Borrelia burgdorferi ELISA was performed; antibodies were not detected, and no treatment was instituted. In the fall, subtle memory loss developed, and he fell twice a few weeks apart. Dad jokingly blamed the falls and the memory loss on “old timer’s disease.” Subsequently, episodes of subtle confusion and more frequent memory loss generated family concern as to what the future might hold. On December 15, he broke his left femur during a fall while climbing 2 stairs to enter our home. Despite having successfully climbed those stairs thousands of times in the past, he would never climb those or any other stairs again.During the summer of 2007, migratory joint pain developed in my (E.B.B.) 86-year-old father, previously an ironworker, farmer, and World War II veteran. Because of occasional tick attachments, a Retrospectively obvious, a pattern of insidious illness characterized by joint pain, memory loss, and incoordination, not recognizable by my father or other family members, had begun before that summer. Medically stable historical problems included coronary artery disease, atherosclerosis, carotid artery occlusion, hypertension, and atrial fibrillation. During the previous year, a normocytic, normochromic, nonregenerative anemia persisted. Despite normal serum iron, total iron binding capacity, ferritin, and vitamin B12 values, anemia was attributed to intestinal blood loss. When examined in May 2007, before anesthesia for endoscopy, mood and affect were appropriate, recent and remote memory were intact, insight and judgment were good. A hiatal hernia, mild antral gastritis, and duodenitis were visualized.When my father was hospitalized December 15, 2007, with a broken femur, a resting pill roll tremor and cogwheel rigidity were suggestive of Parkinson disease. Preoperative neurologic consultation identified severe confusion, inattention, and an inability to answer questions. Short-term memory and problem-solving abilities were decreased. There was mild ptosis of the right eye, normal cranial nerves, mild asterixis, and hand weakness. Laboratory abnormalities included anemia, hypercreatinemia, an elevated aspartate aminotransferase level, and hyperglobulinemia. Due to the severity of the femoral fracture, the femoral head was excised and replaced with a bipolar femoral prosthesis.Candida albicans and herpes zoster. C. albicans esophagitis is known to accompany HIV infection, leukemia, or an unidentified source of immune suppression  cultures were negative. CSF results, including those for special stains, were unremarkable. Serologic results for Treponema pallidum, Borrelia burgdorferi, Rickettsia rickettsii, Bartonella henselae, Bartonella quintana, and HIV were negative. Results of CSF herpes simplex PCR and a test for T. pallidum antibodies were negative.When I was a boy, my father used the expression “something is fishy in Denmark” to imply that something was astray. Based upon historical events, I suspected something was being missed. Laboratory findings included anemia (hemocrit 34%), a normal leukogram , mild hypokalemia, hypoalbuminemia, hyperglycemia, increased serum alkaline phosphatase level, erythrocyte sedimentation rate of 79, and hypergammaglobulinemia. Thoracic radiographs identified mild bilateral pleural effusion. Because an undefined infectious source of immunosuppression seemed plausible, and fever (maximum temperature 38.6°C) occurred 24 hours after neurologic decompensation an infectious etiology was pursued. Nasal methicillin-resistant On January 11, results of magnetic resonance imaging and magnetic resonance angiography were interpreted as a left posterior stroke with no active bleeding. Initial treatment included intravenous acyclovir, fluconazole, vancomycin, ceftriaxone, ampicillin, and dexamethasone.Anaplasma, Ehrlichia, and Rickettsia species were negative. Bartonella 16S–23S intergenic spacer primers  isolated from a southern flying squirrel (Glaucomys volans) and Candidatus Bartonella durdenii (391/422bp) amplified from Orchopeas howardi (GenBank accession no. DQ 336386), a flea found on eastern US gray squirrels (Scinrus carolinensis), and a Bartonella sp.  identified in ground squirrels (Spermophilus danricus) in the People’s Republic of China. The novel rodent Bartonella sequence obtained from my father’s blood had an 18-bp insert at positions 2047 or 334 in EU294521 and DQ336386, respectively. Previously, our laboratory had never worked with rodent Bartonella species and had never amplified a 300-bp internal transcribed spacer region amplicon from >3,000 animal or human blood samples. After several unsuccessful cloning attempts, the CSF amplicon was most similar (393/394 bp) to B. henselae (NC-005956). Blood and CSF, cultured by using Bartonella α Proteobacteria growth medium (BAPGM) (Bartonella species.Because I direct the Intracellular Pathogens Research Laboratory (IPRL) at the North Carolina State University College of Veterinary Medicine, aseptically obtained blood and CSF samples were kindly provided for testing. Results of PCR , hyperglobulinemia (3.6 g/dL), and increased alkaline phosphatase activity (169 IU/L). Urinalysis abnormalities included proteinuria