Child maltreatment has consistently been found to be associated with attention deficit/hyperactivity disorder (ADHD). However, the robustness of this association and the direction of the link between maltreatment and ADHD remain unclear. We used data from the Environmental Risk (E-Risk) Longitudinal Twin Study, a cohort of 2232 British twins, to investigate the associations between exposure to abuse/neglect and ADHD in childhood and in young adulthood, and to test their robustness and specificity. We also aimed to test longitudinal associations between abuse/neglect and ADHD from childhood to young adulthood, controlling for confounders. Results indicated strong associations between abuse/neglect and ADHD in childhood and also in young adulthood. In childhood, the association was concentrated among children with comorbid conduct disorder. Longitudinal analyses showed that childhood ADHD predicted abuse/neglect in later years. This association was again concentrated among individuals with comorbid conduct disorder. Abuse/neglect in childhood was not associated with later ADHD in young adulthood after adjusting for childhood ADHD. Our study does not provide support of a causal link between child abuse/neglect and adult ADHD but highlights the possibility of a long-term effect of disruptive behaviors on the risk for experiencing abuse/neglect. These findings emphasize the need for clinicians treating people with ADHD, especially those with comorbid conduct disorder, to be aware of their increased risk for experiencing abuse/neglect. Interventions aimed at reducing risks of abuse/neglect should also focus on the environment of individuals with disruptive behaviors. The disIn the present study, we used prospectively-collected measures from a longitudinal cohort study of twins to examine the association between exposure to abuse/neglect  in childhood and adolescence, with ADHD up to age 12 and at age 18. First, we examined the associations between abuse/neglect and ADHD diagnoses in childhood and in young adulthood separately. We tested the robustness of these associations by also analysing ADHD symptom scales and by controlling for potential confounders. We also explored the specificity of these associations by looking at bullying and domestic violence. We further examined whether the association was concentrated specifically among ADHD participants with comorbid conduct disorder. In addition, we investigated twins’ differences in abuse/neglect and ADHD to control for familial confounding. Second, we investigated the longitudinal associations between abuse/neglect and ADHD from childhood into young adulthood.22.1Participants were members of the Environmental Risk (E-Risk) Longitudinal Twin Study, which tracks the development of a birth cohort of 2232 British children. The sample was drawn from a larger birth register of twins born in England and Wales in 1994–1995 . Full de2 = 0.86; p = 0.65), age-5 IQ scores , age-5 behavioral or emotional problems , or rates of childhood ADHD . At age 18 years, participants were asked to identify two individuals who know them well to act as co-informants; 99.3% of participants had co-informant data.Follow-up home visits were conducted when children were aged 7 (98% participation), 10 (96%), 12 (96%), and 18 years (93%). Home visits at ages 5, 7, 10, and 12 included assessments with participants and their mothers (or primary caretaker). With parents’ permission, questionnaires were mailed to the children’s teachers, who returned questionnaires for 94% of children at age 5 years, 93% of those followed up at age 7 years, 90% at age 10 years, and 83% at age 12 years. The home visits at age 18 included interviews only with participants (n = 2066). There were no significant differences between those who did and did not take part at age 18 years in socioeconomic status when the cohort was initially defined  children had experienced no severe victimization; 423 (20.7%) had 1; 127 (6.3%) had 2 or more severe victimization experiences by age 12.2.2.1.2These measures have been described previously . In brieabuse/neglect in adolescence included exposure to maltreatment, sexual victimization and neglect to match the variable of childhood abuse/neglect. In our study sample, 16.9% of the participants have experienced moderate (some) abuse/neglect in adolescence and 5.9% have experienced severe abuse/neglect in adolescence. A total of 42.4% have been exposed to some peer/sibling victimization and 15.3% have been exposed to severe peer/sibling victimization. Finally, 6.5% have been exposed to some family violence and 12% have been exposed to severe family violence.For this study, Adolescent poly-victimization. Adolescent poly-victimization was derived by summing all seven victimization experiences coded as severe (“4” or “5”): 1321 (64.8%) adolescents had experienced no severe victimization; 391 (19.2%) had 1; 325 (15.9%) had 2 or more severe victimization experiences.2.2.22.2.2.1We ascertained ADHD diagnosis on the basis of mother and teacher reports of 18 symptoms of inattention and hyperactivity-impulsivity derived from DSM-IV  diagnost2.2.2.2We ascertained ADHD at age 18 years based on private structured interviews with participants regarding 18 symptoms of inattention and hyperactivity-impulsivity according to DSM-5 criteria . Partici2.3Participants’ parental socio-economic status (SES) was measured via a composite of parental income , education (highest mother/father), and occupation (highest mother/father) when they were aged 5, and was categorized into tertiles .IQ at age 5 was measured using a short form of the Wechsler Preschool and Primary Scale of Intelligence—Revised  other forms of victimization, including bullying and domestic violence; and (2) a cumulative measure of victimization (poly-victimization). We tested whether the association was concentrated among ADHD participants with comorbid conduct disorder, and additionally in young adulthood, with other forms of psychopathology. We further controlled for familial confounders by examining correlations between twins’ differences scores of poly-victimization and ADHD total symptom scale. For these analyses, we used continuous variables of exposure to violence and ADHD symptoms to maximise variation in both measures. We conducted the analyses with DZ and MZ twins together, and then only with MZ twins to control for all genetic confounding.Regression analyses were conducted in Stata 14.1 . No inteTo examine the longitudinal associations between abuse/neglect and ADHD from childhood to young adulthood, we used structural equation modelling (SEM) procedures of Mplus 7.11 . We test33.1Our findings indicate higher rates of children meeting diagnostic criteria for ADHD among those who were exposed to abuse/neglect compared to children who were not exposed to abuse/neglect , panel aThe association between abuse/neglect and ADHD in childhood extended to other forms of childhood victimization: children who were frequently bullied or were exposed to repeated phases of domestic violence had greater odds for ADHD diagnosis . FurtherADHD was highly comorbid with conduct disorder in our sample: 118 (47.8%) children with ADHD had comorbid conduct disorder. Prevalence of exposure to abuse/neglect among sub-groups of children with ADHD, with or without comorbid conduct disorder, is presented in Fig. S1 (panel a). We found that the risk for being exposed to abuse/neglect was concentrated among children with ADHD and comorbid conduct disorder (Table S1).3.2Similar to childhood, we found an over-representation of young adults with ADHD among those who were exposed to abuse/neglect between 12–18 years, as well as an over-representation of those who experienced abuse/neglect among young adults with ADHD , panel bAs in childhood, the association between abuse/neglect in adolescence and young adult ADHD extended to other forms of victimization: participants who reported being severely victimized by peers or being exposed to severe family violence in adolescence had increased odds for young adult ADHD . FurtherSimilar to childhood, young adults with ADHD often had comorbid conduct disorder (36.2%), but also other forms of psychopathology (68.7%). Prevalence rates of exposure to abuse/neglect among sub-groups of young adults with a diagnosis of ADHD, with or without comorbidity, are presented in Fig. S1 (panel b and c). We found that the odds for moderate and severe abuse/neglect in adolescence were elevated among adults with ADHD and comorbid conduct disorder, as were the odds among those with ADHD alone (Table S1). We found similar elevated odds of abuse/neglect among the adults with ADHD and other forms of psychopathology, while the odds decreased but remained significant for those with ADHD only.3.3We examined differences between twins on poly-victimization and ADHD symptoms. In childhood, we found a modest association between twins’ difference scores on poly-victimization and difference scores on ADHD total symptoms scale . This indicates that within a twin pair, the twin who had higher score on poly-victimization also had more ADHD symptoms. This association became not significant when repeated with MZ twins only , indicating that the association between poly-victimization and ADHD symptoms in childhood was accounted for by genetic factors. In young adulthood, we found a modest association between twins’ difference scores on poly-victimization and difference scores on ADHD symptoms . This association remained when repeated with MZ twins only, thus controlling for shared environment as well as genetic factors . This finding indicates that the association between poly-victimization and ADHD symptoms in young adulthood is partly environmentally-driven.3.4B = 0.09, p = 0.115; B = 0.13, p = 0.011). This longitudinal association was robust to adjustment for the stability of being exposed to abuse/neglect up to age 18, for ADHD from childhood to young adulthood, and also for concurrent associations between abuse/neglect and ADHD in childhood and in young adulthood. When we controlled for age-5 IQ and parental SES . When we further controlled for conduct disorder in childhood . This finding indicates that the longitudinal association between ADHD and later abuse/neglect is specific to those participants with comorbid conduct disorder in childhood.Abuse/neglect in childhood was not associated with ADHD in young adulthood taking into account sex . In addition, referring to this time-period enabled us to gather detailed information regarding the exposure to victimization throughout adolescence. Second, due to our relatively small sample size, we had limited statistical power when looking at twins’ differences among our group of MZ twins. A larger sample size would facilitate further examination of twins’ differences in twin pairs discordant for abuse/neglect. Third, the E-Risk sample is composed of twins, so the results may not generalize to singletons. Reassuringly, the prevalence of childhood abuse/neglect as well as the prevalence rates of victimization exposure between 12 and 18 years in our sample matches recent UK general population estimates . The pre4.5We provided additional evidence regarding the robustness of the associations between maltreatment and ADHD, emphasising the important role of comorbid conduct disorder. We also showed that this association is not limited to childhood and not specific to abuse/neglect. Our findings highlight the possibility of a long-term effect of disruptive behavior on the risk of experiencing violence victimization, rather than the other way around. Although our study does not support previous causal