Salmonella Infantis strains, isolated from human sources in Tehran hospitals from 2008 to 2010.The aims of this study were to investigate antibiotic resistance pattern and molecular characterization of Salmonella Infantis in clinical isolates in Iran.  Infection caused by Salmonella is one of the major public health problems. Despite the clinical importance of Salmonella enteric subsp. enteric serovar Infantis in humans, there is no information available about the common clones of S. Infantis strains were identified by conventional microbiological and serological testing. The antimicrobial susceptibility of the S.Infantis isolates was determined using the disk diffusion method. The genetic relatedness and the dominant clones of S. Infantis strains were detected by the Multi Locus Sequence Typing (MLST) and pulsed-field gel electrophoresis (PFGE) techniques. S. Infantis isolates represented multidrug-resistant patterns. PFGE revealed high genetic similarity among S. Infantis strains. While, MLST indicated high-clonal similarity among strains, where all S. Infantis strains were assigned to the ST32 sequence type. More than 80% of the S. Infantis in clinical isolates using MLST. The genetically closed MDR S. Infantis clones were responsible for the apparent endemic occurrence of salmonellosis, caused by this Salmonella serovar, in Tehran. This is the first study in Iran conducted to determine the sequence types of  Gastrointestinal tract infection is still one of the most serious public health issues in many geographic areas and is endemic in most countries including Iran .Salmonella is an important cause of gastrointestinal tract and food-borne infections worldwide. Infections caused by multi-drug resistant Salmonella spp. are increasing in many countries, including Iran , S. Infantis is more prevalent in poultry than in other animals  S. Infantis has been reported in Iran and other countries. This has caused problems for the clinical and veterinary sectors . It was conducted in accordance with the CDC (Centers for Disease Control and Prevention) Pulse Net protocol (www.cdc.gov/pulse.net). The restriction patterns were compared using Dice-coefficient online program and grouped by the Unweighted Pair-Group Method with Arithmetic Mean (UPGMA). S. Infantis isolates were clustered based on the similarity with a coefficient higher than 90%.The clonal relatedness of Multilocus sequence typingS. Infantis strains were selected for the MLST analysis. These isolates were chosen based on PFGE patterns, antibiotic resistance profiles, location of sampling, and type of specimens. S. Infantis genomic DNAs were extracted using a commercial extraction kit . The internal fragments of seven housekeeping genes; aroC, dnaN, hemD, hisD, pure, sucA, and thrA of S. enterica were amplified using specific primers as described in the online MLST database (http://mlst.warwick.ac.uk/mlst/dbs/ Senterica). All the PCR products were subjected to sequencing  and the sequencing data were recorded in salmonella MLST database. Phylogenetic analysis was performed by eBURST. A total of 15 Salmonella Infantis isolatesSalmonella strains (94 isolates) were recovered from stools, whereas the remaining strains were isolated from urine, blood or other biological fluids.The study included 6050 patients who had been admitted with enteritis symptoms to Baqiyatallah, Mofid, and Children’s Medical Center in Tehran, during 2008–2010. Of these, 110 patients of which 49 (44.5%) were females and 61 (55.5%) were males showed symptoms of salmonellosis. Salmonella infection was diagnosed based on clinical presentations and laboratory confirmation. The majority of the patients (80%) were less than 12 years of age. Most of the Salmonella isolates, 34.5% (38 strains) were identified as Salmonella Infantis. Thirty one S. Infantis strains (89%) were recovered from pediatric patients under 12 years and seven strains were isolated from patients over 12 years.Of 110 Antimicrobial susceptibility testing (S/TE/D/N/NA/K/CRO/CTX/CAZ/AM) was observed in 45% (n = 14) and 13% (n = 4) of S. Infantis isolates, respectively. Antibiotic resistance patterns of isolates are present in The results of the antimicrobial susceptibility testing were shown in Pulsed-Field gel electrophoresisS. Infantis strains isolated from Tehran hospitals. The isolates were divided into three different PFGE types or pulsotypes—A, B, and C  was detected among Multi-Locus sequence typingS. Infantis isolates were assigned to sequence type 32 (ST32), using MLST. The allele profiles of the aroC, dnaN, hemD, hisD, pure, sucA, and thrA genes were 17, 18, 22, 17, 5, 21, and 19 in ST32, respectively. ST32 was located in the eBURST group number 31 (eBG1) or clonal complex 31.All the S. Infantis clinical samples collected from the hospitals in Iran. The genetic relationship between these strains was also elucidated by PFGE and MLST techniques.The present study provides evidence of the presence of high frequency of MDR, observed in S. Infantis in human samples was 28%, which is higher than the rates reported earlier from Tehran, Iran by Tajbakhsh et al. (8%) and Hamidian et al. (5.4%)  of S. Infantis in broiler farms located in three northern provinces of Iran  ,13. Howe of Iran .S. Infantis from human infections ranged from 2-7% between the years 1980 and 2009—several years even represented the third- or fourth-most prevalent serotype  of MDR  in Iran , 13. TheInfantis , 24, 25.hoxazole . Furtheribiotics .S. Infantis strains in Iran. Rahmani et al. revealed highly similar PFGE patterns in S. Infantis strains isolated from poultry, indicating clonal relatedness across different geographical locations in Iran (S. Infantis strains, isolated from different sources worldwide. Epidemiological evidences confirmed clonal distribution of S. Infantis isolates. Dionisi et al. showed high similarity among S. Infantis isolated from different sources in Italy. Cluster analysis concluded that isolates with same resistance patterns belonged to a large cluster with > 90% genetic similarity (et al. found two large related clusters of S. Infantis isolates in various European countries—of which the Austrian and Polish MDR clones of a cluster are identical with, or closely related to, the main Hungarian clone (et al. showed that two major closely related genotypes of S. Infantis were isolated from broiler, meat, and pork in Germany (There is only one published study that used PFGE for molecular typing of  in Iran . The PFGmilarity . Nógrádyan clone . Hauser  Germany .S. Infantis strains studied may have descended from a common precursor that is responsible for the contamination in humans in Tehran.Additionally, MLST showed a high clonal similarity among all the strains assigned to the same ST32. This is consistent with the previous results found by others from different sources worldwide -28. Our S. Infantis isolated from human sources in Tehran. The genetically closed MDR S. Infantis clones are responsible for the apparent endemic occurrence of salmonellosis, in Tehran. Further molecular epidemiology investigations are required to assess the linkage and clonal relatedness of the S. Infantis strains isolated from different sources, such as humans, food and animals, in a different period of time and region in Iran.The findings of this study provide further information on the molecular epidemiology of


Cockroaches are found as the most common urban pests of tropical countries, prompting economic and serious health risk problem for humans by carrying microbes and allergens, acting as vector for various pathogens of diseases. The present study was conducted from April 2013 to March 2014 in various human dwelling localities of urban area of district Lahore, Pakistan.Periplaneta Americana (P. amercana), Blattella germanica (B. germanica), Blatta orientalis , and Blatta lateralis  were collected and identified from the study site.Cockroaches were collected randomly by hand, food baited and sticky traps throughout the year. Four species of cockroaches (B. germanica was the most dominant indoor species with highest diversity indices in study areas. Overall cockroach species diversity was highest in July–September, 2013 with highest Simpson index of diversity and Shannon index as well. P. americana was found second broadly distributed in the study area followed by B. orientalis and B. lateralis were intermediately distributed in residential areas and narrowly distributed in hospitals. Residential areas and hospitals were highly infested with B. germanica followed by P. americana. Population index of B. germanica for hospitals was double than residential areas. B. lateralis was observed as displacing B. orientalis in outdoor habitat through competing with its habitat and food sources.The infestation rate of different species depends on availability of food sources, sanitary conditions and climatic conditions. Cockroach infestation can be controlled with knowledge about their biology and behavior, attention to sanitation and effective use of commercial insecticides. Out of the 4600 species of cockroaches only 50 species have been reported as pests of human structures and dwellings worldwide. They randomly infringe in human habitation and never cause threat to indoor structures . MorpholBlattella germanica (B. germanica)  . Poor sarmanica) .Periplaneta americana (P. americana) (American cockroach), P. australasiae , Periplaneta brunea (P. brunnea) (Burmeister) (Large brown cockroach), Periplaneta fuliginosa (P. fuliginosa), Blatta orientalis  , Neostylopyga rhombifolia (N. rhombifolia) (Stool) The Harlequin cockroach, Blatta (Shelfordella) lateralis   (Turkistan cockroach) and Hebardina concinna (H. concinna) (Dehaan)  .B. germanica (German cockroach), Blattella lituricollis (B. lituricollis)  , Supella longipalpa  (Fabricius) (Brown