with occasional hyaline casts.Bartonella infection, intravenous doxycycline, rifampin, and gentamicin were administered, and total parenteral nutrition was instituted. Again, shortly after initiation of antimicrobial drugs, a seizure occurred. Bartonella spp. were not amplified or isolated from a 1-mL blood sample obtained 4 days after initiation of treatment with antimicrobial drugs. During the next 3 weeks, while intravenous antimicrobial drugs were administered, our father again remained encephalopathic, with frequent hallucinations, severe agitation, and near-constant mental confusion. On March 14, intravenous methylprednisolone for potential immune-mediated vasculitis elicited no improvement in mental status. Similar to the previous treatment course, improvement in mental status, coherent communication, and renewed ability to recognize family members occurred during the fourth hospitalization week.Lorazepam was administered to control the agitation and restlessness. The warfarin dose was increased and heparinization initiated for a potential cerebrovascular accident. Due to the prior documentation of Bartonella DNA sequence was again amplified from his blood, but no bacteria were isolated.On April 4, Dad was discharged to our home and oral antimicrobial drugs (doxycycline and rifampin) were dispensed. Despite all efforts by medical professionals, members of our family, and our tough 86-year-old father, protracted illness and prolonged hospitalizations had resulted in mental and physical debilitation, severe muscle wasting, and profound weakness. More important, he had lost his desire to live. After discharge, there was minimal neurologic improvement. Before the availability of April 4 IPRL test results, he began to refuse all medications. The identical rodent pm, around quitting time for an old iron worker. My mother and youngest brother were at his side. After his death, blood culture results from another sample obtained by the hospice nurse on April 28, 2008, became available. B. vinsonii subsp. berkhoffii genotype II was amplified and sequenced from the enrichment BAPGM blood culture. As direct extraction of DNA from blood was negative, growth of viable bacteria in liquid culture was implicated , a flying squirrel (Candidatus Bartonella volans), and a groundhog (Candidatus Bartonella monaxi) isolate, the most similar GenBank sequence was Candidatus Bartonella volans. There was no perfect match with these 3 isolates. However, sequences from Dad’s blood clustered with a squirrel Bartonella subgroup. This observation supports the presence of a novel Bartonella species on the eastern shore of Maryland, an as yet undefined animal reservoir, and an unknown arthropod vector.Following our father’s death, I recalled a small, 0.5-cm, raised, firm lesion within his right eyebrow that developed during the summer of 2007 and would spontaneously hurt or burn, causing him to rub or squeeze the lesion. The mass disappeared after he began taking antimicrobial drugs in 2008. Retrospectively, I suspected a rodent flea bite above the eye had transmitted a novel Bartonella sp. and B. vinsonii subsp. berkhoffii supports the unexpected failure of 2 intensive courses of intravenous and oral antimicrobial drugs to eliminate these fastidious, intravascular bacteria. During the first two hospitalization periods, there was similar and progressive improvement in neurologic signs and mental capabilities that began during the fourth week of antimicrobial drug administration. Pre-enrichment BAPGM growth of B. vinsonii subsp. berkhoffii from blood obtained 4 days before death supports persistence of viable organisms. Recently, antimicrobial drug resistance genes have been characterized in B. bacilliformis, B. henselae, and B. quintana by in vitro serial passage (Regardless of the mode(s) of transmission, repeated molecular documentation of a novel rodent Bartonella spp. by antimicrobial drugs in immunocompetent patients may be more difficult to achieve than is currently appreciated , which suggests that a bacteremic cat might facilitate transmission of this subspecies. My parents had an old (≈21 years of age) exclusively outdoor barn cat that would occasionally scratch. The cat could not be tested because it died in 2007. B. henselae and B. clarridgeae have been transmitted experimentally by transfusion to cats  patients, and atypical CSD manifestations are more likely to develop in elderly patients  most nurses are absolutely amazing, caring, and dedicated professionals; 2) in human medicine, unlike veterinary medicine, no physician claimed or accepted the responsibility to be my father’s doctor; and 3) for many reasons, I found the human healthcare system to be frayed, if not broken. Whether blame lies with the insurance companies, our litigious society, the profit-based motivations of hospital administration, the increased complexity of medical technology, or the medical education of physicians, it really does not matter. As my father would say, “It is no way to do business.” I have taught internal medicine at a College of Veterinary Medicine for 32 years. During that time, every sick animal on our medical service had at