inferences regarding the relationship between child maltreatment leading to adult ADHD, it emphasises the complexity of establishing causality. Additional research using prospective longitudinal designs is important to examine whether our findings can be replicated. Another factor to be considered when examining the direction of the association from ADHD to abuse/neglect is the presence of ADHD symptoms among the parents of a child with ADHD. Since ADHD is a heritable condition , it is pOur study also has clinical implications. First, our findings emphasize that clinicians treating people with ADHD, and especially those with comorbid conduct disorder, should be aware that their patients are at heightened risk for current and future maltreatment and of other forms of violence victimization. This indicates the importance of conducting an evaluation of concurrent and past victimization during routine assessment and treatment planning of people with ADHD. Second, our findings suggest that along with interventions focusing on children’s ADHD and conduct disorder symptoms there is a need to provide guidance and support to carers. Knowledge about behavioral problems might help to better understand the potential challenges they are facing. Teaching them various strategies that can be used in order to facilitate behavior and function might give them more effective ways to deal with the child’s behavior. Third, our findings suggest that while maltreatment may not directly cause ADHD, maltreatment and ADHD are associated and mental health professionals and clinical services that are in contact with children, adolescents and adults who experienced maltreatment should be aware of their higher risk of having ADHD.Medical Research Council (UKMRC grant G1002190). Additional support was provided by National Institute of Child Health and Human Development (grant HD061298), National Society for Prevention of Cruelty to Children (NSPCC) and Economic and Social Research Council (ESRC), The Avielle Foundation, and by the Jacobs Foundation. Adi Stern is supported by The Haruv Institute’s Post-Doctoral Students Fellowship and by the Humanitarian Trust Fellowship. Helen L. Fisher is supported by an MQ Fellows Award (MQ14F40). Louise Arseneault is the Mental Health Leadership Fellow for the UK Economic and Social Research Council (ESRC).The E-Risk Study is funded by the


Bartonella spp. cause persistent bacterial infections in mammals. Although these bacteria are transmitted by blood-feeding arthropods, there is also evidence for vertical transmission in their mammalian hosts. We aimed to determine: (i) the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival.Microtus arvalis, 76 Microtus oeconomus and 17 Microtus agrestis. In total, 115 embryos were isolated from 21 pregnant females. In the following year 11 pregnant females were kept until they had given birth and weaned their pups (n = 62). Blood smears and PCR targeting the Bartonella-specific rpoB gene fragment (333bp) were used for the detection of Bartonella. Bartonella DNA was detected in 66.8% (145/217) of the wild-caught voles. Bartonella infection was detected in 81.8% (36/44) of pregnant female voles. Bartonella-positive individuals were identified among the embryos  and in 54.8% (34/62) of pups. Congenitally acquired Bartonella infections and co-infection with B. microti had no impact on the survival of pups over a 3-week period post partum. Among 113 Bartonella sequences, four species were detected: Bartonella taylorii, Bartonella grahamii, Bartonella doshiae and a Bartonella rochalimae-like genotype. Bartonella taylorii clade B was the dominant species in wild-caught voles (49%), pregnant females (47%), their embryos (85%), dams (75%) and pups (95%).We sampled 124 Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.High prevalence of The online version of this article (10.1186/s13071-018-3047-6) contains supplementary material, which is available to authorized users. Bartonella cause persistent infections in erythrocytes and endothelial cells of mammalian hosts. Infection may contribute to the development of an important vector-borne disease: bartonellosis [Candidatus” species, 17 are recognized as causes of increasing numbers of human and animal bartonellosis cases [Bartonella-derived endocarditis cases diagnosed in humans has increased, attributable in some measure to the increasing awareness of these bacteria by medical practitioners [Bacteria of the genus nellosis , 2. Of titioners , 4.Bartonella henselae, Bartonella clarridgeiae, Bartonella rochalimae, Bartonella vinsonii berkhoffi) but also with several species  for which different rodent species constitute the main hosts [Apodemus and Peromyscus spp.) and voles (Myodes and Microtus spp.) [Bartonella infections in rodents differs, but may reach 60–70% or even 90% in susceptible host species [Bartonella spp. and genotypes have been described to date [Zoonotic bartonellosis in humans is associated with several species infecting cats and dogs (i.e. in hosts –7. Many us spp.) , 7–11. P species , 12–21.  to date , 7, 22 a to date –26.Sigmodon hispidus) and deer mice (Peromyscus leucopus) in the USA [Bartonella birtlesi has been experimentally demonstrated in BALB/c mice in which 76% of fetal resorptions were culture positive for B. birtlesi, and vascular lesions were observed in the maternal placenta [Bartonella DNA was detected in 69% of fetuses of Bartonella-infected wood mice (Apodemus sylvaticus) from the Barcelona region in Spain [Myodes glareolus in the UK [Bartonella [There is also evidence for efficient vertical transmission of bartonellae in different rodents. The first study on vertical transmission was conducted on naturally infected cotton rats  the prevalence and diversity of Bartonella spp. in a Microtus spp. community; (ii) whether vertical transmission occurs from infected female voles to their offspring; (iii) the effect of concurrent Babesia microti infection on the success of vertical transmission of Bartonella; and (iv) the impact of congenital infection on pup survival.In our previous studies in rodent hosts, we observed an interesting pattern of host age-related prevalence suggesting the existence of vertical transmission , 33, 34.Bartonella in embryos dissected from naturally infected voles, since this should completely eliminate the possibility of vector-borne transmission to the embryos. To eliminate the possibility that the tissues of the embryos may have been contaminated by maternal blood, we also maintained in captivity naturally infected pregnant female voles, completely deprived of ectoparasites, until a suitable period after parturition when individual sampling of the blood of the pups was possible. Thus we assessed the prevalence of congenitally transmitted Bartonella infection in the pups and evaluated the impact of congenital infection on pup survival. Finally, we identified the bacterial species infecting wild-caught voles and offspring through their distinct molecular signatures. The obtained results expanded the existing knowledge on the prevalence, vertical transmission and species composition of Bartonella in wild living rodents in Poland.To achieve the aims, we determined the presence of Microtus oeconomus) to dry grassland . Individuals of Microtus agrestis were trapped in the intermediate zones. Trapping took place in summer (August and early September) in 2013 and 2014. Traps were set for at least 5 consecutive nights. All animals , within an extensive forest and old field system adjacent to Lakes Śniardwy and Łuknajno. A detailed description of trapping sites is provided in Tolkacz et al. . In shorM. oeconomus and M. agrestis [1 and M2 and the second upper molar (M2) [In 2013, the autopsies were carried out under terminal anaesthesia . Voles wagrestis . We conflar (M2) . EmbryosIn the summer of 2014, all captured voles were live-processed as described in Tolkacz et al. , includiBartonella DNA was used for the identification of infection in all adult voles , embryos and pups. Between 20 (from the live-processed voles) and 200 μl  of whole blood were collected into 0.001 M EDTA and frozen at a temperature of -20 °C before DNA extraction. Embryos were isolated from the uterus and individually autopsied following two washes in sterile water, to minimize contamination with maternal blood. We autopsied 115 embryos from 21 litters   and Hemacolor  staining kits, according to each manufacturer’s instructions. Specific amplification of Smears from all captured animals and pups were examined under oil immersion (×1000 magnification) for parasitaemia estimation. A total of 200 fields of vision were scanned by experienced researchers and the number of infected RBC (iRBC) was recorded. Parasitaemia was expressed as the mean number of iRBC/200 fields of vision.Bartonella species/variants were performed by amplification and sequencing of the 333 bp gene fragment of RNA polymerase β-subunit (rpoB) obtained in a single-step PCR or in a nested PCR . The primers and thermal profile used in this study have been described previously [The detection and genotyping of eviously , 37.rpoB) was amplified with specific primers: 1400F (5'-CGC ATT GGC TTA CTT CGT ATG-3') and 2300R (5'-GTA GAC TGA TTA GAA CGC TG-3') [In the first step of the nested PCR, the 825 bp gene fragment of RNA polymerase β-subunit (C TG-3') . The PCRBartonella acomydis extracted from wild rodents from Egypt [In the second step (nested reaction) and in the single-step PCR, the forward primer rpoF (5'-GCA CGA TT(C/T) GCA TCA TCA TTT TCC-3') and the reverse primer rpoR (5'-CGC ATT ATG GTC GTA TTT GTC C-3') were used . The PCRom Egypt .http://www.ncbi.nlm.nih.gov/genbank/).PCR products were subjected to electrophoresis on a 1.5% agarose gel, stained with Midori Green stain . Selected PCR products from voles trapped in 2013 and 2014, all pregnant females and dams, and at least two embryos/pups per litter (if available), were sequenced by a private company . DNA sequence alignments and analyses were conducted using MEGA v.7.0. Consensus sequences were compared with sequences deposited in the GenBank database , host species , host age  and host sex  as factors. Subsequent analyses were carried out for each host species separately.The statistical approach adopted has been documented comprehensively in our earlier publications , 38–40. Bartonella in embryos, we implemented ‘female infection’ as a binary factor (i.e. infected/uninfected mother). For analysis of the prevalence of Bartonella in pups, we implemented pup survival as a binary factor . In order to test the hypothesis that co-infection with Babesia microti in females/dams may facilitate congenital transmission of Bartonella to their embryos/pups, we fitted models with B. microti infection of female/dam and embryo/pup as an additional factor . Beginning with the most complex model, involving all possible main effects and interactions, those combinations not contributing significantly to the explanation of variation in the data were eliminated stepwise, beginning with the highest-level interaction. A minimum sufficient model was then obtained, for which the likelihood ratio of χ2 was not significant, indicating that the model was sufficient in explaining the data.For analysis of the prevalence of Bartonella, litter size, mean weight of pups, etc.), which are reported with standard errors of their means (SE). The abundance of Bartonella infection was calculated as the number of infected red blood cells (iRBC) in 200 fields of vision (×1000 magnification). When samples were only positive by PCR, an intensity of 0.001 iRBC/200 fields was implemented