banded cockroach), Blattella vaga (B. vaga) Hebard (Field cockroach), Blattella asahinai (B. asahinai) Mizukubo (Asian cockroach), Jacobsonina erebis (J. erebis), Symploce pallens  (Smooth cockroach) Symploce sphaerica (S. sphaerica), Symploce miyakoensis (S. miyakoensis), Symploce okinerabuensis (S. okinoerabuensis), Symploce paramarginata (S. paramarginata) and Symploce evidens (S. evidens). Family polyphagiadae include Polyphaga aegyptica (P. aegyptica) and Polyphaga saussurei (P. saussurei). Family Blaberidae include Rhyparobia maderae (R. maderae) (Madeira cockroach). Family Pycnoscelidae include Pycnoscelis surinamensis (Linnaeus) (P. surinamensis) the Surinam cockroach. Family Oxyhaloidae include Nauphoeta cinerea (Olivier) (N. cinerea) the lobster cockroach , Ascaris lumbricoides (A. lumbricoides), Trichuris trichura (T. trichura), Taenia spp and identified protozoans are Cyclospora spp, Entamoeba histolytica (E. histolytica), Entamoeba coli (E. coli), Balantidium coli (B. coli) and Isospora belli (I. belli) , Mucor, Rhizopus spp, Aspergillus fumigans (A. fumigans) and Penicillium spp. (The parasitic organisms identified from cockroaches include helminthes and protozoans. The helminthes include . belli) . Cockroaium spp. .Klebsiella pneumoniae (K. pneumoniae), Salmonella spp, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Enter-obacter cloacae (E. cloacae), Citrobacter freundii (C. freundii), Enterobacter aerogenes (E. aerogenes) and Proteus mirabilis (P. mirabilis) which are potential pathogens. There was no significant difference between the overall bacteria load on the external surface in cockroaches found in the food-handling establishments (60.08%) and households (39.92%) , Shigella dysenteriae (S. dysenteriae) and toxigenic strains of E. coli retained in the gut of cockroaches for up to several days (Cockroach associated bacteria include (39.92%) . Pseudomral days .Present study was conducted with the objective to report species composition, diversity, abundance, richness and population dynamics of cockroaches in urban areas of district Lahore. Lahore features a five season semi-arid climate ie foggy winters (15 Nov–15 Feb) with few western disturbances causing rains, pleasant spring (16 Feb–15 April), summer (15 April-June) with dust rain storms and heatwave periods, rainy monsoon (July–16 September) and dry but pleasant autumn (16 September–14 November) (Punjab Meteorology Department 2014). Due to the high population density in the city, 5000 tons per day of solid waste is produced in Lahore that is collected and disposed in one landfill site at Mehmood Booti and sewage is drained into river Ravi (Lahore Waste Management Company Report 2014). There are few scattered researches were conducted about species composition and abundance in last years. Economic importance of this domestic pest should be recognized. However, they are notorious as vector of nosocomial infections, contaminating and deteriorating food and consumables, carry allergens of asthma and resistant to insecticidal sprays.Study site is the urban area of district Lahore lying between 31°15′–31°45′ N and 74°01′–74°39′ E, 217m elevated from sea level with total population of 7,566,000. Samples were collected from different hospitals, shopping malls/stores, institutes and residential areas in different seasons around one year.Cockroaches were observed and collected randomly from April 2013 to March 2014 with the help of sticky traps, food baited pitfall traps and manual catching by hand. Specimens were collected from 20 different sites including hospitals  shopping malls/stores , institutes/office , houses . Global Positioning System of study sites are listed in Average monthly temperature and humidity data were obtained from the Punjab Meteorological Department, Lahore and comparatively analyzed with population density of cockroaches.The observations were tabulated and data were statistically analyzed using Microsoft excel. Relative Abundance, species richness and evenness of each species of cockroaches was calculated. Diversity of different cockroach species on the Simpson and Shannon indices was worked out according to P. americana) They found in sewers and basements especially around pipes and drains.American Cockroach : They found beneath the mulch, leaf litter, stones and debris outdoors, garbage, filthy materials that is going to decay.Oriental Cockroach (B. germanica): They found in kitchens, storage areas especially where food being prepared or stored.German Cockroach :Turkestan Cockroach  were identified during the entire sampling period.Turkestan cockroaches are native to large area of the Middle East extending from Libya Eastward to Central Asia including Afghanistan, Pakistan, Uzbekistan and southern Russia . They weB. germanica was the most dominant species belonging to family Blattelidae comprising of 45% of the total catch followed by P. americana (35%) belonging to family Blattidae. These two species comprises 80% of the total specimens collected. Other two species include B. orientalis (9%) and B. lateralis (11%) of the total specimen trapped. These findings are compatible with the studies of From this study it was revealed that B. germanica, followed by P. americana (33.50%) while B. orientalis and B. lateralis are 9.96% and 9.75% respectively  and −0.15434 (Shannon index). During sampling the higher number of males in food bait traps and sticky traps was not surprising as the males were active predators and roam in search of mates. Females were low in number because they conserve energy for reproduction and found less active and common. One of the many reason is may be the traps were baited with female sex attractants which results in maximum male capturing in traps.During the first trimester of study  the minimum and maximum average temperature was 23.8 °C and 37.6 °C, respectively and relative humidity was 41% . The autectively . DiversiB. germanica was followed by P. americana (35.81%) while, B. orientalis and B. lateralis comprised 8.11% and 11.7% respectively  and −0.1566 (Shannon index). During sampling the higher number of nymphs of late instar were found. Now they had gone through metamorphosis to change into adults and are ready for reproduction. In most species time for late instar to turn into females is longer than males.During the second trimester of study  the minimum and maximum average temperature was 24.8 °C and 34.1 °C, respectively and relative humidity was 74% . Summer ectively . DiversiB. germanica was most dominant species (44.62%) followed by P. americana, B. orientalis and B. lateralis respectively. In third trimester of study , . Most dominant species B. germanica (40.82%) was followed by P. americana (37.02%) and B. orientalis and B. lateralis comprise 7.17% and 14.97% respectively  Shannon index value was −0.52419 and Simpsons index of diversity was 0.6687ectively . OverallP. americana and B. orientalis as outdoor species of cockroaches but they can intrude into indoor environment through severage pipes and crevices in harsh seasons. While B. germanica and B. lateralis always found in indoor environment  it will increases their likelihood to colonize that habitat. When an organism experience high or low temperature in their environment they produce certain proteins called “heat shock proteins (HSP)” in their cells which allows recovery on a cellular level. These HSP are found in many organisms from bacteria to mammals .Organisms have the ability to increase or decrease their core temperature in response to environmental temperatures . CertainB. asahinai had expanded its range Northward from Florida. They concluded that visual and bucket sample population began increasing in late May and reached their zenith in late August or early September.In this study, cockroache’s species distribution was observed almost all around the year though environmental temperature may effect on distribution of some outdoor species. Cockroaches are highly adapted for diverse land environment especially dry harsh environment. Cockroaches were not more noticeable in cold months in third and fourth trimester but probably they have physical and behavioral adaptation which helps them to withstand in extreme low temperature on land. This study coincides with the findings of B. germanica followed by apartments and villas. The infestation rate of the cockroaches was related to the residential types.The present study found indoor species of cockroaches including German and Turkestan cockroach were more prominent in bedrooms, kitchen, stores, hospital wards, office stores and rooms adjacent to canteen. Their high prevalence in these areas can be related to ideal shady, enriched food and cool environment which is more favorable to increase their population. Other outdoor species American and Oriental cockroaches are outdoor species and found near to sanitary pipes, washrooms, filthy habitat, adjacent gardens of houses, sewerage pipes and kitchen exit pipe where plenty food is available. Late nymphs and adults were more numerable in collection site where fewer females were captured. Environmental conditions like temperature and humidity also do affect the life cycle and developmental stages of cockroaches. Findings of our study are also in conformity to Most of the urban areas in district Lahore especially human dwellings are infested with cockroach population. Their presence in human associated environment and species abundance pose them a critical threat for human health and indicate poor infrastructure of sanitary and waste disposal. The infestation rate of different species depends on availability of food sources, sanitary and climatic conditions. Cockroach infestation can be controlled with knowledge about their biology and behavior, attention to sanitation and effective use of commercial insecticides.