least 2 doctors (1 being a student), who were directly responsible for the animal’s care, for frequent communications with the owner, and for communications with the referring veterinarian. In our increasingly complex hospital environments, every patient needs a personal advocate or designated doctor to represent his or her interests.Bartonella infection and complex disease expression, provides documentation for an as yet uncharacterized zoonotic rodent Bartonella sp., and offers disconcerting evidence supporting antimicrobial drug ineffectiveness and clinical evidence supporting the concept that persistent infection with >1 Bartonella spp. may lead to immunosuppression and opportunistic infections with organisms such as herpes zoster and C. albicans. As in his life, Dad would want this story to benefit others after his death. We hope that it does.Some years ago in a conversation with my mother I suggested that the term natural death may well represent an oversimplification of the processes that end a person’s life. My father and our family were substantially affected during his illness. Each day, from initial hospitalization until his death, there was at least 1 family member at his side. In the eyes of family and friends, my father was a great man in so many respects. He was a loving husband, a caring father, diligent worker, and a friend and supporter to many persons. Potentially, his illness illustrates a complex interaction between intravascular


Attention deficit hyperactivity disorder (ADHD) is a neurocognitive behavioral developmental disorder most commonly seen in childhood and adolescence, which often extends to the adult years. Relative to a decade ago, there has been extensive research into understanding the factors underlying ADHD, leading to far more treatment options available for both adolescents and adults with this disorder. Novel stimulant formulations have made it possible to tailor treatment to the duration of efficacy required by patients, and to help mitigate the potential for abuse, misuse and diversion. Several new non-stimulant options have also emerged in the past few years. Among these, cognitive behavioral interventions have proven popular in the treatment of adult ADHD, especially within the adult population who cannot or will not use medications, along with the many medication-treated patients who continue to show residual disability. Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder that is defined by persistent and maladaptive symptoms of hyperactivity/impulsivity and inattention  , cerebellum and parietal cortex. Research has indicated that some of these brain regions are slightly smaller or have decreased activation in people with ADHD.Pediatric ADHD commonly co-occurs with multiple psychiatric disorders including mood, anxiety and disruptive behavioral disorders ,10. LikeResearch on ADH) has been published at an exponential rate during the past 30 years . Within There are both pharmacological and non-pharmacological treatments for ADHD for both children and adults. Pharmacological approaches to treatment are the most common, and typically consist of stimulant medication, such as methylphenidate, dexmethylphenidate, mixed amphetamine salts and lisdexamfetamine dimesylate (LDX). However, non-stimulants such as atomoxetine, clonidine and guanfacine have also been found to be efficacious in treating ADHD. In addition to medication, there are also non-pharmacological treatments. Many of the drugs discussed in this section are currently licensed for use in North America, but not in other countries.Treatment for children with ADHD includes parent and teacher training in effective behavior-management techniques aimed at reducing the problem behaviors associated with ADHD. CBT is a skill-based approach commonly used for adults with ADHD and there are some preliminary data showing its efficacy.For most patients with ADHD, stimulants remain the first choice for medication management, as meta-analyses of existing research have shown that they are more efficacious than non-stimulant medications . VariousResearch has continued to suggest that osmotic-release oral system (OROS) methylphenidate lessens ADHD symptoms throughout the day and has greater adherence, thought to be associated with the convenience of once-daily dosing -41. DexmComorbid anxiety was not found to affect stimulant efficacy in a recent study , and somThe commonest side-effects of stimulants  have also continued to receive recent research interest. Some research suggests that it is difficult to predict which children with ADHD will have adverse effects, based upon demographic and clinical characteristics . AlthougWithin the past 3 years, concerns have continued to rise regarding the abuse of stimulants and/or drug diversion -66. SomeIn summary, stimulant medication is often the first choice for medication management of ADHD. Research has shown that stimulant medication is an effective treatment for many of the symptoms associated with ADHD. However, there are some concerns about diversion of these medications for misuse and abuse, and some rare but serious cardiovascular side-effects can occur with the