into the quantitative statistical analysis.General linear models (GLMs in SPSS v.21) were used for comparison of mean parameters (abundance of Bartonella-positive pups/litter was correlated with the litter size using the Spearman’s rank correlation test (SPSS v. 21). Fisher’s exact test (INSTAT software) was used to compare the percentage of infected pups between Bartonella-negative and Bartonella-positive females.The success of vertical transmission to each litter, calculated as the fraction of M. arvalis; 76 root voles, M. oeconomus; and 17 field voles, M. agrestis. Prevalence of Bartonella spp. infection by year of study, host species and sex is provided in Table Bartonella spp.’ infection. In total, a positive product of the PCR reaction and microscopy was obtained for 66.8% (95% CI: 62.4–71.0%) of voles in the community. The highest prevalence of Bartonella spp. was detected in M. arvalis , lower and similar in M. oeconomus  and in M. agrestis , thus prevalence did not vary significantly between the three host species  . In M. arvalis, prevalence was high in juvenile voles , highest in young adults  but lowest in the oldest adults  . In M. oeconomus, prevalence increased gradually with host age  but the difference in prevalence between these age classes was not significant. In M. agrestis, the effect of age was not significant as our sample only included one juvenile individual of this species, which was found to be infected (100%). Prevalence was 50% in young adults and 60% in adult field voles (ns).Age-related effects on prevalence differed among host species  = 2.2, P = 0.114)  and lowest in juvenile voles. In M. agrestis, we observed highest abundance in adult individuals and lowest in juveniles and young adults.Similarly, host age did not have a significant effect on abundance, although the mean values for age classes followed the same sequence as for prevalence among Bartonella-positive females. We excluded 12 litters because pregnancy was still at a very early stage and the embryos were too small to enable reliable isolation of fetal tissues. Six litters from Bartonella-negative mothers were available as a control group for analysis of vertical transmission  , then 83.9% (95% CI: 70.8–92.4%) in M. arvalis females, but only one female of three pregnant M. agrestis  was found to be Bartonella-positive  from Bartonella-positive females and 6 litters from Bartonella-negative females; Fig. We were able to analyze the prevalence of infections in 115 embryos from 21 litters . No Bartonella DNA was detected in 30 embryos of the six Bartonella-negative females , in comparison to 47.1%  of positive embryos recovered from 15 Bartonella-positive females (Fisher’s exact test: P < 0.0001). Among Bartonella-positive pregnant females, nine were M. arvalis, five M. oeconomus and one M. agrestis  were found in 80% (95% CI: 53.4–94.3%) of these litters.Prevalence of Bartonella positive females, in which at least one embryo was Bartonella-positive, was similar among the three host species . Among nine Bartonella-positive females of M. arvalis, positive embryos were identified in 77.7% (95% CI: 44.2–95.9%) of litters but the overall prevalence among embryos was 38.3% (95% CI: 22.9–56.0%)  of litters and 60% (95% CI: 41.6–76.4%) of embryos of five Bartonella-positive M. oeconomus. In the one litter of the Bartonella-positive M. agrestis female, 50% (95% CI: 19.3–80.7%) of embryos were Bartonella-positive. Thus, not all embryos carried by infected females were positive for Bartonella, and the success of vertical transmission (fraction of positive embryos) of Bartonella spp. differed between litters even within host species: in three litters from infected mothers no positive embryos were identified, in another three all tested embryos were Bartonella-positive and prevalence of Bartonella spp. was within the range 14–75% for the remaining nine litters.The proportion of litters from ies Fig. , ns. HowBartonella DNA was detected in 54.8% (95% CI: 43.9–65.3%) of pups born to 11 Bartonella-positive dams  of pups were positive, in comparison to 60% (95% CI: 45.0–72.2%) positive pups from nine M. arvalis dams  and among positive litters, the percentage of Bartonella-positive pups varied within the range 14–100%  = 6.33, P = 0.015).The abundance of B. microti in wild voles. In the minimal sufficient model derived from analysis of a model that included B. microti infection, only the presence of Bartonella infection in females/dams had a significant effect on Bartonella infection in embryos/pups . Babesia infection in females/dams had no effect: the success of vertical transmission of Bartonella was 32.4%  in embryos/pups of Babesia-negative females/dams and 44.3%  in embryos/pups of Babesia-positive females/dams, and the difference was not significant. Focusing on the infection status of offspring, we correlated the success of vertical transmission of Babesia in the litters (fraction of Babesia-infected litter) with the success of vertical transmission of Bartonella in the litter (fraction of Bartonella-infected litter), for offspring of co-infected females/dams (n = 10), but no positive correlation was found. Finally, we tested a model for the prevalence of Bartonella and Babesia in embryos/pups, but again, no significant association was found.In our previous study  on the sM. arvalis, 6 pups of M. oeconomus) died 1–2 days after birth. Both M. arvalis pups and dam (ID: 2014/107) were Bartonella-positive by PCR  among Bartonella-negative pups (ns).All the other pups delivered by 9 Bartonella infection of the dam on the litter size could not be reliably analyzed as there were no litters from Bartonella-negative dams.The effect of Bartonella infection on the weight of pups at the age of three weeks in a model controlling also for host species and sex. Although the mean weight of M. oeconomus pups was significantly higher than for M. arvalis pups: 17.56 ± 1.07 and 15.12 ± 0.52 g, respectively  = 5.31, P = 0.027), no impact of Bartonella infection was found (ns). The mean weight was similar for Bartonella-positive and Bartonella-negative pups (16.68 ± 0.73 and 15.45 ± 0.71g) (ns).We also tested the effect of congenital Bartonella sequences from 108 voles (62 adult voles and 46 embryos and pups), including 5 voles for which two different sequences were obtained in independent sequencing events  Bartonella rpoB sequences were analyzed. Among these, 65 were derived from naturally infected voles, including pregnant females and dams  and 48 were obtained from embryos or pups . Selected representative sequences (including “mother” isolates starting with “M” and embryos/pups isolates marked with mother “M” and offspring “D” numbers) are presented on the phylogenetic tree in Fig. We obtained 113 B. taylorii  and then B. grahamii . Additionally, two sequences of B. doshiae were identified. We also identified three sequences of a B. rochalimae-like variant  isolated from M. oeconomus and M. arvalis, respectively, from the Mazury Lake District. The second genotype was most similar (99–100% of homology) to B. taylorii clade B , previously isolated from bank vole M. glareolus and M. arvalis, respectively, from the Mazury Lake District [B. doshiae sequences were obtained from wild-caught voles, from M. arvalis and M. oeconomus displaying highest similarity (98–100%) to B. doshiae from M. arvalis (GenBank: GU338939) from the study area [B. doshiae has been deposited in GenBank under the accession number MF357900.Among the udy area . One genDistrict  Fig. 2)B. taylorudy area . A repreM. arvalis female and two embryos) which showed highest similarity (98–99%) to a unique Bartonella sequence found in M. glareolus in France (GenBank: JX846416). These sequences constituted a sister group to the B. rochalimae sequences obtained from clinical cases in South and North America. We thus consider them to be a B. rochalimae-like variant , pregnant females  and their embryos  and also among dams  and their pups    and in 7.4% (2/27) of embryos , in pregnant females  but not in any of their embryos, among dams  and in one pup   Tables , and altBartonella sequences obtained from wild-caught voles, 31 were from M. arvalis, 26 from M. oeconomus and 8 from M. agrestis. The B. taylorii clade B genotype was identified in 45% of sequences derived from M. arvalis , in 50% of sequences derived from M. oeconomus  and in 63% of sequences derived from M. agrestis .Among 65 B. taylorii clade A genotype was identified in 29% of isolates from M. arvalis , in 4% of sequences derived from M. oeconomus  and in three sequences from M. agrestis .The Bartonella grahamii was identified in 19% of isolates from M. arvalis  and in 42% of sequences derived from M. oeconomus .Bartonella doshiae was identified in two female voles  and the B. rochalimae-like variant was found in one pregnant M. arvalis female (co-infection with B. taylorii clade B).Bartonella genotypes infecting mother voles and their corresponding offspring. Sequences of appropriate quality were obtained for 15 pairs. In 12 cases the same species and genotypes were identified in females/dams and their embryos/pups  were identified in one individual. In this litter two embryos were co-infected with the B. rochalimae-like variant and with B. taylorii clade B, but the third embryo was only infected with B. taylorii clade B.The final step was the correlation of B. taylorii clade B genotype was isolated from embryos, although B. grahamii (2 cases) or B. taylorii clade A were identified in their mothers.Only in three cases were different genotypes found in female-embryo pairs Table . In threBartonella genotype identified in offspring was B. taylorii clade B, which was found in 85% of 27 embryos and in 95% of 21 pups. There were two other females co-infected concurrently with two Bartonella species, but in each of these cases only one of the genotypes was found in the offspring's tissues . Furthermore, our data indicate that vertical transmission of Bartonella spp. is unlikely to be associated with the successful vertical transmission of the piroplasm B. microti, which was also endemic in these vole populations. Although quite high genetic diversity was identified among the Bartonella sequences (4 species comprised of 5 main genetic clades), the B. taylorii clade B genotype was the most successful in vertical transmission, being the dominant genotype recovered from Bartonella positive embryos and pups.In the present study, we report a high prevalence of infection with Bartonella spp. among a multi-species sympatric Microtus vole community. We provide novel data to complement our long-term monitoring of Bartonella infections and our earlier work at these same study sites in the Mazury Lake District. The first study on Bartonella in voles from this region was carried out in 1997–2000 [M. arvalis. Subsequently, in the period 2004–2006, the second study incorporated M. arvalis and M. oeconomus populations [Bartonella in more recent years (2013–2014) and focus on the question of why prevalence of Bartonella spp. in this community of three vole species  is consistently so high among the youngest animals which are unlikely to have experienced encounters with hematophagous vectors (and hence unlikely to have been infected after birth).Our study is among the first to dissect the complex interactions involved in the transmission and circulation of 997–2000  and focuulations , 14, andulations , 17. BuiBartonella spp. in this period of 17 years was lowest in the first period  in the Microtus community in the period 2013–2014 . This much higher prevalence (up to four times higher than previously recorded) can be explained