Porphyromonas gingivalis  is a well-known pathogen in periodontitis. This study investigated the pathogenic effects of P.gingivalis on autoimmune arthritis in vivo. Collagen-induced arthritis (CIA) mice were intraperitoneally injected with W83 and 2561 strains of P.gingivalis. Infection with P.gingivalis exacerbated arthritis score in CIA mice. Synovial inflammation and bone destruction in CIA mice infected with P.gingivalis were more severe than in uninfected CIA mice. Both W83 and 2561 strains were more pro-arthritic after arthritis symptom was fully activated. Interestingly, only W83 strain was arthritogenic before autoimmune reaction initiated. Citrullination was detected in synovial tissue of CIA mice and CIA mice inoculated with P.gingivalis, but not in normal control mice. The citrullinated area was greater in P.gingivalis-infected CIA mice than in non-infected CIA mice. This study showed that P.gingivalis exacerbated disease in a mouse model of autoimmune arthritis and increased the expression of citrullinated antigens in the synovium. The arthritogenic effects of P.gingivalis were at least in part, dependent upon the bacterial strain with or without fimbriae expression, route and time of infection. P.gingivalis-mediated citrullination may explain the possible link between periodontal disease and RA.Epidemiological studies show an association between rheumatoid arthritis (RA) and periodontal disease.  Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation and destruction . ProductCitrullination is an important post-translational modification that is ubiquitous in normal physiology. It is a process which arginine is conversed into citrulline in proteins. Peptidylarginine deaminase (PAD) is the enzyme that is responsible for this process ,7. PAD ePorphyromonas gingivalis (PPAD) is the only active form of bacterial PAD [Porphyromonas gingivalis  is a gram-negative, anaerobic bacterium. P.gingivalis is usually found in oral cavities, which induce periodontal disease such as periodontitis [PAD is present in most organisms, yet, PAD expressed by rial PAD . Porphyrdontitis . The phydontitis . Ammoniadontitis .P.gingivalis, citrullination, and RA was actively investigated [P.gingivalis, PPAD converts peptidyl-arginine into peptidyl-citrulline and T cells are activated by bacterial lipopolysaccharides. These T cells then activate B cells, leading to production of anti-citrullinated protein/peptide antibodies (ACPA) specific for citrullinated peptides. Citrullinated peptide-ACPA immune complexes are then formed and trigger inflammation in the joints of RA patients. During this process, citrullinated peptides originating from P.gingivalis and host peptides citrullinated by PPAD act as autoantigens that exacerbate autoimmune responses associated with RA [P.gingivalis and humans are reported [Because of these characteristics, the relation between stigated ,25–27. I with RA –31. Severeported ,32,33. Preported ,34,35. Preported .P.gingivalis in autoimmune arthritis. The arthritogenic effects of P.gingivalis was confirmed by bacterial strain (i.e. W83 and 2561), route, and time point of inoculation. We conducted clinical and histological analyses of a collagen-induced arthritis (CIA) mouse model infected with P.gingivalis. CIA mice infected with P.gingivalis showed increased expression of enolase, fibronectin, and citrullinated antigens in the synovial region. This study may provide a possible link between P.gingivalis and RA.Based on these observations, we aimed to examine the pathogenic role of All procedures involving animals were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experimentation provided by the Institutional Animal Care and Use Committee of the School of Medicine of The Catholic University of Korea. Experiments were performed according to the ARRIVE guidelines . The stuMice were each subdivided into 6 groups. Each group was composed of 5 mice. Non-treated mice were used as normal control group. To induce CIA, mice were immunized intradermally using 2mg/mL Bovine type II collagen  emulsified 1:1 with 2mg/mL Complete Freund’s adjuvant . 6-week-old male DBA/1J mice  were intradermally injected at day 0 with 100mL of CII/CFA emulsion. CII emulsified 1:1 with Incomplete Freund’s adjuvant  was injected into mice as a booster immunization 21 days after the first immunization.Development of arthritis was monitored and scored in a blinded manner. Disease severity was scored three times a week. The severity of arthritis in each front and hind paw was scored from 0 to 4 . A representative arthritis score was determined by summing the scores of all four paws .Porphyromonas gingivalis W83  and Porphyromonas gingivalis 2561 , Fusobacterium nucleatum  were used in this experiment. P.gingivalis strains W83, 2561 and Fusobacterium nucleatum (F.n) were grown in anaerobic conditions.9 colony-forming units (CFU) of bacteria in 30 μl PBS with 2% carboxymethyl cellulose . Bacteria were administered to the oral cavity of each mouse through a feeding needle two times a week.Oral infection group mice were infected before and after CIA induction. For oral infection, mice were inoculated with 1×109 CFU bacteria in a total volume of 100 μl. Intraperitoneal infection group mice were infected before and after CIA induction as well.For Intraperitoneal infection, mice were injected with 1×10The total anaerobic bacteria count was determined using the McFarland turbidity standard protocol. Bacteria were directly swabbed from the gingival surface of CIA mice using a small sterilized brush, and then soaked in tryptic soy broth medium , and incubated in an anaerobic basket with an anaerobic gas pack . Alveolar bone loss was examined morphometrically by measuring the distance between the cementoenamel junction (CEJ) and alveolar bone crest (ABC), as previously described ,13. Brie2 inhalation. The joint tissues from the hind paws were fixed in 4% paraformaldehyde and decalcified in 10% EDTA bone decalcifier prior to embedding in paraffin. Paraffin sections were then prepared (5μm thickness) and stained with hematoxylin and eosin, safranin O, and toluidine blue.Mice were sacrificed by COThe inflammation score and joint destruction score were measured using the procedure of Huckel et al. by three individual researchers in a blinded manner ,38. InflTartrate-resistant acid phosphatase (TRAP) staining was performed using a commercial kit , with hematoxylin as the counterstain. TRAP-positive cells with three or more nuclei were counted as osteoclasts.2O2 prepared in PBS. Staining was performed using Mouse on Mouse Basic Kit. Tissue sections were incubated with Mouse on Mouse (M.O.M) mouse IgG blocking agent to block nonspecific antibody binding. Anti-CCP (12G1) antibody  was diluted in M.O.M Diluent buffer and incubated overnight at 4°C. IgG isotype control antibody  was used to confirm nonspecific antibody binding IgG. The sections were then incubated with Biotinylated Anti-Mouse Ig Reagent and an avidin-biotin complex. Chromogenic reactions were visualized with DAB Peroxidase (HRP) Substrate Kit . Sections were counterstained with hematoxylin.To confirm the presence of citrullinated proteins, joint tissues were stained with anti-CCP (12G1) antibody  . Joint tTo detect the synovial proteins , anti-vimentin antibody , anti-fibronectin antibody , anti-enolase antibody  were diluted at a concentration of 1: 100 in 5% normal goat serum containing 1% PBA. Slides were incubated at 4°C overnight. The sections were then incubated with Biotinylated Anti-Mouse Ig Reagent and an avidin-biotin complex. Chromogenic reactions were visualized with DAB Peroxidase (HRP) Substrate Kit . Sections were counterstained with hematoxylin.The co-localization of vimentin, fibronectin, and enolase was confirmed by fluorescence staining. Staining was performed using M.O.M Fluorescein Kit . Tissue sections were incubated with M.O.M mouse IgG blocking Reagent. Anti-CCP (12G1) antibody was diluted at a concentration of 1:100 in M.O.M Diluent buffer and was incubated at 4°C overnight. Fluorescein Avidin DCS was diluted in PBS and incubated at RT for 10 min. For serum blocking, 10% normal goat serum  containing 1% PBA was incubated at RT for 30 minutes. Anti-vimentin antibody , anti-fibronectin antibody , anti-enolase antibody  were diluted at a concentration of 1: 100 in 5% normal goat serum containing 1% PBA. Slides were incubated at 4°C overnight. Alexa Fluor 594 goat anti-rabbit IgG (H+L) antibody  was diluted at a concentration of 1:200 in PBS and incubated for 40 minutes at RT. Nucleus staining was performed using 4',6-diamidino-2-phenylindole .Automated image analysis techniques were used to quantify the number of TRAP-positive cells. To quantify TRAP staining and IHC staining, slide labeled with TRAP and ACPA were scanned using the Leica SCN400 slide scanner . Using TissuemorphDP version software , the percentage of surface area per osteoclast (≥3 nuclei) and then DAB surface area were quantified. Assays were performed in triplicate.All experiments were repeated three or more times. The results are shown as mean and standard error of the mean. Error bars represent the standard error of the mean. Statistical analysis was performed and graphs were drawn using GraphPad Prism 5 (GraphPad). The