use of stimulant medication. In addition to stimulant medication, some non-stimulant medication has been shown to be efficacious for the treatment of ADHD Table .Some children may not respond to stimulant medications, or may not be able to tolerate the stimulant medications due to side-effects . Thus, several non-stimulant medications are also used for ADHD pharmacotherapy. Modafinil  and reboSeveral reviews on the use of atomoxetine have been published recently ,77, and The α-2-adrenergic agonists clonidine and guanfacine have long been known to be of some assistance in treating ADHD . RecentlResponse to single-agent treatment for ADHD often falls short of full remission. Recent studies have shown that adding clonidine to methylphenidate , extendeTreatment of ADHD can result in alleviation of comorbid depression, anxiety, oppositional defiant disorder and/or aggression . HoweverA number of reports have been published about the use of alternative and complementary medicines in the treatment of ADHD. In a small study, traditional Chinese medicines were found to compare favorably with methylphenidate . PositivThe need for treatment in adults with ADHD has been debated in the past, with reports of suboptimal response, diversion and abuse. There is a growing body of research on the treatment, and recent years have seen the creation of evidence-based guidelines . Meta-anNon-stimulant options are also similar to pediatric ADHD options, although bupropion and modafinil are used more often in adults than in children with ADHD. However, the only non-stimulant with FDA approval for adults with ADHD is atomoxetine.The potential for diversion and misuse may be greater in adults than in children, as parents might control the medications for the latter. Stimulant misuse seems to be more common in those with comorbid alcohol-, drug- and cigarette-related problems, and those with higher levels of ADHD symptoms . In addiIn pediatric ADHD, a combined treatment approach generally consists of pharmacotherapy and some form of psychosocial intervention, generally consisting of training parents in behavioral management, consultation with teachers/school personnel and individual work with the child . For exaIn adults, a combined treatment approach similarly typically consists of pharmacotherapy and psychosocial intervention. However, unlike pediatric ADHD, there is some evidence that CBT interventions are efficacious  is often needed for patients with comorbid disorders, and is sometimes indicated when ADHD is the only presenting problem. Adjunct psychosocial treatments are often useful, but these should be targeted to patients based on a needs assessment.Possibly as a function of the disorder itself, non-adherence to treatment regimens is high in ADHD . In addiIn sum, despite considerable advances in our understanding and treating ADHD, the disorder remains difficult to manage. Poor treatment adherence and psychiatric comorbidity clearly complicate treatment and negatively affect outcomes.Cognitive neuroscience has permitted a greater understanding of ADHD. Recent research and novel drug developments have provided new treatment options for adolescents and adults with ADHD. New stimulant formulations have made it possible to tailor treatment to the duration of efficacy required by patients and to help mitigate the potential for abuse, misuse and diversion. Although they tend to be less efficacious than stimulants, new non-stimulant options also allow for extended duration of treatment without the adverse consequences associated with stimulant therapy. Progress in non-medical therapies now provides several options for patients who cannot or will not use medications, and for the many medication-treated patients who continue to show residual disability.Diagnostic and Statistical Manual of Mental Disorders, Fourth edition (DSM-IV)) are effectively treated. Future treatment development should aim at developing both psychosocial and medical treatments for these areas of difficulty. Future treatment research should also work to define and achieve optimal treatment outcomes for people with ADHD. Although current treatments are effective for achieving substantial symptom reduction in most patients, more work is needed to achieve full symptom reduction, and to reduce the burden of ADHD-associated disabilities.Looking toward the future, research will need to address several unmet needs. Many treated people with ADHD continue to have problems with executive functioning and deficient emotional self-regulation. These problems persist in many patients even when the core ADHD symptoms (as outlined in the There are also diagnostic challenges for clinicians that could be addressed by future research. ADHD symptoms, especially hyperactive-impulsive symptoms, tend to decline through adolescence into adulthood, so that the adult presentation of ADHD differs somewhat from the childhood presentation. Helping clinicians understand these differences, and how such differences should affect the application of diagnostic criteria requires more work.Ideally, medical and psychological treatments should be tailored to the underlying pathophysiology of the