primarily by the fact that sampling was undertaken exclusively in late summer, rather than additionally in the spring when prevalence is generally lower, and by the use of a nested PCR technique for the detection of infection. Seasonal variation in prevalence of Bartonella has been reported previously, and prevalence values in late summer and autumn have been found to be at least twice as high as those observed earlier in the year in most studies [The overall prevalence of  arvalis ). Here w studies , includi studies . Nested  studies –44.Bartonella in the Microtus community and in each vole species may be attributable to changes in the quality and character of the microhabitat affecting vector populations. Fleas are the main vectors of Bartonella spp. among rodents [M. arvalis (81%) inhabiting the dry upper parts of fallow land, and lowest in root voles (68%) inhabiting the narrow strips surrounding ponds on the grassland, which are often totally submerged during periods of intense rainfall [Bartonella recorded in 2006 was accompanied by the highest recorded infestation by fleas (84% infested rodents in 2006 versus 34% in 2004) [It is also possible that this marked increase in the prevalence of  rodents , 16, 26 rainfall . Additioin 2004) . We suspBartonella in Microtus spp. with which we can directly compare our data. Perhaps the closest was completed on M. agrestis in the UK [Bartonella spp. in autumn (58%) was very similar to that observed in our field voles, although in spring prevalence fell to just over half this level (34%). A similar prevalence was recently found in M. arvalis in Slovakia  but in her (83% ). Overal1%  but iher , fois level %. A simiBartonella spp. infections in Microtus hosts is dependent to some extent on vertical transmission, most likely through the transplacental route as supported by the high fraction of PCR-positive embryos found in this study. This idea was inspired by our consistent observation of a ‘reversed’ age effect in the rodent host, with juvenile individuals more often infected than adults [M. arvalis, with the lowest prevalence among mature adults, as in our first study in this area in 1998 and 2000 [Bartonella spp.  was very similar in all three host species: 50% for M. arvalis, 52% for M. oeconomus and 50% for M. agrestis. Comparing these values with the prevalence of Bartonella in juvenile voles , it is not unreasonable to conclude that the high proportion of Bartonella infections recorded among this youngest age class is likely to have been as a consequence of transmission in utero, through the placental route and hence congenital or vertical in its nature.The main focus of the present study was to test the hypothesis that the perpetuation of n adults , 9, 23. and 2000 . InteresBartonella in the current study corresponds well with findings in some other studies from different geographical regions. In North America, 80% of litters and 47% of cotton rat (S. hispidus) embryos/newborns tested positive for Bartonella and 100% of litters and three out of three tested white-footed mouse (P. leucopus) embryos were found to be Bartonella-positive [Bartonella-positive wood mice (A. sylvaticus) were positive [B. birtlesi [Thus, in addition to the well-established vector-borne route of infection, vertical transmission may also act as an important additional mechanism facilitating the circulation of these bacteria in naturally existing rodent populations. Interestingly, although we noted a high rate of transmission to litters (80–100%) from infected females, not all offspring of infected mothers became infected, with the proportion per litter ranging from 0 to 100% of the embryos/pups becoming infected. The proportion of litters and embryos/pups from infected females that became infected with positive . In the positive . A highebirtlesi  but a mubirtlesi .Bartonella may, therefore, depend to some extent on host species, but there is also evidence that it may depend on the species/genotype of bacteria involved. The pioneering study of Kosoy et al. [Bartonella were identified by gltA gene sequencing, first provided evidence that the efficiency of congenital transmission varied significantly between Bartonella genotypes. In the study all Bartonella genotypes found in positive embryos/newborns were identical to genotypes in their corresponding mothers, but only two genotypes were successfully transmitted between females and their offspring: genotype A in cotton rats and genotype D in white-footed mice [The success of vertical transmission of y et al. , in whicted mice . No posiBartonella by sequence analysis of a short fragment of the rpoB gene, in both females/dams and their corresponding embryos/pups. Four main genetic groups were identified: B. taylorii clades A and B, B. grahamii and a B. rochalimae-like variant. In five litters born to infected dams, no infected offspring were noted, and among these we identified B. taylorii clade A (2 litters) and B. taylorii clade B (3 cases) , thus supporting the results of our study [We also genotyped  clade B  cases (T) Tables . However clade B  cases . However, in root voles, the pattern was reversed (mean abundance 0.4 iRBC in wild-caught voles versus 2.5 iRBC in pups). We found no evidence that the success of vertical transmission of Bartonella may be associated with successful vertical transmission of B. microti. The exact mechanism of vertical transmission of these pathogens in rodents is not entirely understood; however, in the case of Bartonella, bacteria have been found in cultured placental tissues and lesions [B. microti was higher than the success of transmission of Bartonella spp., reported here, ranging between 70–81% [To the best of our knowledge, our study is the first to evaluate the impact of congenital infections of  microti , we foun lesions , 28. Eacn 70–81% .Bartonella rpoB sequences, the most common species was B. taylorii, with clade B genotype more frequently detected than clade A, followed by B. grahamii. Rare genotypes were those of B. doshiae and the B. rochalimae-like variant. This pattern is observed among other studies of rodent communities in Europe, in which B. taylorii and B. grahamii are profoundly dominant in rodents [B. taylorii is genetically the most variable species, and this may be related to adaptation to different host species [rpoB sequences recorded in the current work were very similar to genotypes obtained from the same rodent community in earlier surveys in 2007–2009, thus confirming their successful circulation in the rodent populations in this region for at least a decade and likely longer [B. taylorii and B. grahamii constituted the great majority of identified Bartonella species/genotypes [M. agrestis in the UK, the distribution of Bartonella species/genotypes markedly differed: of the 886 isolates, 264 were B. doshiae, 248 B. grahamii, 324 B. taylorii and 50 of an unidentified Bartonella genotype . Iny longer , 50. Addenotypes , 34. In  . This cB. rochalimae. Although B. rochalimae is believed to infect rodents [Bartonella sequence obtained from a bank vole from France  and as a result, the occurrence of different genotypes in a single litter. Thus our results show that mixed infections of Bartonella species/genotypes can occur in adult voles, with the B. taylorii genotype more ‘detectable’ or dominant in adult voles and in offspring. However, the observation that Bartonella species/genotypes differ in the success of vertical transmission or in pathogenicity for rodent hosts, requires further study.The final step to complete our study on vertical transmission was to identify and compare the species/genotypes of Bartonella spp. infection maintained in Microtus spp. community is followed by a high rate of vertical transmission of several rodent species of Bartonella in three species of naturally infected voles, M. arvalis, M. oeconomus and M. agrestis. Congenitally acquired Bartonella infection does not affect the survival of pups. Co-infection with B. microti does not affect the effectiveness of the vertical transmission of Bartonella in voles. Bartonella taylorii clade B was found to be the dominant species in wild-caught voles, including pregnant females and dams, and in their offspring, and was also found to be the most successful in vertical transmission.High prevalence of Additional file 1:Table S1. Accession numbers of representative Bartonella spp. 313 bp gene fragment of RNA polymerase β-subunit (rpoB) amplified in this study from blood samples from voles near Urwitałt, north-east Poland, in summer 2013 and summer 2014. (DOCX 18 kb)Additional file 2:Figure S1. Mean water level in Lake Śniardwy (a) and in the Vistula River (b) in the period 1999–2014. (DOCX 17 kb)


Bartonella henselae, can be responsible for the observed association. However, no direct evidence for the role of the Bartonella infection in this association has been published until now. In this preregistered case-controls study performed on 250 healthy subjects tested earlier for the presence of anti-Toxoplasma IgG antibodies, we searched for the positive association between presence of anamnestic anti-Bartonella IgG antibodies and depressiveness measured with Beck II inventory, depression subscale of neuroticism measured with N-70 questionnaire, and self-reported health problems. We found that that Bartonella seropositivity was positively correlated with Beck depression only in Toxoplasma-seronegative men and negatively correlated with health in Toxoplasma-seronegative women. Bartonella seropositivity expressed protective effects against Toxoplasma seropositivity-associated increased neuroticism in men while Toxoplasma-seropositivity expressed protective effects against Bartonella seropositivity-associated health problems in women. A comparison of the patterns of association of mental and physical health problems with Bartonella seropositivity and with reported cat-related injury suggests that different factor, possibly infection with different pathogen transmitted by cat related-injuries than the B. henselae, is responsible for the observed association of cat related-injuries with depressiveness and major depression. The existence of complex interactions between Bartonella seropositivity, Toxoplasma seropositivity, and sex also suggest that the effect of symbionts on the host's phenotype must by always studied in the context of other infections, and separately for men and women.Several recent studies have demonstrated the association of cat-related injuries with major depression and with depressiveness in the general population. It was suggested that cat-scratch disease, the infection with the bacterium  Bartonella, can be responsible for this association. These bacteria are present in blood and other bodily fluids of about 50% of cats in many parts of world  “Effects of bartonellosis on depression and health,” osf.io/agvbj. The experimental setup, four hypotheses to be tested and statistical tests for testing these particular hypotheses, as well as the most important follow up tests had been registered before the sera were examined for the presence of anti-Toxoplasma and other pathogens. These samples are kept frozen in −18°C. For the purpose of the current project, we selected a sample of 250 subjects (we tried to find the same number of Toxoplasma seropositive and Toxoplasma-seronegative subjects of similar age) who completed an anamnestic questionnaire containing 20 health-related questions, Beck Depression Inventory , and N-70 questionnaire . These sera were examined for the presence of anamnestic titres of anti-Bartonella antibodies. The study was approved by Institutional Review