T-test was applied to analyse non-parametric quantitative datasets, and the one-tailed p-value was calculated. *, P<0.01; **, P<0.005; and ***, P<0.001 indicated statistical significance.P.gingivalis infection affects the incidence or severity of CIA. P.gingivalis was inoculated orally and intraperitoneally with both W83 and 2561 strain. W83 is a strain lacking fimbriae expression, while 2561 is known to have several types of fimbriae on its membrane. To determine whether the time of infection affects CIA, we examined two different time points: before the first CII immunization (Pre-infection) and after the boosting CII immunization (Post-infection). Each group was infected twice a week with different strains of P.gingivalis  (S 2). F.n is a bacterial strain with mutant PAD. The W83 infected CIA mice resulted in an increase in arthritis scores whereas the F.n infected CIA mice arthritis score was similar to that of CIA mice. Through this observation we can assume that the increased inflammation in the CIA mice paw was affected by PPAD.In the case of intraperitoneally inoculated mice, the W83 strain resulted in a higher arthritis score than 2561 in the Pre-infection setting . Whereasswelling . To confP.gingivalis infection aggravated the pathology associated with CIA. Histology revealed that inflammatory cell infiltration and joint destruction in infected CIA mice was more severe than in CIA mice  that are prone to citrullination in the joint tissue was examined. The expression of these proteins in the arthritic synovium of Post-infected groups was higher than that in non-infected CIA mice . The areTo confirm if these proteins were citrullinated, we detected the co-localization of citrulline and vimentin or fibronectin. Co-localization of citrulline peptides and vimentin were increased in all CIA induced mice . The expP.gingivalis infection aggravated arthritis and increased citrullination in RA mouse model. Infection with P.gingivalis increased synovial inflammation in CIA mice, which is consistent with the findings of previous studies [P.gingivalis was higher than that in non-infected CIA mice. In this study, we demonstrated that P.gingivalis affects citrullination in the joint synovium in vivo.The present study showed that  studies –46. HoweP.gingivalis, the causative agent of periodontitis, is an eligible candidate because it citrullinates peptides via bacterial PPAD. Citrullination is a critical process in RA pathogenesis because citrullinated peptides induce pro-inflammatory responses [Epidemiological studies consistently report an association between periodontal disease and RA; thus many researchers have attempted to identify a pathophysiological connection between the two diseases –27,47–49esponses ,51 and aesponses . Murine esponses .P.gingivalis promotes citrullination in the synovium; however, evidence suggests that the bacterium directly citrullinates the host proteins in RA. P.gingivalis DNA has been detected in the synovial fluid and tissues of patients with RA [in vitro, implying that P.gingivalis generates citrullinated antigens in RA patients. Marsez et al. reported that PPAD plays a critical role in the development and progression of CIA [P.gingivalis induces autoimmune experimental arthritis via a mechanism that is independent of citrullination  [P.gingivalis.It is unclear how  with RA . Moreoven of CIA . A recengnaling) ; thus fuP.gingivalis (i.e. 2561 and W83). W83 is afimbriated strain and 2561 is a fimbriated strain of P.gingivalis. The bacteria were delivered through two route; orally or intraperitoneally. Several studies showed significant results through oral infection of P.gingivalis [P.gingivalis was delivered after immunization, both strains showed increase arthritis score than that of CIA controls  expression in the knee joint. Also, citrullinated forms of these proteins were increased in infected mice compared to that of CIA control mice.P.gingivalis induced production of citrullinated antigens and aggravated both the clinical and histological signs of arthritis. Therefore, P.gingivalis-mediated citrullination may be the link between periodontal disease and RA.Here, we showed that infection of CIA mice with the periodontal pathogen. S1 FigP.gingivalis (n = 5). Pre and Post P.gingivalis were oral infected twice per week throughout the experimental period. The score for each paw ranged from 0 (no swelling) to 4 (erythema and severe swelling encompassing the ankle and foot); the scores for all four paws were summed to generate a representative arthritis score.  (C) Representative image of jaw bone from mice in each group. (D) Methylene blue staining image of the jaw bone to examine the CEJ-ABC distance. (E) The distance was measured. Arthritis severity score of NC mice (n = 5), CIA mice (n = 5), and CIA mice infected with (TIF)Click here for additional data file.S2 FigFusobacterium nucleatum (F.n).The arthritic index was compared between normal, CIA control mice and mice infected with W83 or (TIF)Click here for additional data file.S1 FileExperiments were performed according to the ARRIVE guidelines.(PDF)Click here for additional data file.


Schistosoma infection may be associated with Plasmodium falciparum infection or related reduction in haemoglobin level, but the nature of this interaction remains unclear. This systematic review synthesized evidence on the relationship of S. haematobium or S. mansoni infection with the occurrence of P. falciparum malaria, Plasmodium density and related reduction in haemoglobin level among children in sub-Saharan Africa (SSA).It has been suggested that 2 were used to assess heterogeneity and the Egger test was used to examine publication bias. The summary odds ratio (OR), summary regression co-efficient (β) and 95% confidence intervals (CI) were estimated using a random-effects model. Out of 2,920 citations screened, 12 articles  were eligible to be included in the systematic review and 11 in the meta-analysis. The 12 studies involved 9,337 children in eight SSA countries. Eight studies compared the odds of asymptomatic/uncomplicated P. falciparum infection, two studies compared the incidence of uncomplicated P. falciparum infection, six studies compared P. falciparum density and four studies compared mean haemoglobin level between children infected and uninfected with S. haematobium or S. mansoni. Summary estimates of the eight studies based on 6,018 children showed a higher odds of asymptomatic/uncomplicated P. falciparum infection in children infected with S. mansoni or S. haematobium compared to those uninfected with Schistosoma . The increase in odds of asymptomatic/uncomplicated P. falciparum infection among children infected with Schistosoma remained significant when subgroup analysis was conducted for S. haematobium  and S. mansoni  infection. However, the density of P. falciparum infection was lower in children co-infected with S. haematobium compared to those uninfected with Schistosoma . The mean haemoglobin level was higher among children co-infected with S. haematobium and P. falciparum than those infected with only P. falciparum A systematic review in according with PRISMA guidelines was conducted. All published articles available in PubMed, Embase, Cochrane library and CINAHL databases before May 20, 2015 were searched without any limits. Two reviewers independently screened, reviewed and assessed all the studies. Cochrane Q and Moran’s IS. mansoni or S. haematobium co-infection may be associated with increased prevalence of asymptomatic/uncomplicated P. falciparum infection in children, but may protect against high density P. falciparum infection and related reduction in haemoglobin level.The current review suggests  Schistosoma co-infection is essential to inform decisions on appropriate control strategies for schistosomiasis and malaria in SSA. In this systematic review and meta-analysis, we synthesized evidence on the nature of relationship of S. haematobium and S. mansoni infection with the prevalence/incidence of P. falciparum infection, density of the parasite and related reduction in haemoglobin level among children in SSA. We searched all published articles available in PubMed, Embase, Cochrane library and CINAHL databases before May 20, 2015 without any language restriction. We found five cross-sectional and seven prospective cohort studies eligible to be included in the systematic review, and 11 of these studies were included in the meta-analysis. A summarized analysis of the study findings showed that S. haematobium and S. mansoni infection is associated with an increased odds of asymptomatic/uncomplicated P. falciparum infection. However, density of P. falciparum infection decreased and haemoglobin level increased during S. haematobium co-infection.A clear understanding of the epidemiology of malaria during  Plasmodium falciparum  is responsible for most malaria cases and deaths due to the disease in SSA  and subgroup analysis further minimized the risk of heterogeneity among studies evaluating the effect of S. mansoni infection on the odds of P. falciparum infection (I2: 0.0%).This is the first meta-analysis to study association of idelines . Studiesstimates , 36, 41.ect size . Hence, ne study , the hetPlasmodium and Schistosoma infection