patient. Theoretically, this should be possible by using the scientific literature on the neurobiology of ADHD with treatment outcome studies, as it is possible that patients with specific brain-imaging abnormalities or genetic variants would have different responses to treatments. To date, most of this work has been done in the area of pharmacogenetics which, although promising, cannot yet guide treatment choices -134In summary, although the science of ADHD and its application to diagnosis and treatment have made great strides, more work is needed to improve the lives of patients and families affected by the disorder.ADHD: attention deficit/hyperactivity disorder; CBT: cognitive behavioral therapy; LDX: lisdexamfetamine dimesylate; OROS: osmotic-release oral system; SNRI: Serotonin-norepinephrine reuptake inhibitorKevin M. Antshel*, Teresa M. Hargrave, Mihai Simonescu, Prashant Kaul, Kaitlin Hendricks and Stephen V. FaraoneStraight Talk About Your Child's Mental Health.KMA, TMH, MH, PK, and KH report no biomedical financial interests or potential conflicts of interest. SVF has in the past year received consulting fees and has been on Advisory Boards for Eli Lilly, Ortho-McNeil and Shire Development and has received research support from Shire and the National Institutes of Health; in previous years, has received consulting fees or has been on advisory boards or has been a speaker for Shire, McNeil, Janssen, Novartis, Pfizer, Ortho-McNeil and Eli Lilly; in previous years has received research support from Eli Lilly, Shire, Pfizer and the National Institutes of Health, and has a published book with Guilford Press: All authors have been involved in drafting the manuscript or critically revising it or important intellectual content, and have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/9/72/prepub


Although hydrocarbon degrading cultures are abundant in nature, complete biodegradation of oil is rarely achieved even under favorable environmental conditions due to the structural complexity of oil and culture specificities. Moreover, the interaction among cultures in a consortium, substrate interaction effects during the degradation and ability of specific cultures to alter the bioavailability of oil invariably affect the process. Although SEB has the potential to increase the degradation rate of oil and its constituents, there are numerous challenges in the successful application of this technology. Success is dependent on the choice of appropriate surfactant type and dose since the surfactant-hydrocarbon-microorganism interaction may be unique to each scenario. Surfactants not only enhance the uptake of constituents through micellar solubilization and emulsification but can also alter microbial cell surface characteristics. Moreover, hydrocarbons partitioned in micelles may not be readily bioavailable depending on the microorganism-surfactant interactions. Surfactant toxicity and inherent biodegradability of surfactants may pose additional challenges as discussed in this review.Surfactant enhanced bioremediation (SEB) of oil is an approach adopted to overcome the bioavailability constraints encountered in biotransformation of nonaqueous phase liquid (NAPL) pollutants. Fuel oils contain  Widespread use of petroleum hydrocarbons, generation of petroleum wastes in large quantities, and their recalcitrance lead to the accumulation of these pollutants , 2. PersSurfactants are amphiphilic in nature. They lower the interfacial tension at the oil-water interface and the surface tension of water and thus favor mass transport of hydrocarbons from the oil phase into the aqueous phase. Primarily, three mechanisms are responsible for enhancing bioavailability by affecting the distribution of hydrocarbons , 8, thatMore recently, it has been highlighted that surfactants not only affect the distribution of oil but also affect the microbial cell surface properties, and such interactions may have both beneficial and detrimental effects on the biodegradation of hydrocarbons –13. It iMicroorganisms degrading complex petroleum mixtures, such as diesel, crude oil, gasoline, heavy oil, and lubricating oil, are abundant in nature. However, complete mineralization of such complex mixtures is normally not attained. Some microorganisms may degrade aliphatic hydrocarbons and some may only degrade aromatic hydrocarbons, while some other microorganisms may have the capability to degrade both aliphatics and aromatics –21. Comm4–106 hydrocarbon degrading microorganisms per gram. Hence, the remediation of oil contaminated area can be done by the enrichment of local microorganisms without any external seeding or use of genetically engineered microorganisms. However, complete mineralization is possible only when the microorganisms specific for utilizing the different components of oil are present in the contaminated site. Alkanes are degraded by most microorganisms. Cycloalkanes and aromatics, such as polynuclear aromatic hydrocarbons (PAHs), are degraded only by specific microorganisms. Such specificities of the microorganisms and the presence of the contaminants at concentrations toxic to some microorganisms may hinder the