Board of Faculty of Science, Charles University (No: 2016/16).Data were collected during several projects studying the effects of latent toxoplasmosis on human behavior, personality and health that were performed at the Faculty of Science, Charles University over the past 10 years. During these projects, about 3000 subjects completed various questionnaires in an electronic or paper form and provided a sample of blood for serological testing for In the anamnestic questionnaire , the conToxoplasma infection status of the subjects. ELISA assay cut-point values were established using positive and negative standards according to the manufacturer's instructions. In CFT, the titre of antibodies against Toxoplasma in sera was measured in dilutions between 1:4 and 1:1024. The subjects with CFT titres between 1:8 and 1:128 were considered Toxoplasma-seropositive. Only subjects with clearly positive or negative results of CFT and IgG ELISA tests were diagnosed as Toxoplasma-seropositive or Toxoplasma-seronegative, whilst subjects with different results of these tests, or ambiguous results, were retested or excluded from the study. Frozen samples of sera after 1: 64 dilution were also tested for the presence of anamnestic titres of anti-Bartonella IgG antibodies using Indirect Immunofluorescent Assay . All tests were performed in the reference laboratories of the National Institute of Public Health, Prague.The complement-fixation test (CFT), which determines the overall levels of IgM and IgG antibodies of particular specificity, and Enzyme-Linked Immunosorbent Assays (ELISA)  were used to detect the Toxoplasma (Bartonella) seropositivity on the probability of being Bartonella (Toxoplasma) seropositive. ANCOVA tests with a BDI-II score  as an output variable were used to search for the effects of the predictors Bartonella, age, sex, size of place of living in childhood, Toxoplasma, and the sex-Bartonella, Toxoplasma-infection-Bartonella, and sex-Toxoplasma-Bartonella interactions. The effects of these predictors on six (preregistered) or all seven N-70 variables were studied with MANCOVA. In parallel, the effects of Bartonella and Toxoplasma seropositivities were also measured with nonparametric partial Kendall test that allow controlling for one confounding variable, here age  was controlled with the Benjamini-Hochberg procedure  values, while healthy participants had low (negative) values of this index. A logistic regression was used for searching for effects of the predictors age, sex, size of settlement in which a subject spend its childhood, and Toxoplasma seropositivity” to “Toxoplasma,” and “Bartonella seropositivity” to “Bartonella” in the description of our statistical models. For example, 3-way interaction Toxoplasma seropositivity-Bartonella seropositivity-sex interaction is described in a more condensed way as Toxoplasma-Bartonella-sex interaction. Also, the statistical relations between  dependent and  independent variables are called “effects” in the Result section of the paper, despite the real causality relation between the these variables can be different or even non-existing (as expanded upon in the Discussion).Terminological notes: we abbreviated “Bartonella, Toxoplasma-infection-Bartonella) we always additionally analyzed the full model with all three 2-way interactions  to show the robustness of our results and to follow recommendation of many statisticians concerning the necessity to analyze full models. However, all inferences have been done exclusively on the basis of results of the preregistered tests. We always used more conservative two-sided, instead of the preregistered one-sided variant of tests because the observed effects of the Bartonella-seropositivity were in the opposite directions in men and women.We had to substitute the preregistered ANCOVA test for depression subscale from N-70 with non-parametric tests as this variable failed in the Levene's test of equity of errors. In addition to the preregistered tests with two 2-way interactions (sex-https://doi.org/10.6084/m9.figshare.5852511.All data are available at: S.D. = 7.00) and women  in the whole set  = −0.316) or between Bartonella-or Toxoplasma-seropositive and seronegative male or female subjects . Most subjects (31.6% men and 25.0% women) spent their childhood in Prague, 12.0% men and 15.2% women in settlements with < 1000 inhabitants, 17.4% men and 13.9% women in settlements with 1–5 thousands of inhabitants, 26.1% men and 29.8% women in settlements with 5–50 thousands of inhabitants, 10.9% men and 4.4% women in settlements with 50–100 thousands of inhabitants, 8.7% men and 5.1% women in settlements with more than 100 thousands of inhabitants, except Prague. A logistic regression with age, sex, size of settlement in which a subjects spent their childhood as the covariates showed no association between the Bartonella seropositivity (dependent variable) and the Toxoplasma seropositivity  and no association between the Bartonella seropositivity and the covariates. Analogical analysis with the Toxoplasma seropositivity as the dependent variable showed that the probability of being Toxoplasma-seropositive decreased with the increasing size of the settlement in which a subject spent his or her childhood ; other associations of Toxoplasma seropositivity with covariates were nonsignificant.The final set contained data from 92 men and 158 women, see Table Four hypotheses have been preregistered before the start of the present study.Bartonella-seropositive express higher levels of depression measured with N-70 inventory than the Bartonella-seronegative subjects when sex, age, toxoplasmosis and size of place of living are controlled.Subjects who have been diagnosed as Bartonella-seropositive) and 92 men (32.6% Bartonella-seropositive). The Levene's test of equity of errors showed that a preregistered ANCOVA test cannot be done ; therefore only the nonparametric partial Kendall correlation tests with age as a covariate were performed. Figure Bartonella-Toxoplasma interaction. The expected higher depression in Bartonella-seropositive subjects was observed only in Toxoplasma-seronegative men. However, even here the association was not significant . Toxoplasma-seronegative women showed negative nonsignificant association while Toxoplasma-seropositive men showed negative nonsignificant association and Toxoplasma-seropositive women no association between Bartonella and depression. Nonsignificantly lower depression was observed in Toxoplasma-seropositive men , Toxoplasma-seronegative women , and Toxoplasma-seropositive women –all in comparison with corresponding Bartonella-seronegative subjects.The N-70 inventory was completed by 156 women  and 82 men (34.1% Bartonella-seropositive). An ANCOVA test with a BDI-II score as the output variable and Bartonella, age, sex, size of place of living in childhood, Toxoplasma, and the sex-Bartonella, Toxoplasma-Bartonella and sex-Toxoplasma-Bartonella interactions as the independent variables indicated a significant association of depression with the Toxoplasma-Bartonella interaction . Similar results, namely the significant Toxoplasma-Bartonella interaction  and sex-Toxoplasma-Bartonella interaction , also provided the full model with three 2-way and one 3-way interactions. Again, the expected positive Bartonella-depression interaction was observed only in Toxoplasma-seronegative men. In all three other subpopulations, the Bartonella-seropositive subjects expressed lower or the same BDI-II depression scores than the Bartonella-seronegative subjects, Figure Toxoplasma-seronegative men , non-significantly lower depression in Toxoplasma-seropositive men , and non-significantly higher depression in Toxoplasma-seronegative , and Toxoplasma-seropositive women –all in comparison with corresponding Bartonella-seronegative subjects.The BDI-II questionnaire was completed by 148 women  and 92 men (32.6% Bartonella-seropositive). An ANCOVA test with the health score as the output variable and Bartonella, age, sex, size of childhood home, Toxoplasma, and the sex-Bartonella, Toxoplasma-Bartonella and sex-Toxoplasma-Bartonella interactions as the independent variables indicated significantly worse health scores in women than in men  and also a significant association between health score and sex-Bartonella interaction . Similar results, namely the effects of sex , and the sex-Bartonella interaction  also provided the full model with three 2-way and one 3-way interactions. The Figure Bartonella seropositivity had a negative association with health in women and positive association with health in men. Nonparametric partial Kendall correlation tests (age controlled) showed non-significantly better health status of the Bartonella-seropositive subjects in Toxoplasma-seronegative men , and in Toxoplasma-seropositive positive men  and also non-significantly worse health status in Toxoplasma-seronegative women , and in Toxoplasma-seropositive women –all in comparison with corresponding Bartonella-seronegative subjects.The anamnestic questionnaire was completed by 158 women . The results of univariate ANCOVA analyses showed that this interaction was significant for the anxiety , obsession , hysteria , hypochondria , vegetative lability , and psychasthenia ; for the direction of these associations see Figure Bartonella-Toxoplasma-sex interaction  and the effects of this interactions on obsession , hysteria , vegetative lability , and psychasthenia . The results of nonparametric tests partial Kendall correlation tests performed separately for Toxoplasma-seropositive and Toxoplasma-seronegative male and female subjects are shown in Table A MANCOVA analysis performed with six N-70 facets (except depression) as well as all seven N-70 subscales provided qualitatively the same result; therefore, only the result of the preregistered MANCOVA test with six subscales will be reported. The independent variables were sex, age, size of settlements where the responders spent their childhood, the Bartonella seropositivity, Toxoplasma seropositivity, and Bartonella-sex, Bartonella-Toxoplasma, and Bartonella-Toxoplasma-sex interactions as the independent variables. The result showed no effect of the Bartonella-Toxoplasma-sex interaction. At the same time it showed a significant effect of the Bartonella-Toxoplasma interaction on heart disorders , recurrent pain , and frequency of being tired . The sex-Bartonella- interaction was significant for cardiovascular disorders , frequency of being tired , and frequency of taking antibiotics within the past three years . The main effect of the Bartonella seropositivity was observed only for variable psychiatric problems, . No significant effect of Toxoplasma seropositivity was observed. The results obtained with nonparametric tests  as the dependent variables and sex, age, size of settlements where the responders spent their childhood, Bartonella seropositivity and depression, we computed partial Kendall correlations of Bartonella seropositivity with responses of subjects to 21 questions of BDI-II, separately for Toxoplasma-seropositive and Toxoplasma-seronegative male and female subjects, Table Bartonella seropositivity in Toxoplasma-seronegative men. In Toxoplasma-seropositive men, the responses to five questions, namely 7-self-dislike, 11-agitation, 12–loss of interest, 13–indecisiveness, 14–worthlessness, and 15–loss of energy correlated negatively with Bartonella seropositivity, i.e., the men infected with both Bartonella and Toxoplasma reported lower levels of depression than men infected only with Toxoplasma.To show which problems are responsible for the observed associations of