could also affect the present results [S. haematobium and P. falciparum co-infection did not examine the study participants for infection with soil-transmitted helminths. Therefore, children without S. haematobium infection may have had other helminth infections that could have further confounded the results [Schistosoma infection. This made it difficult to clearly evaluate the potential effect of classes of intensity of Schistosoma infection on the current result. Finally, the lack of sufficient data precluded performing a meta-analysis to estimate the effect of Schistosoma co-infection on the risk of acquiring P. falciparum infection and related anaemia. We did not find any studies which examined the relationship between Schistosoma and P. vivax infection or severe malaria that were eligible in the present review.Limitations of the original studies related to the diagnosis of  results ,45. In a results ,47. MoreS. mansoni or S. haematobium co-infection may increase susceptibility of children for asymptomatic/uncomplicated P. falciparum infection. However, S. haematobium co-infection may protect against high P. falciparum density and related reduction in haemoglobin level. Findings on the effect of Schistosoma infection against the risk of P. falciparum infection are heterogeneous.The present review suggests that S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigP. falciparum infection between children who were infected and uninfected with Schistosoma in SSA.Odds ratio against standard error of odds ratio for eight studies, which compared the prevalence of asymptomatic/uncomplicated (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Babesia microti is an emerging tick-borne pathogen and the causative agent of human babesiosis. Mathematical modeling of the reproductive rate of B. microti indicates that it cannot persist in nature by horizontal tick-host transmission alone. We hypothesized that transplacental transmission in the reservoir population contributes to B. microti persistence and emergence in North American rodent populations.Peromyscus leucopus were collected from Connecticut and Block Island, Rhode Island and analyzed using a highly specific quantitative PCR (qPCR) assay for infection with B. microti.n = 103) of mice were infected with B. microti. Females exhibited significantly higher parasitemia than their offspring (P < 0.0001) and transplacental transmission was observed in 74.2% of embryos (n = 89). Transplacental transmission of B. microti is thus a viable and potentially important infectious pathway in naturally infected rodent species and should be considered in future theoretical and empirical studies.In April, 100%  contains supplementary material, which is available to authorized users. Babesia microti (Apicoplexa: Sporozoea) is a zoonotic intraerythrocytic apicomplexan parasite and is responsible for almost all cases of human babesiosis in the United States  and two locations on Block Island, Rhode Island  for two trapping sessions from April 26 - May 2 (hereafter referred to as April) and July 23–31, 2016. In each trapping session, animals were trapped for two consecutive nights using Sherman live traps  baited with peanut butter, oats, and sunflower seeds. Traps were arranged in nine 200 m transects with one trap placed every 10 m for a total of 180 traps at each location, except for NI where 100 m transects were used for a total of 90 traps.Adult Animals were removed from traps, morphological characteristics  were collected, and attached larval and nymphal ticks were counted and removed from the ears and body. All animals were euthanized with an overdose of isoflurane and necropsied immediately in the field. All organs and tissues were preserved in liquid nitrogen, blood samples were dried on Whatman FTA cards , and the carcasses were wrapped in Whirl-Pak bags  and frozen in liquid nitrogen; embryos of pregnant females remained within the carcass. All samples were transferred to -80 °C for long term storage upon return to the laboratory. All animal procedures were in accordance with guidelines approved by the Columbia University Institutional Animal Care and Use Committee (IACUC no. AC-AAAL3656).P. leucopus is 22–28 days, embryos were separated into three developmental age categories , and DNA concentration was analyzed using a spectrophotometer .B. burgdorferi using a quantitative PCR (qPCR) specific for a unique 69 bp segment of the 16S rRNA gene: forward primer (5'-GGC GGC ACA CTT AAC ACG TTA G-3'), reverse primer (5'-GCT GTA AAC GAT GCA CAC TTG GT-3'), probe (6FAM-TTC GGT ACT AAC TTT TAG TTA A-MGBNFQ) [B. burgdorferi is an extracellular bacterium and primarily observed in tissues, we restricted our screening of B. burgdorferi to only adult ear punch biopsy samples.For adult samples, a 3 mm ear punch biopsy was collected from each mouse and DNA was extracted using the QIAcube HT DNA extraction system (Qiagen). DNA concentration was measured for each sample using a spectrophotometer (Denovix Inc). Each ear punch biopsy sample collected from adults was then tested in duplicate for the presence of -MGBNFQ) . SamplesB. microti using a qPCR designed specifically for detecting a 104 bp section of the 18S rRNA gene of B. microti: forward primer (5'-AAC AGG CAT TCG CCT TGA AT-3'), reverse primer (5'-CCA ACT GCT CCT ATT AAC CAT TAC TCT-3'), probe (6FAM-CTA CAG CAT GGA ATA ATG A-MGBNFQ) [Babesia microti is primarily an intracellular erythrocytic protozoan and therefore we analyzed embryos, embryonic tissues, and adult dried blood samples for the presence of B. microti. Because blood samples could not be obtained from embryos, a 25 mg sample of heart tissue was also collected and analyzed from a subset of adult mice for direct comparison to embryos and embryonic tissues.For adult individuals, DNA from dried blood samples was extracted from FTA cards using the QIAcube HT DNA extraction system or by hand using the Qiagen DNeasy Blood and Tissue kit following the manufacturer’s protocol (Qiagen). DNA concentration was analyzed using a spectrophotometer (Denovix Inc). Embryos, embryonic tissues, and adult blood samples, were screened in duplicate for the presence of -MGBNFQ) . BabesiaB. burgdorferi and B. microti into pUC57-Kan plasmids . A dilution series (106–1 copy number dilutions) for each pathogen was developed by combining a single uninfected I. scapularis nymph (courtesy of the CDC) and a known amount of plasmid DNA followed by DNA extraction [Average cycle threshold (CT) and quantity values were collected from each run and mean infection prevalence  was calculated for each location. qPCR standards were constructed by separately cloning the aforementioned targeted regions of traction , 15. Quatraction , 16, 17;Babesia microti positive samples, determined via qPCR, were then subjected to a standard PCR using a specific primer set to amplify 437 bp of the B. microti18S rRNA gene (forward PIRO-A: 5'-AAT ACC CAA TCC TGA CAC AGG G-3' and reverse PIRO-B: 5'-TTA AAT ACG AAT GCC CCC AAC-3') [B. microti sequences deposited in the GenBank database and previously described as human-infecting Clade 1 and nonhuman-associated Clade 2 and Clade 3 pathogens  [ctively) . Alignmectively)  and usinctively) .B. burgdorferi were evaluated using a Fisher’s exact test. A Student’s t-test was used to compare the number of nymphs collected from April mice, individuals infected with B. burgdorferi and/or B. microti in April mice between males and females, between CT and BI, and to compare female infection to embryo infection overall and between trapping sessions. A nonparametric Kruskal-Wallis test was performed to assess differences in B. microti infection in the stages of embryonic development and the average number of embryos in each developmental stage. A pairwise Wilcoxon test was used to determine the variation between each stage of development. Finally, an analysis of variance (ANOVA) was completed to determine differences in parasitemia between the three stages of embryonic development. Geometric means of duplicate samples were used in all calculations.Infection prevalence was calculated for sex of adults, embryonic tissue type, and embryonic stage of development. Significant differences between the proportion of individuals infected and not-infected with n = 6) and their embryos (n = 26) collected in July were necropsied and analyzed for the presence of B. burgdorferi and B. microti using qPCR. In the April sampling session, a total of 63 P. leucopus mice were collected from the Connecticut (CT) locations and 40 from the Block Island (BI) locations . Overall, 12.62%  of mice were infected with B. burgdorferi and 100% with B. microti. The average B. burgdorferi log mean copy number per pg total DNA (MCN) did not vary significantly between CT and BI  and females . No significant differences in B. microti MCN were observed between CT and BI  and females .All mice collected in April and only pregnant females , including three and five mice collected on BI in April and July, respectively. All 12 pregnant females from CT were collected in April. One pregnant meadow vole (Microtus pennsylvanicus) was collected on BI in July and was included in some of the analyses. Four pregnant mice and the vole tested positive for B. burgdorferi (25.0%). All blood samples collected from adult pregnant females tested positive for the presence of B. microti via qPCR (100%) and no significant differences were observed between CT and BI females. The number of embryos per female (mouse only) ranged from 2–6 with an average of 4.5 embryos per female . The average number of embryos in Week 1 (n = 3.4) was not significantly different from the number of embryos in Week 2 , however there were significantly more Week 3 embryos compared to Week 1 , but not compared to Week 2 , whereas Week 3 embryo infection was lowest (56.10%); the average infection prevalence among all embryos was 74.16% . Embryos from Week 1 (MCN = 2.55 ± 0.17) and Week 2 (MCN = 2.49 ± 0.18) displayed significantly higher parasitemia than embryos from the Week 3 stage of development . Other reproductive tissue types  also tested positive for B. microti  than their embryos  showed significantly higher parasitemia than pregnant females from July . Conversely, July embryos  exhibited a significantly higher MCN than embryos from April . To assess differences in MCN among different sample types, we also compared heart tissue  and blood samples  collected from a subset of adult mice and found that they did not differ significantly (P = 0.2757).Females displayed significantly higher 18S rRNA gene of B. microti were obtained for six individuals including each embryo, adult female uterine arteries and veins, uterus, placenta, and embryonic sac . Most sampled sequences aligned nearly identically to the reference sequence for Clade 1 (AY144696), except for one sample of adult female arteries (2033-Arteries). The overall pairwise mean distance and sequence diversity was 0.005 for all embryonic samples (0.001 when 2033-Arteries was excluded) including the reference sequence for Clade 1 (AY144696) and 0.014 (0.011 when 2033-Arteries was excluded) when reference sequences from Clade 2 (AY144701) and Clade 3 (AY144690) were added. Unique B. microti sequences generated from this study have been deposited in GenBank .Sequences in the forward and reverse directions of the B. microti occurs in wild, naturally-infected populations of P. leucopus and M. pennsylvanicus in Connecticut and on Block Island, RI, USA, with 74% transmission efficiency from mother to fetuses. Although studies have previously reported transplacental transmission of Babesia canis canis in canines [B. microti in BALB/c laboratory mice [B. microti occurring in naturally infected rodent populations [B. microti in two species of vole  in Poland, finding 81% infection occurring in embryos and 90% in pups. Here, we report transplacental transmission in P. leucopus and a third species of vole, M. pennsylvanicus.Our study demonstrates for the first time that transplacental transmission of  canines , 23, B. ory mice , and humory mice , 26 onlyulations . Tolkaczulations  reportedB. microti in April, in contrast to only 12.6% of individuals infected with B. burgdorferi. These mice could have been infected by nymphal I. scapularis in the spring or could have survived and maintained the infection through the winter months (chronic infection). Because nymphal I. scapularis were found on 50.8% of mice collected in CT and 65.0% of mice collected on BI, it is possible these mice all became infected by questing nymphs once they emerged from their winter burrows. However, because a higher proportion of nymphal I. scapularis are typically infected with B. burgdorferi  than B. microti  in the New England area  [B. microti infection prevalence in mice requires an alternative explanation. A combination of vertical transmission and chronic infection with B. microti through the winter months would amplify B. microti even before most ticks emerged from diapause, typically in mid to late May [B. burgdorferi. The duration of B. microti infection in naturally infected (via tick vector) P. leucopus is unclear; however, chronic infection has been shown to persist in laboratory P. leucopus for seven months and possibly longer [B. microti was also observed for approximately five months in naturally infected voles from Russia [Peromyscus; consequently, mice could either become re-infected or B. microti could become reactivated every transmission season resulting in chronic infection and an increase of the pathogen in the population via vertical transmission. Reactivation of certain pathogens has been shown to occur during stressful life events (i.e. migration in birds) [B. microti and provide an additional explanation for prolonged infection in wild P. leucopus.All individuals were infected with y longer , 29. Chrm Russia . In thesn birds) , 32. SeaP. leucopus in our study region starts around March, while nymphal tick activity typically starts in April or May, although both events are highly dependent on seasonal variation in temperature [B. microti parasitemia in P. leucopus occurs 14 days after infection via tick bite [B. microti infection via ticks. The lower average number of Week 1 compared to Week 3 embryos may be explained by the higher likelihood of reabsorption of some or all embryos when the female is inseminated simultaneously or before infection. Reabsorption of embryos was suggested in a study using BALB/c laboratory mice which found that females inseminated in the acute phase of B. microti infection (0–12 days) did not produce any offspring [Neospora caninum in BALB/c laboratory mice [B. microti after insemination or during later stages of gestation and the pathogen had not fully disseminated to all embryos when the female was euthanized. Experimental manipulation of the timing of infection and insemination is required to fully characterize the affect B. microti may have on developing fetuses and pathogen persistence.The relative timing of insemination and infection in the mother may be important determinants of the survivorship and infection outcome of fetuses , 33. Theperature . Furtherick bite . Given tffspring . ReabsorPlasmodium spp. [Toxoplasma gondii [Trypanosoma spp. [Plasmodium spp., B. microti sporozoites invade host red blood cells and are able to breach the placental barrier to infect a female’s offspring [Females exhibited significantly higher parasitemia compared to their offspring. Although the different tissues investigated could partially account for this difference, we found no significant differences in MCN in a subset of samples of adult heart tissue and blood. Decreased parasitemia in embryos may thus be due to limited placenta permeability. Although the placenta is known to function as a barrier to potentially harmful microbes and pathogens , multiplium spp. , as wella gondii , Trypanooma spp. , and sevoma spp. , 41. Simffspring ; exactlyB. microti in female voles and their offspring, although the mechanisms for these differences were not explained. This was not the case in our study, with sequences from P. leucopus and M. pennsylvanicus revealing high similarity between females and fetuses and overall. Low genome-wide sequence diversity was also observed in other studies [B. microti represents a rich species complex consisting of three distinct clades in the United States using the 18S rRNA gene; however genetic diversity within each clade is relatively invariant [Ixodes species, locations known to be endemic for human babesiosis, and exhibit the least amount of genetic diversity. Whereas strains in Clade 2 were isolated from carnivores and those in Clade 3 were found to infect rodents; neither Clade 2 nor 3 have been found to infect humans. Sequences from this study most closely aligned with the reference sequence from Clade 1 and these sequences showed a low sequence diversity of 0.001. One of our sequences (2033-Arteries) exhibited higher diversity than predicted by previous studies; this might have occurred due to multiple infections or interference from other pathogens during Sanger sequencing (resulting in a noisy chromatograph) and most likely does not represent a unique B. microti species.Tolkacz and colleagues  found di studies , 44. Somnvariant . StrainsB. microti is a potentially important pathway for infection in wild rodent species, P. leucopus and M. pennsylvanicus, which may partially explain B. microti emergence and geographic expansion. In endemic areas, transplacental transmission may enhance early season amplification of B. microti, contributing to higher infection prevalences in both hosts and vectors. Additional enhancement mechanisms have been described, including increased transmission of B. microti to ticks feeding on hosts coinfected with B. burgdorferi based in field and laboratory studies [B. microti maintenance and spread in small rodent populations in areas with limited I. scapularis occurrence. For instance, in areas of Alaska, California, Colorado, Florida, Maine, and Montana that lack I. scapularis, small rodent populations  were infected with both human-infecting and non-human-associated strains of B. microti [I. angustus, I. muris, I. spinipalpis) or other exophilic ticks (I. pacificus) may contribute moderate enzootic persistence in certain areas [I. scapularis (a ‘bridge’ vector to humans) invades a region where B. microti had been enzootically maintained, as has been shown for B. burgdorferi [Our field-based results indicate