LPS). Here, we investigated the possible involvement of apoptosis in this premature sperm death. METHODS: Highly motile preparations of sperm from normozoospermic patients were co-incubated for 6 h with extracted LPS from C. trachomatis serovars E and LGV. Three different methods were used to determine if LPS-treated sperm underwent apoptosis, including: (i) flow cytometry; (ii) measurement of ADP:ATP ratios; and (iii) measurement of mono- and oligonucleosomal DNA fragments. Caspase activity was also investigated by fluorimetry and by use of a pan-caspase inhibitor and caspase-3 inhibitor. RESULTS: All three methods used for detection indicated that C. trachomatis LPS induced some apoptosis in sperm after 6 h when compared with a staurosporine (apoptosis-positive) control. Moreover, a greater degree of apoptosis was seen with C. trachomatis serovar E than with serovar LGV. It was also shown that C. trachomatis LPS-induced apoptosis of sperm could be blocked with a pan-caspase inhibitor and a caspase-3 inhibitor. Moreover, by using a fluorogenic substrate, apoptosis was shown to be caspase-mediated. CONCLUSIONS: In general it is believed that apoptosis does not occur in C. trachomatis-infected host cells. However, using three different methods, our findings clearly indicate that co-incubation of sperm with C. trachomatis LPS results in cellular death which is in part due to apoptosis and is caspase-mediated. These findings provide an explanation as to how C. trachomatis can mediate premature death in human sperm.

Chlamydia trachomatis-induced death of human spermatozoa is caused primarily by lipopolysaccharide.

Elementary bodies (EBs) of Chlamydia trachomatis serovar E are more toxic to sperm than those from serovar LGV. In this study, lipopolysaccharide (LPS) was prepared from the EBs of both serovars and incubated with human spermatozoa at concentrations that matched the LPS concentration of EBs. The effects of EBs and LPS on sperm motility, viability and acrosomal status were then determined. Sperm motility was measured by computer-assisted sperm analysis and the hypo-osmotic swelling test was used to determine the proportion of dead cells. Acrosomal status was examined using a standard mAb assay. Over a 6 h incubation, LPS from both serovars resulted in a marked reduction in sperm motility (and a concomitant increase in the proportion of dead spermatozoa) in a manner similar to that seen in response to EBs of serovar E. In addition, when sperm were incubated with a range of doses of EBs and LPS, probit analysis revealed that the greater spermicidal effects of EBs from serovar E (when compared with serovar LGV) were not observed when sperm were incubated with LPS from the two serovars. This suggests that the more potent effect of EBs of serovar E cannot be explained entirely by differences in the composition of LPS. Interestingly, Escherichia coli LPS was required in doses 500 times more concentrated than chlamydial LPS in order to kill a similar proportion of sperm, suggesting that bacterial LPSs may differ in their spermicidal properties. However, that chlamydial LPS was spermicidal was demonstrated by the use of polymyxin B (a polycationic antibiotic known to neutralize LPS effects), confirming that the effects observed were primarily a result of LPS activity.

Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death.

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.

Coincubation of human spermatozoa with Chlamydia trachomatis in vitro causes increased tyrosine phosphorylation of sperm proteins.

Elementary bodies (EBs) of the obligate intracellular bacterium Chlamydia trachomatis are responsible for the first step of attachment to host cells. We have studied the effects of EBs on human sperm protein tyrosine phosphorylation, which is important to sperm function. Indirect immunofluorescence using antiphosphotyrosine antibodies showed that serovar E, but not LGV, caused increased tyrosine phosphorylation which was localized to the sperm tail region. Immunoblotting revealed that serovar E caused a marked increase in tyrosine phosphorylation of 80- and 95-kDa sperm proteins, whereas serovar LGV caused increased phosphorylation of only the 80-kDa moiety. Considering the importance of tyrosine phosphorylation for sperm capacitation and other aspects of sperm function, we conclude that EBs may affect these events.