Bartonella-Toxoplasma-sex association on depression, we also included health status as another covariate into the ANCOVA test . The health score had a strong effect on the BDI-II depression score . However, the strength of the association of depression with Bartonella-Toxoplasma-sex remained approximately the same  as was detected in the model without the health score . The same analysis for depression measured with N-70 could not be done due to the highly significant result of Levene's test of equality of errors .To see whether the deteriorated health status played a role in the effect of Bartonella-Toxoplasma-sex association on the neuroticism estimated using N-70 inventory, we also included health status as another covariate into the MANCOVA test and following six ANCOVA tests . The results showed that health status had a strong effect on total neuroticism  and on five of six of it's subscales, the anxiety , obsession , hysteria , hypochondria , vegetative lability , and psychasthenia . Again, the strength of association between neuroticism and Bartonella-Toxoplasma-sex was approximately the same as it was detected in the model without the covariate health—.To see whether deteriorated health status played a role in the effect of Bartonella-seronegative and Bartonella seropositive subpopulations). Analogically, we searched for the associations between bartonellosis and health-related variables on the whole population (not on Toxoplasma-seronegative and Toxoplasma-seropositive subpopulations). Table Toxoplasmosis is known to have negative effects on the health of infected subjects. To check whether these effects can also be detected in our dataset, we performed nonparametric analyses searching for the association between toxoplasmosis and health-related variables on the whole population  and men . The Beck depression score of the antidepressants-taking women was higher  while that of the antidepressants-taking men was lower  than the score of their non-drug-taking peers. No such correlations or trends were observed between taking antidepressants and the subscale of depression measured with N-70  and no association was observed between scores of depression and taking anxiolytic drugs . The correlation between the Beck depression score and the N-70 depression score was insignificant both for women , and for men .Twenty-eight (20.7%) of 130 women and eight of (13.9%) of 67 men reported using antidepressants and twenty-seven (20.6%) of 131 women and five (7.6%) of 66 men reported using anxiolytics. A strong correlation between taking antidepressants and anxiolytics was observed in both women  was observed only in Toxoplasma-seronegative men and only when measured with the 21 questions of BDI-II test of depression (hypothesis 2), not with the 10 items of the depression subscale from N-70 (hypothesis 1). The men seropositive for both Bartonella and Toxoplasma expressed lower level of depression than the subjects seropositive only for the Bartonella. This contrasted with the expectations of hypothesis 4, but agreed with recently published data, see below. No difference in the depression scores between Bartonella-seropositive and Bartonella-seronegative subjects was observed in women. Worse physical health, especially more frequent cardiovascular and dermatological problems, of Bartonella-seropositive subjects was observed only in Toxoplasma-seronegative women. Similarly, the Bartonella- and Toxoplasma-seropositive men reported better health, especially less frequent dermatological and cardiovascular problems, than the men seropositive only with Bartonella (which again contrasted with the expectations of hypothesis 4).Our results suggest that the Bartonella seropositivity in both sexes, the stronger one in women. In reality, while our data show a relatively strong negative association between Bartonella seropositivity and physical health in women, the strong positive association with depression was observed only in (Toxoplasma-seronegative) men.Our results seem to contradict the outcomes of two previous studies. The first study revealed a very strong association between having suffered a cat-related injury and being diagnosed with unipolar depression . The assBartonella seropositivity, in Toxoplasma-seropositive men was also observed (but not commented) in the previous study (Toxoplasma-seropositive men was 18 (C.I.95 = ±5), while this score for 31 scratched Toxoplasma-seropositive men was 14 (C.I.95 = ±3.5). Corresponding scores for Toxoplasma-seronegative men were 10.2 (C.I.95 = ±2.1) for 88 non-scratched men and 12.8 (C.I.95 = ±2.4) for 90 scratched men. These scores are rather similar to those shown in Figure Bartonella antibodies was used as a proxy of the Bartonella seropositivity in the past and the present study, respectively.The second study  showed aus study . Figure The third study  showed aToxoplasma-Bartonella interaction), used a case-control study design with a similar number of Toxoplasma-seropositive and Toxoplasma-seronegative subjects while the earlier cohort studies analyzed data drawn from general populations of internet users. Typically, the Toxoplasma-seronegative subjects are much more common than those Toxoplasma-seropositive subjects (who seem to be protected against negative effects of Bartonella) in a general population, especially among young people. Lower representation of Toxoplasma-infected individuals in the participants of the cohort studies explains why being injured by a cat, the proxy of Bartonella seropositivity, has been shown to be positively associated with depression and with the probability of reporting having been diagnosed with major depression in all three cohort studies.It must be reminded that the present study, in order to test the prediction of the hypothesis 4  had a positive, not negative, effect on the Beck depression score, compare the Figures B. henselae, could be responsible for the observed positive association between the cat-related injuries and depression.The alternative explanation of the observed pattern is that the men infected with both 2) of Bartonella seropositivity increased rather than decreased when the effect of health was controlled. This suggests that the association between Bartonella seropositivity and depression is the result of some relatively specific effect, rather than being a non-specific side effect of the impaired health of Bartonella seropositive subjects.The strength of the statistical effect (ηBartonella seropositivity and depression (observed only in Toxoplasma-seronegative men) was much stronger when measured with the Beck inventory, which was used in the previous study  was high enough for the reliable detection of main effects of infections and their 2-way interaction. However, for a reliable analysis of 3-way interaction, collecting a larger dataset would be highly desirable. It is also known that toxoplasmosis affects Rh-positive and Rh-negative subjects differently –41. For Toxoplasma has been shown to affect hosts' behavior by the experimental infection of laboratory animals  and the infection by Bartonella.It is not possible to decide based on associations detected with observational studies what is the cause and what is the effect.  animals –45; no sToxoplasma infection has been explained by the opposite reactions of men and women on the chronic stress that could be associated with life-long infection (Bartonella seropositivity on physical health and the positive effect of Bartonella seropositivity on the mental health of Toxoplasma-seronegative women, and the positive effect of Bartonella seropositivity on both physical and mental health in Toxoplasma-seropositive men, suggest that the stress-coping hypothesis alone cannot explain all observed phenomena and that the physiological state of men and women could really react differently to some specific environmental factors, including pathogen infections.At the present time, the effects of various symbionts on a phenotype of humans is the subject of a growing number of studies. Our results, however, suggest that rather than main effects of particular symbionts, the interaction of different species symbionts, parasites and mutualists, and their interactions with genotype and environment should be always studied. As a minimum, the effect of symbionts should be always studied separately on men and women as they usually react differently, often in an opposite way, to the infection. The opposite behavioral reaction to the nfection , 47. HowThis study was carried out in accordance with the recommendations of European Communities guidelines. The protocol was approved by the by Institutional Review Board of Faculty of Science, Charles University. All subjects gave written informed consent in accordance with the Declaration of Helsinki.JF designed the study and performed the statistical analyses. PB analyzed the sera. All authors participated on interpretation of results and writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aims to investigate dietary and nutritional biochemistry profiles of attention-deficit/hyperactivity disorder (ADHD) and to explore their potential relationship by path analysis. We enrolled 216 children with ADHD and 216 age-, height- and gender-matched controls from 31 elementary schools in Taiwan. Dietary intake of the participants was assessed using a food frequency questionnaire (FFQ). Fasting blood samples were collected to determine the serum levels of multiple nutritional markers. Moreover, we employed a structural equation model (SEM) to link diet, nutritional markers and ADHD. Compared to healthy control, ADHD children had significantly lower serum levels of vitamin B12, folate, vitamin B6, ferritin concentration, and monounsaturated fatty acids (MUFA), but higher levels of serum saturated fatty acids (SFA), n-6/n-3 fatty acid ratio, and inorganic phosphorous concentration. Children with ADHD had more intake of nutrient-poor foods such as high sugar and high fat foods, and had less intake of vegetable, fruit, protein-rich foods than their counterpart. SEM analysis showed that the poor nutritional biochemistry profiles linked the association between unhealthy dietary patterns and ADHD. In conclusion, an unhealthy dietary pattern may be a predecessor of the poor nutritional biochemistry status, and managing diet and nutrition conditions should be considered to improve ADHD symptoms in children. Attention-deficit/hyperactivity disorder (ADHD), a psychiatric disorder commonly found in children and adolescents, is characterized by inattention, hyperactivity, and impulsivity . This diData from Nutrition and Health Survey in Taiwan 2001–2002 showed that children with a dietary pattern of more free sugar-rich foods and/or fried foods, but less nutrient-dense foods  had poorer overall school performance compared to their counterparts . SimilarA critical question is whether poor nutritional status plays a role in ADHD etiology. A study indicated that nearly half of the ADHD children had suboptimal nutrition, while only 11.1% of typically developed non-ADHD children revealed such a condition . StudiesWe hypothesized that the pathophysiology of ADHD may be related to nutritional imbalance and involved in multiple dietary components which is in part a result of an unhealthy dietary pattern. Carrying out a case-control study and applying a path analysis, we aim to not only document the dietary and nutritional biochemistry difference between the ADHD and the control children, but also explore the potential inter-relationships among diet, nutritional biochemistry, and ADHD.This study first received approval from the Medical Research Ethical