that transplacental transmission of  studies , 11, 35  studies . Transpl microti –50. Somein areas  in a ‘crgdorferi , 51.B. microti, studies in a laboratory setting are required to specifically quantify the relative efficiencies of B. microti transmission routes, including tick-to-host transmission, vertical transmission in relation to insemination and infection timing, chronic infection, and xenodiagnoses and transmission efficiency of infected offspring. More extensive field studies quantifying the role of vertical transmission and chronic infection of B. microti should then be conducted to assess how these transmission pathways influence this pathogen’s persistence and transmission, in comparison to B. burgdorferi. As winter temperatures in these areas continue to increase each year [B. microti through chronic infection and vertical transmission.To confirm the relevance of transplacental and vertical transmission in ach year , overwinach year , 54, furB. microti occurs with high efficiency in wild rodent populations in New England. This non-vector mediated transmission mode may result in significant pathogen amplification, in particular if combined with chronic infection in the host and increased overwintering survival in warming climates.We demonstrate that transplacental transmission of Additional file 1:Table S1. Information for each pregnant female and infection status of her embryos collected in April and July, 2016. Site: LG, Lake Gaillard; OL, Old Lyme; RH, Rodman’s Hollow; NI, North Island. Age of embryos: W1, Week 1; W2, Week 2; W3, Week 3. (DOCX 19 kb)Additional file 2:Table S2.Babesia microti infection prevalence in reproductive tissues . (DOCX 14 kb)Additional file 3:Figure S1.Babesia microti sequence alignment from embryos and tissues of the 18S rRNA gene for six females (labeled by a four digit identification number) including three reference sequences representing the three Babesia clades . (DOCX 136 kb)


Background: Prostate cancer has been known as one of the most common malignancy in the men and it is therefore very important to prevent and treat this cancer. In this study, the anticancer effects of 20 species of medicinal plants in Iran, especially those grown in Chaharmahal and Bakhtiari province, were investigated on prostate cancer cell lines to identify potential natural alternatives for the development of prostate cancer anticancer drugs.Methods: The plants were gathered from Chaharmahal va Bakhtyari and their aerial parts extracted through maceration method using ethanol 70%. Anti-proliferative activity of extracts on PC-3, DU145 and HDF cell lines was evaluated by MTT assay 48 hours after treatment.Euphorbia szovitsii Fisch. & C.A.Mey. and Achillea wilhelmsii had anti-proliferative activity more than other plants on PC-3. Also IC50s for Urtica dioica, Euphorbia szovitsii Fisch. & C.A.Mey. and Medicago sativa were lower amount among the examined plants on Du-145.Results: Euphorbia szovitsii Fisch. & C.A.Mey., U. dioica and Medicago sativa with good anti-proliferative activity can serve as an effective source of natural products to develop new antiprostate cancer drugs.Conclusion: According to our result,  TheseGiven the treatments that are currently being used to fight prostate cancer and associated complications, drug resistance especially in metastatic prostate cancers, ever-increasing costs of common treatments, and the increasing incidence of prostate cancer in both developing and developed countries, it is necessary to discover newer therapeutic approaches with higher efficacy to reduce the incidence and mortality of prostate cancer. In this regard, it is necessary to find cytotoxic plants against various cancers, especially prostate cancer, which, despite lower side effects, can replace chemotherapy and difficult treatments, and also be used for treatment-resistant cases -17. ThesTaken together, our aim was to investigate certain species of medicinal plants in Iran, especially those growing in Chaharmahal va Bakhtiari province in Iran, whose effects have not yet been studied on prostate cancer cell lines but they are used as anticancer agents according to public beliefs and Iranian traditional medicine books, or whose anti-inflammatory and antioxidant effects were confirmed in studies so that they may be used to produce more efficient and novel drugs to treat prostate cancer.2.2.1.Stachys inflate, Salvia multicaulis Vahl, Hertia angustifolia, Sophora alopecuroides, Haplophyllum perforatum, Moriera spinosa Boiss., Teucrium orientale L. subsp. taylori. (Boiss.), Achillea wilhelmsii, Urtica dioica, Plantago lanceolata, Euphorbia microsciadia Boiss., Medicago sativa, Satureja bachtiarica, Acanthophyllum glandulosum Bung. ex Boiss, Onosma sericeum, Parietaria judaica, Phlomis persica, Ziziphora clinopodioides, Echinophora platyloba D.C, and Euphorbia szovitsii Fisch. & C.A.Mey included in this study.The plants were collected locally in different points of Chaharmahal va Bakhtiari province in Iran in May-Sep 2015 and botanically authenticated by Dr. Shirmardi  and Miss S. Khademian . 2.2./ml.The herbal samples were cleaned, shade dried in, pulverized to powder in a mechanical grinder and macerated in ethanol (70%) at room temperature for 72 hours. In the next step, the hydro alcoholic extracts were concentrated by a rotary evaporator under reduced pressure. Samples were dissolved in DMSO % 0.1  -23. Fina2.3.The following cancer cell lines were used for this study: PC-3 and DU145 (prostate cancer cell lines) and HDF  as non-cancer cell line. Cells were obtained from National Cell Bank of Iran .Cells were cultured in RPMI1640  with 1% penicillin-streptomycin and 10% FBS (Sigma) in a humidified atmosphere with 5% Co2 at 37°C throughout the assay.2.4./ml) was added to the wells which were incubated for 4 h. In the next step, supplements were eliminated, and the formazan crystals were dissolved in DMSO. The absorbance of the plates were determined at 570 nm with a reference wavelength of 630 nm in an enzyme linked immunosorbent assay (ELISA) reader.3--2,5 diphenyl tetrazolium bromide assay (MTT assay) was used for evaluating cell viability. The cells  cells were seeded in 96-well plates and incubated at 37°C. After 24 h of incubation, when cells reached more than 80-90% confluence, the medium was removed and the cells were treated with fresh medium containing various concentrations of plant extracts to be tested (10 μg/ml-1 mg/ml). After 48 h, the supernatant liquids were eliminated and a medium including MTT solution ] × 100. The IC50 value (the concentration with 50% cell inhibition) was calculated 2.5.The dose-response curves of the plants were fitted by means of the computer program GraphPad Prism 6.0 , and IC50 was defined by regression analysis.3.In this study, 20 species of Iranian medicinal plants were investigated. The most of the plants were from Lamiaceae family ./ml in MTT assay were considered inactive.Anticancer activity (IC50) of the 20 medicinal plants on DU-145 and PC-3 has been shown in tables 2. Extracts with IC50>300 μgEuphorbia szovitsii Fisch. & C.A.Mey.  activity in prostate tissue [So far, the preventive and anticancer effects of many medicinal plants, as well as the effects of their derivatives on different cell lines, have been studied. In a review (2015), the effects of 34 Iranian medicinal plants tested for their anticancer effects on different cell lines and with animals and human subjects were reported . U. dioi studied . In our e stress . Aqueouse tissue .Medicago sativa, as one of the other plants in our study, has good anti-proliferative activity on DU-145 cell line. In the previous study, anticancer effect of M. sativa on multidrug-resistant tumor cells lines has been reported. M. sativa could induce apoptosis in these cells lines [M. sativa have been indicated to have antitumor activity against different cancer cell lines such as leukemia, cervix and breast cancer [U. dioica and M. sativa on cancers suggest them as effective medicinal plants in the prevention and treatment of cancers.ls lines . Also sot cancer , 29. TheThymus vulgaris, Taverniera spartea, Camellia sinensis, Ferula gummosa, Allium sativum, Curcuma longa and Zingiber officinale are other species in Iran, which are similar to the studied plants studied in the current work in terms of plant families or chemical compounds, and their anticancer effects on prostate cancer have also been investigated [stigated -35. HoweEuphorbia szovitsii Fisch. & C.A.Mey, that is a species of the Euphorbia genus, was found to have better antitumoural effects on both prostate cancer cell lines than other plants under study. The plants from the Euphorbia genus have traditionally been used to treat inflammation and tumors [Euphorbia szovitsii Fisch. & C.A.Mey has an acceptable potential for anticancer effects on prostate cancer.In the present study, d tumors , 37. Besd tumors -45. The 5.E. szovitsii Fisch. & C.A.Mey. with good anti-proliferative activity on both prostate cancer cell lines and U. dioica and M. sativa with good anti-proliferative activity on DU-145 cell line can serve as an effective source of natural products to develop new anti-prostate cancer drugs.According to our results, The authors declare no conflict of interest.