Committee, Institute of Biomedical Sciences, Academia Sinica (AS-IBMS-MREC-92-01). The subject’s recruitment and study procedures have been described elsewhere . We mailWe obtained information about the education levels, family income, lifestyle, and disease history of the students and their parents through a questionnaire interview. Meanwhile, the body weights and heights of the participating children were measured by trained technicians. We asked parents to fill out a food frequency questionnaire (FFQ) to assess the dietary intake of the child in the previous year .We used food frequency questionnaires  filled bThe food preference of children was collected by directly interviewing children. The food items of the questionnaire were composed of 20 items which were the same as the items in the food frequency questionnaire. There were three levels of preference: disliking, neutral on, and liking the food .Approximately 10 mL venous blood was drawn from each student, which was carefully kept in darkness as much as possible in the process of centrifugation and aliquoting. All the serum and plasma samples were stored at −70 °C until analysis. The plasma pyridoxal 5′-phosphate (PLP) concentration was measured using high performance liquid chromatography (HPLC) . Plasma The G-Power 3 software was used for estimating sample size in this matched case-control study. Given the significance level (α) was set at 0.05 and odds ratio (OR) was set as 2.5, 131 cases and 131 controls were required to achieve a power (1-β) of 0.8, and 211 cases and 211 controls were required to achieve a power of 0.95.p < 0.05 were considered statistically significant. We performed group comparisons between cases and controls using Chi-square tests for categorical variables and t-tests for continuous variables.All statistical analyses were carried out using the statistical software package SPSS, version 24.0  and (IBM SPSS Amos 24.0). Variables being offered as either mean (±standard deviation) or frequency (%), and To sort out interrelations among large numbers of dietary and nutritional markers, we used principal component analysis (PCA) to identify latent components from either nutritional biochemistry markers or from food frequency variables. Varimax rotation was applied and orthogonal components with Eigen value greater than 1.0 were chosen. Loading scores were used to weight the chosen biochemical or food frequency variables within each PCA component to generate factor score and the variables with loading scores greater than 0.50 were designated as the major contributors of the components. Subsequently, the relationships between ADHD, the dietary factors and nutritional biochemistry factors were determined using the General Linear Model to control for parents’ education levels and balanced budget status.This study applied Pearson’s correlation to analyze the mutual relationships between the nutritional biochemistry factors and food factors yielded by PCA. Moreover, this study used a structural equation modeling (SEM) to find causal relationship and the strength of the relationships between food frequency, nutritional biochemistry markers, and ADHD. SEM, which is better than traditional methods  in analyzing complex causal relationships among variables . The maxThe ADHD children and the control subjects were comparable with respect to age, height, weight, body mass index (BMI), and gender distribution . CompareFood consumption frequency data were also compared between the ADHD children and the controls . SignifiFor the four PCA-yielded dietary factors, factor 1 primarily consisting of all nutrient-poor foods was labelled “Nutrient-Poor Foods”. Factor 2 contained vegetable and fruit and was named “Vegetable-Fruit”. The main component of Factor 3 was meat, poultry and fish, and was therefore labelled “Flesh foods”. Factor 4 is comprised soy milk and eggs and was named “Soymilk-Egg”. The relationships between ADHD, the dietary factors and nutritional biochemistry factors were shown in r = −0.24, p < 0.001) and “Minerals” . Vegetable-Fruit revealed a positive correlation to “B-Vitamins”  and “Minerals” . “Flesh food” intake was also positively correlated with “B-Vitamins”  and “Minerals” . Finally, “Soymilk-Egg” consumption had a positive correlation with “Minerals” , and negative correlation to n-6/n-3 saturated fatty acids ratio . “MUFA/SFA Fatty Acids” did not have significant correlations to any diet factor.Correlation coefficients among the four biochemistry factors and the four dietary factors were shown in β = −0.06, p = 0.005), “B-Vitamins” , “Minerals: ferritin/Pi”  were significantly and inversely associated, but “n-6/n-3 FA Ratio”  was positively associated with having an ADHD diagnosis.The results of the path analysis of diet, nutritional biochemistry, and ADHD are provided in Compared to healthy control children, ADHD children showed significantly lower blood levels of vitamin B12, folate, and vitamin B6. In addition, ADHD children exhibited lower serum ferritin concentration, but higher serum inorganic phosphorous (Pi) concentration. Furthermore, ADHD children had significantly higher SFA proportion and n-6/n-3 fatty acid ratio, but lower MUFA proportion.With respect to vitamin status, previous studies have suggested a role of vitamin B12 and vitamin D in the pathogenesis of mental disorders in childhood and adolescence ,15. In aFor mineral status, ADHD children exhibited lower serum ferritin concentration, but higher serum Pi concentration. Evidence has shown that ferritin deficiency is associated with ADHD in children ,17,33. AFurthermore, ADHD children had significantly lower MUFA proportion in blood lipids, but slightly higher SFA and PUFA proportions. The advantages of MUFA over SAF has been widely documented . The ω-6It is important to understand the diet-biochemical profile-ADHD interrelationships. We found ADHD children prefer nutrient-poor foods , but not nutrient-dense foods . Their dietary pattern is consistent with the food preference. Previous studies have revealed that a higher intake of sweets, fried food, and salt is positively associated with ADHD, but a balanced diet is inversely associated ,10. Our The path analysis suggests that the nutrient-poor diet leads to suboptimal blood nutritional biochemistry which in turn contributes to the ADHD development. We found the B6, folate and B12 (B-vitamin factor) inadequacy were related to the lower intake of vegetable/fruit and protein abundant foods, and correlated to more intake of nutrient-poor foods. In addition, increased intakes of vegetable-fruit, flesh foods, and eggs contributed to a better iron status and lower phosphate concentration in blood, whereas nutrient-poor foods had an opposite effect. Being short of aforementioned whole foods and excessive in low-nutrient-density foods may compromise children’s vitamin and mineral status, and further link to ADHD characteristics. This also supports the potential inter-relationship among diet, biochemical profile, and behaviours among children. In the future, a clinical trial is warranted to untangle the direction of relationship; that is, to clarify whether modifying dietary pattern from nutrient-poor to nutrient-dense foods can correct children’s vitamin and mineral status, and further improve children’s ADHD symptoms.Our study has some inherent limitations due to its nature of the case-control study design and bottle-neck in dietary data assessment. First, although we performed a SEM analysis, our study could not conclude the causal relationship between poor dietary patterns, nutritional status, and ADHD. Second, some may argue the inferior nutritional biochemistry profiles might result from the higher activity levels in ADHD children. However, we found the biochemistry profile is consistent with that food consumption assessed using the food frequency questionnaires. Compared to the control group, ADHD children preferred less nutrient-dense foods  and more nutrient-poor foods . It is nThe results of this study revealed that nutritional risk factors of ADHD include lower serum levels of B-vitamins as well as imbalance between iron status and inorganic phosphorus, between MUFA and SAF and between n-3 and n-6 PUFAs. Moreover, poor nutrition states may play a mediating factor between the link of unhealthy diet habit and ADHD. Further investigation is needed to clarify whether nutrition deficiency may contribute to abnormal brain function, and further lead to ADHD symptoms among children. Managing dietary and nutrition conditions may be trialed and considered as adjunct therapy for improving ADHD symptoms in children.


We hypothesized that efficient delivery of MKH might be critical to regulate HCC proliferation. The discovery of a suitable prodrug targeting HCC in terms of delivery and activation could reduce the clinical dose of MK-4 and maximize efficacy and safety. We previously showed that MKH dimethylglycinate (MKH-DMG) enables effective delivery of MKH into HCC cells and exhibits strong antitumor effects compared with MK-4. In this study, we prepared anionic MKH hemi-succinate (MKH-SUC) and non-ionic MKH acetate (MKH-ACT), in addition to cationic MKH-DMG, and evaluated MKH delivery profiles and antitumor effects in vitro. MKH-SUC showed the highest uptake and the most efficient release of MKH among the examined compounds and exhibited rapid and strong antitumor effects. These results indicate that MKH-SUC might have a good potential as an MKH delivery system for HCC that overcomes the limitations of MK-4 as a clinical chemopreventive agent.Hepatocellular carcinoma (HCC) shows poor prognosis owing to its very frequent recurrence even after curative treatment. Thus, an effective and safe long-term chemopreventive agent is strongly in demand. Menahydroquinone-4 (MKH) is an active form of menaquinone-4 (MK-4, vitamin K Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors in the world. Despite so-called curative treatments, such as surgical resection or liver transplantation, the long-term prognosis of HCC is very poor because of a high relapse rate and frequent incidence of intrahepatic metastasis ,2. Soraf2) may play a role in suppressing the growth and recurrence of HCC both in vitro and in vivo  vs. normalized response-variable slope analysis of GraphPad Prism, version 6.0 .Cell viability was determined using a previously reported method . Brieflyg, 1 h) in isotonic phosphate buffer (pH 7.4). Stock solutions of each ester were made in the same manner as the cell viability assay, and the solutions were diluted with 0.1% w/v albumin in PBS. The enzymatic reactions were initiated by adding 5 μL of the diluted of each ester into amber test tubes containing 95 μL of preheated S9 fractions. A BCA protein assay  was used to determine protein concentrations, and the final S9 concentration was adjusted to 1.0 mg of protein/mL in PBS. The solutions were incubated at 37 °C, and at appropriate times, the aliquots from the reaction were combined with an equal volume of methanol and three times volume of n-hexane. The samples were vortexed for 2 min and centrifuged at 1750× g for 10 min. The upper layer (n-hexane) was collected and evaporated under nitrogen. The residue was reconstituted with 100 μL of methanol, sonicated for 10 s and subjected to LC-MS/MS analysis as described below.The hydrolysis of the esters was performed at 37 °C in S9 fractions of PLC/PRF/5 and SK-Hep-1 cells after ultrasonic homogenization and centrifugation . Separations were performed on the same column mentioned in the 4.8.1 LC-MS/MS section and monitored at a wavelength of 240 nm. The mobile flow was a binary gradient: (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in methanol. The total flow rate was 0.3 mL/min; 0 min at 80% B, 1 min at 80% B, 4.0 min at 100% B, 7.0 min at 100% B and 8.0 min at 80% B. The Column temperature was 40 °C. The retention times were as follows: MKH-DMG, 9.2 min; MKH-ACT, 8.2 min; and MKH-SUC, 6.6 min.5 cells/well in 6-well plates and allowed to attach for 48 h. Cells were treated with MK-4, MKH-ACT, MKH-DMG or MKH-SUC for 24 h. Western blotting was performed as described previously  refers to the concentration of the substrate and Km is defined as the concentration of substrate at the half-maximal rate of action (Vmax). Data were analyzed by GraphPad Prism (GraphPad Software).For the determination of


Positive family history was significantly evident in patients with epilepsy and ADHD with epilepsy, but not in the control or ADHD groups. Speech development was significantly delayed in the ADHD and ADHD with epilepsy groups. EEG abnormalities were detected in patients with ADHD (12%) and ADHD with epilepsy (68%). Focal frontal activities were significantly detectable in the ADHD (100%) and ADHD with epilepsy (77.8%) groups, whereas focal temporal activity was significantly present in the epilepsy (83.3%) group. Serum ferritin was significantly lower in the ADHD group (110.27 ± 6.64 ηg/ml) when compared to the control (134.23 ± 14.82 ηg/ml), epilepsy (159.66 ± 33.17 ηg/ml), and ADHD with epilepsy (203.04 ± 50.64 ηg/ml) groups. Serum zinc was significantly higher in the ADHD, epilepsy, and ADHD with epilepsy groups , when compared to the control group (144.21 ± 17.40 μg/dl). Serum adenosine deaminase was insignificantly different among the groups. Our results indicate that gender and family history are significant moderators in the aetiology of ADHD and epilepsy or their comorbidity. We also demonstrated that EEG could be central in the assessment of ADHD with epilepsy cases. Serum ferritin and zinc alteration may contribute significantly in ADHD and epilepsy pathophysiology.Attention deficit hyperactivity disorder (ADHD) is among the most prevalent neurobehavioral disorders affecting children worldwide. The prevalence of ADHD is higher in children with epilepsy. Despite the plethora of conducted work, the precise cause of ADHD is not identified yet. We studied here the sociodemographic, clinical, electrophysiological, and biochemical profiles of children with ADHD, epilepsy, and ADHD with epilepsy. Subjects were divided into 4 groups (25 child/group): I—control, II—ADHD, III—epilepsy, and IV—ADHD with epilepsy. Male to female ratio was significantly ( Attention deficit hyperactivity disorder (ADHD) is one the most prevalent childhood neuropsychiatric disorders worldwide in both developed and developing countries. ADHD affects 5–15% of school-age children and was found to persist in 30–50% of these cases through adolescence . ADHD caEpilepsy is a common chronic neurological disorder characterized by seizures. The annual incidence of epilepsy is estimated to be around 40–70/100,000 births/year in developed countries and 100–190/100,000 births/year in developing countries . SeizureAlthough several studies have addressed the neurobiology of ADHD from imaging and genetics points of view, relatively inadequate attention has been given to study the factors or mechanisms that may be involved. In the present investigation, we studied the sociodemographic and electrophysiological data, as well as the serum levels of some biomarkers in children with ADHD and ADHD with epilepsy in comparison to those in control healthy and epileptic volunteers.100 children of comparable age were recruited for the present cross-sectional study. Patients were selected from those attending the Paediatrics Neurology Clinic in Menoufia University Hospital and those admitted to the Paediatrics Department in Suzanne Mubarak Hospital, as well as 25 healthy volunteers who served as controls. 75 selected patients were diagnosed with ADHD, epilepsy, or comorbid ADHD and epilepsy. All the controls and patients provided written informed consents following a comprehensive description of the study. The Ethics Committee of the Menoufia University Faculty of Medicine approved the study. A semistructured form was used to detect several sociodemographic and clinical variables of the patients, and the medical records of the patients were reviewed.Subjects were divided into 4 groups (25 child/group): I—control group, II—ADHD group , III—epilepsy group  classification), and IV—ADHD with epilepsy group .Patients with a history of chronic systemic diseases such as diabetes mellitus and hypertension, other psychological or neurological disorders, ADHD criteria for less than 6 months, uncontrolled epilepsy, or with IQ < 70 were excluded. None of the children were taking nutritional supplements such as antioxidants. All subjects underwent brain magnetic resonance imaging (MRI) evaluation, and all were free of any abnormalities. Delayed speech was assessed by both history taking from the parents and preschool language scale 5 (PSL-5).ηg/ml) and adenosine deaminase (ADA) (U/l) levels were determined by in vitro enzyme-linked immunosorbent assay kits , using an automatic optical reader  [μg/dl. Assay was determined by routine kinetic and fixed-rate colorimetric methods on a Jenway Genova autoanalyser (UK).Whole venous blood was collected via EDTA-lined Vacutainer tubes. The blood was allowed to coagulate for 30 minutes at room temperature. Blood samples were then centrifuged at 2000 rpm for 15 minutes to separate serum samples. Serum was then separated and stored at −80°C. The serum ferritin (Austria) . The serAustria) . Resultst-test was used for the comparison between the means of any 2 groups of patients, while Pearson's chi-square (χ2), Kruskal-Wallis (K), and Fisher's exact tests were used for the comparison between qualitative variables using Statistical Package for Social Sciences (SPSS) software and the probability of chance . p values < 0.05 were considered significant.Results are expressed as mean ± standard error (SE). Student's p > 0.05) between the studied groups. Gender distribution in the ADHD and ADHD with epilepsy groups showed significant increased male numbers , when compared to the corresponding values in the control  or epilepsy  groups. There was insignificant difference (p > 0.05) in gender when comparing the corresponding values in the control and epilepsy groups or when comparing the values in the ADHD and ADHD with epilepsy groups. Positive family history of seizures or neurological diseases was significantly different (p < 0.05) in the epilepsy and ADHD with epilepsy groups when compared to the corresponding values in the control and ADHD groups . However, it was insignificantly different in the ADHD group when compared to the corresponding value in the control group (p > 0.05). There was insignificant difference in consanguinity or developmental history among the studied groups (p > 0.05).The sociodemographic profiles of the studied groups are illustrated in Speech development was significantly delayed in the ADHD  and ADHD with epilepsy  groups, when compared to the corresponding values in the control  or epilepsy  groups. There was insignificant difference between epileptics and control subjects and also between the ADHD and ADHD with epilepsy patients .The distribution of the different subtypes of ADHD in the ADHD and ADHD with epilepsy groups is shown in p > 0.05) between the epilepsy and ADHD with epilepsy groups regarding the types of seizures . However, there was an important difference in the number of patients with generalized myoclonic convulsion which was significantly higher in the ADHD with epilepsy group  when compared to the corresponding value in the epilepsy group (0 patients). The number of patients with complex partial convulsions in the ADHD with epilepsy group was 4 (100%), which was significantly higher when compared to the corresponding value in the epilepsy group (0 patients), while the number of patients with focal secondary generalization convulsions in the ADHD with epilepsy group (0 patients) was significantly lower when compared to that in the epilepsy group .As shown in ηg/ml) was significantly lower when compared to those in the control (134.23 ± 14.82 ηg/ml), epilepsy (159.66 ± 33.17 ηg/ml), or ADHD with epilepsy (203.04 ± 50.64 ηg/ml) groups. Serum ferritin level was significantly higher in the ADHD with epilepsy group when compared to the control subjects (μg/dl), epilepsy (286.74 ± 43.84 μg/dl), and ADHD with epilepsy (229.95 ± 67.34 μg/dl) groups, when compared to the corresponding value in the control group (144.21 ± 17.40 μg/dl). There was insignificant difference in serum Zn between the ADHD, epilepsy, and ADHD with epilepsy groups (p < 0.05) between serum ADA and family history of neurological diseases in the control group. There was also a significant positive correlation between serum zinc and family history of neurological diseases and between serum ferritin and the age in the epilepsy group , while the inattention subtype was the most prevalent in the ADHD with epilepsy group. Our results were in agreement with several published reports . ADHD isEEG is usually normal in most cases with ADHD; therefore, it was expected to find insignificant EEG changes in the ADHD group when compared to controls. But interestingly, three cases in the ADHD group showed abnormal focal epileptogenic activity in the frontal lobe in their EEGs despite negative previous history of seizures. This was consistent with previous reports . EEG epiWe demonstrated here that the mean value of the serum ferritin level was significantly lower in the ADHD group, while on the contrary, the serum ferritin level in the ADHD with epilepsy group was significantly higher when compared to the corresponding values in the control or epilepsy groups. Similar results were reported previously, where the low serum ferritin level was associated with hyperactivity symptoms in children with ADHD . SeveralSerum zinc (Zn) levels were significantly higher in the ADHD, epilepsy, and ADHD with epilepsy groups, when compared to the corresponding value in control group. Zn homeostasis is necessary for the proper development of the brain especially for the cerebellum, stellate, and basket and also for Purkinje and granule cells . Zn may In the present investigation, serum levels of ADA were insignificantly different among the studied groups. ADA is an essential enzyme for the T lymphocyte maturation and function. As the plasma activity of ADA has been suggested to be increased in inflammatory diseases, ADA has been considered as an indicator of cell-mediated immunity . StreptoTo our knowledge, this is the first paper to study a full spectrum of neurobehavioral disorders which can exist together. Our attempt to characterize ADHD, epilepsy, and their comorbidity is aimed at elucidating common or diverse underlying mechanisms. Gender and family history were shown to be key moderator factors. EEG, although not a precise diagnostic tool, should be considered if ADHD is suspected especially if the patients are positive for family history of seizures, prenatal events, history of trauma, or coexistence of other behavioral manifestations. Iron and zinc metabolism may contribute to the pathophysiology of ADHD, epilepsy, or their comorbidity. Accordingly, serum biomarkers may play a promising role in early identification of the diseases.The sample size of the study was relatively small, and more work including larger numbers of the study children should continue. More work on the correlation between risk factors is essential.