Anaplasma phagocytophilum, which causes the disease tick-borne fever (TBF), is the most important tick-borne pathogen in European animals. TBF may contribute to severe welfare challenges and economic losses in the Norwegian sheep industry. The bacterium causes a persistent infection in sheep and several other animal species. The objective of this study was to investigate whether intrauterine transmission occurs in persistently infected sheep. The study included thirteen 5–6-month-old unmated ewes, of which twelve were experimentally infected with A. phagocytophilum (GenBank acc. no. M73220). Four to six weeks later, all ewes were mated, and nine became pregnant. Blood samples were collected from these ewes and their offspring. If the lamb died, tissue samples were collected. The samples were analyzed with real-time PCR (qPCR) targeting the msp2 gene. PCR-positive samples were further analyzed by semi-nested PCR and 16S rDNA sequencing. A total of 20 lambs were born, of which six died within two days. Six newborn lambs (30%) were PCR-positive (qPCR), of which one was verified by 16S rDNA sequencing. The present study indicates that intrauterine transmission of A. phagocytophilum in persistently infected sheep may occur. The importance of these findings for the epidemiology of A. phagocytophilum needs to be further investigated.  Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) may cause disease in several mammalian species including humans. In ruminants, the disease is named tick-borne fever (TBF) [Ixodes ricinus-infested pastures in Europe [er (TBF) ,2. The in Europe  and not n Europe . A. phagocytophilum may cause persistent infection in several animal species [I. ricinus ticks [A. phagocytophilum to their offspring during pregnancy, i.e., if intrauterine infection occurs. This mode of pathogen transmission may have important epidemiological consequences.  species , and she species ,6. This us ticks ,8, so laus ticks . The preA. phagocytophilum strain (GenBank acc. no. M73220), containing approximately 1 × 106 infected cells/mL and originally isolated from a local sheep flock [Thirteen 5–6-month-old unmated ewes of the Norwegian white breed (NKS) were used. Twelve ewes were inoculated intravenously with 0.4 mL of a whole blood dimethyl sulphoxide stabilate of an A. phagocytophilum. After delivery the following spring, blood samples (EDTA) and serum samples from the newborn lambs were obtained on the day of birth (day 0) and thereafter on days 3, 7, 14, 28, and 42. If the lamb died, tissue samples were collected, such as brain, lung, heart, spleen, liver, and heart blood. In addition, birth fluids and colostrum were collected at birth. The newborn lambs from the uninfected ewe were used as controls. All samples were stored at −20 °C before analysis.Rectal temperature was measured daily, and blood samples (EDTA) were collected regularly for the first month after inoculation. Haematology was performed using the ADVIA 120 Haematology System . Thereafter, blood samples, including serum samples, were obtained monthly. The ewes were mated 4–6 weeks post-inoculation (p.i.) with Samples were thawed at room temperature, and 200 µl was used for extraction. DNA was extracted with the MagNA Pure LC Instrument DNA Isolation Kit I  according to the manufacturer’s and instrument’s protocol. The DNA I blood cells high-performance program was used. The eluted DNA samples were stored at −20 °C. DNA from the tissue samples was extracted with the use of the MagNA Pure LC Instrument DNA Isolation Kit II  according to the manufacturer’s and the machine’s protocol. The DNA II tissue external Proteinase K program was used. Twenty-five milligrams of tissue was weighed and homogenized in 160 µL of tissue lysis buffer and 40 µL of Proteinase K. The samples were incubated at 55 °C overnight. One hundred microliters of the homogenized tissue samples was used for extraction, and the eluted DNA samples were stored at −20 °C.2O, Light Cycler 480 DNA SYBR Green I Master mix, Mannheim, Germany and template. The extracted DNA samples and reaction mix were loaded onto a 96-well white plate. The primers  amplify a product of 208 bp covering the conserved N-terminal region of the expression site for the msp2 (p44) gene. Negative and positive controls were included. The positive control was obtained from an inoculated animal during the acute course of infection, whereas the negative control was a reaction mix without any added substance. Samples with a crossing point (Cp) ≤ 40 were validated by Tm analysis in a range of 82–83 °C [A. phagocytophilum DNA were further analyzed by semi-nested PCR and sequenced for verification. The qPCR analysis was performed in a Light Cycler 480 Instrument  as described in previous studies . Briefly82–83 °C . All samrrs (16S rDNA gene) in A. phagocytophilum. Thereafter, the products were used in a second reaction with the same forward primer (16S-F5) and the new reverse primer ANA-R5 (5’TCCTCTAGACCAGCTATA) to amplify a 282 bp fragment. The amplicons from the semi-nested PCR were validated by gel electrophoresis on a 2% agarose gel. Positive PCR products were sequenced directly using BigDye Terminator cycle sequencing chemistry and capillary electrophoresis , and A. phagocytophilum variants were detected by visual inspection of the chromatograms. The finished sequences were compared with databases by a BLAST  search in NCBI’s webpage (http://blast.ncbi.nlm.nih.gov/Blast/). Semi-nested PCR was carried out in a PTC-200 instrument  as earlier described . BrieflyA. phagocytophilum [A. phagocytophilum antigen . Bound antibodies were visualized by fluorescein isothiocyanate (FITC)-conjugated rabbit-anti-sheep immunoglobulin . Sera were screened for antibodies at dilution of 1:40. If positive, the serum was further diluted and retested. A titre of 1.6  or more was considered positive. Sera were analyzed using an indirect immunofluorescence antibody assay (IFA), to determine the antibody titre of an equine variant of tophilum ,15. BrieA. phagocytophilum reacted with a fever (≥40 °C), bacteraemia, and neutropenia during the next two weeks (The unmated ewes infected with wo weeks . All werA. phagocytophilum infection (positive DNA) was only suspected in two lambs, which belonged to the same ewe. One of these lambs was found to be PCR-positive on two occasions: days 7 and 28 (A. phagocytophilum infection was found in different tissues from four lambs (67%), of which three belonged to the same ewe (A. phagocytophilum by semi-nested PCR and sequencing of the 16S rDNA gene (data not shown). The ewes were found to be PCR-positive at different occasions after the primary infection, especially during the period of delivery (data not shown). A total of 84 blood samples from newborn live lambs were investigated, and indication of an 7 and 28 . Birth fsame ewe . In summA. phagocytophilum were detected in all newborn live lambs, except for the precolostral samples  newborn lambs were PCR-positive (qPCR) for Anaplasma DNA. However, further investigation is needed in order to confirm a viable A. phagocytophilum infection in these newborn lambs.The present study indicates that intrauterine transmission of A. phagocytophilum may cause a persistent infection in different animal species, of which sheep can remain infected 25 months after the initial infection [A. phagocytophilum-infected sheep is not known, but antigenic variation of major surface proteins has been proposed to be the key feature to allow persistence [A. phagocytophilum shows a cyclic bacteraemia in infected sheep, characterized by varying numbers of organisms in the blood [Several microorganisms have evolved effective ways to escape the host’s immune system and can in this way persist in the host . As alresistence ,18. A. phe blood . Howeverhe blood ,19,20. A. phagocytophilum variant [A. phagocytophilum infection has to be further investigated. In the present study, all inoculated lambs reacted with a fever typical for TBF . Thereaf variant . In a pr variant ,22,23,24A. phagocytophilum are usually transmitted by ticks, but other pathways have been reported. For instance, human infection has been recorded by blood transfusion [A. phagocytophilum has been observed in cattle during the acute phase of the infection [A. marginal, has been reported to be transmitted to calves via placenta in chronically infected cows [nsfusion  and aftensfusion . Howevernsfusion . Incidennsfusion ,29. In anfection ,31. It mted cows . A. phagocytophilum was transmitted to one newborn lamb during pregnancy [A. phagocytophilum DNA in the original samples. The present result indicates that qPCR is more sensitive than semi-nested PCR in the detection of A. phagocytophilum, as has earlier been reported [In the acute phase of the infection, a human isolate (NY-18) of regnancy , howeverreported .A. phagocytophilum cycling and intrauterine transmission has to be further elucidated. Three pregnant animals had an indication of blood infection both right before and after delivery, and the newborn lambs from these ewes were all PCR-positive. Individual variations in the clinical and immunological response and cyclic bacteraemia have been previously observed ,19. HoweA. phagocytophilum infection causes stillborn and weak-born lambs is an open question. As already mentioned, three weak-born lambs were born 14 days before the estimated lambing time, which is beyond the expected time of survival after an early delivery [Of the six PCR-positive newborn lambs, all except two lambs died . Whetherdelivery . A. phagocytophilum-positive lambs may have been infected before day 70. However, this has to be confirmed in future studies. Newborn lambs could have been infected by A. phagocytophilum by ingesting fetal fluid and colostrum, but both fluids were PCR-negative in the pregnant ewes. In addition, three of the PCR-positive newborn lambs died before any colostral intake. In sheep, immunoglobulins are not normally transferred to the fetus via the placenta; a newborn lamb will therefore obtain maternal antibodies by ingesting colostrum during the first 48 hours . Fetal lA. phagocytophilum in the present study has been previously used in different experimental trials [A. phagocytophilum affecting sheep cause intrauterine infection remains an open question.The variant of l trials . Howeverl trials . WhetherA. phagocytophilum DNA was detected. In order to confirm a viable A. phagocytophilum infection in newborn lambs, tick transmission or blood inoculation studies using susceptible lambs should be performed. Future studies will hopefully reveal if intrauterine transmission plays a role in the epidemiology of A. phagocytophilum.In conclusion, persistently infected sheep may carry the pathogen from one grazing season to another. The present study indicates that the bacterium can be transmitted from ewe to offspring during pregnancy. However, only