degradation process. Further, the interrelationship of the microorganisms involved also affects the degradation process. During the degradation of crude oil, Van Hamme and Ward [Rhodococcus sp. strain F9-D79 and Pseudomonas sp. strain JA5-B45 enhanced the degradation of crude oil only slightly over that achieved by the individual cultures. Thus, culture specificities and interrelationship amongst the cultures of a consortium determine the fate of oil degradation in a contaminated site.Since hydrocarbons constitute a ubiquitous class of natural compounds, microorganisms degrading them, such as bacteria, fungi and algae, are widely distributed in nature. Rosenberg  estimateand Ward  observedMicrobial degradation of petroleum hydrocarbons may also be affected by interaction effects of the multiple substrates present in complex hydrocarbon mixtures . This hao- and p-xylene (BTEX compounds) [Several models have been proposed to predict the type of interaction in such complex substrate systems. Most models have been tested with only two substrates, and their applicability to larger mixtures has been assumed without validation. However, a few models have been proposed and tested for multicomponent mixtures with more than 2 components. Some examples are growth of a mixed culture on benzene, toluene, ethylbenzene, and mpounds)  and the mpounds) . Guha etmpounds)  proposedmpounds)  found thAn important factor affecting oil biodegradation is the accessibility of the petroleum hydrocarbons to the microorganisms. Bioavailability is of concern due to the aqueous and nonaqueous biphasic nature of the system. The utilization of hydrocarbons by microorganisms can take place either through direct interfacial uptake by the attachment of microbial cells to the nonaqueous phase liquid- (NAPL-) water interface or after mass transfer of the NAPL to the aqueous phase . BiodegrSurfactant aided biodegradation is affected by a complex interplay of factors as illustrated in It is essential that an appropriate surfactant is selected for remediation of a particular type of petroleum waste in any scenario. The surfactant structure, its hydrophile-lipophile balance (HLB) number, dose, and its mechanism of action, that is, emulsification versus micellar solubilization, may affect the outcome of SEB. Surfactants are mainly classified into four types depending on the charge on the hydrophilic head group, that is, cationic, anionic, nonionic, and zwitterionic . In syntMycobacterium sps. and Rhodococcus erythropolis, rhamnolipids produced by Pseudomonas sps., Sophorolipids produced by Candida apicola; lipopolysaccharides produced by Acinetobacter calcoaceticus (RAG1), and phospholipids produced by Thiobacillus thiooxidans. Biosurfactants are mostly anionic or nonionic. Chemically, the hydrophilic head group of biosurfactants may consist of a carbohydrate, peptide, amino acid, phosphate, carboxylic acid, or alcohol while the hydrophobic tail may consist of fatty acids, hydroxy fatty acids, or α-alkyl-β-hydroxy fatty acids. For total petroleum hydrocarbons (TPHs) associated with soil, biosurfactants such as rhamnolipids and surfactin have been found to remove TPH at higher rates compared to the synthetic surfactants [In addition to synthetic surfactants, biosurfactants secreted by microorganisms may also be utilized for bioremediation of oil and petroleum hydrocarbons. Several microbial species are known to produce biosurfactants, and the chemical structure of these biosurfactants is reported to vary widely . These ifactants . BiosurfBurkholderia multivorans (NG1) as illustrated in Burkholderia multivorans (NG1) depicted negligible degradation of aliphatic hydrocarbons in the absence of surfactants. In the presence of the emulsifying surfactants, Triton X-100 and Igepal CA-630, significant degradation of aliphatic hydrocarbons was observed. In contrast, in the presence of Tween 80, the Burkholderia sp.illustrated much lower degradation since Tween 80, characterized by a higher HLB value, did not cause emulsification. The NAPLs differed from each other in the naphthalene: n-hexadecane ratio such that for NAPL A1 it was 1 : 1 and for NAPL A2 it was 1 : 3. Song and Bielefeldt [elefeldt  also recelefeldt  demonstrπ electrons can also form weak bonds with oxygen containing head groups in the shell region of nonionic surfactant micelles. In cationic surfactants, PAHs may form bonds with the cationic head groups and may thus exist both at the micelle water interface and in the micellar core. Micellar solubilization of naphthalene and pyrene was found to be affected by the charge on the hydrophilic group, the hydrophobic chain length, and the geometry of micelles. For comparable hydrophobic chain lengths, nonionic surfactants typically showed higher solubilization. However, pyrene solubilization in ionic surfactants, such as sodium dodecyl sulphate , cetyltrimethyl ammonium bromide  and dodecylethyldimethyl ammonium bromide , was found to be enhanced in the presence of naphthalene [Surfactant type not only affects the mechanism through which bioavailability is enhanced but also affects the structure of the micelles and the solubilization of components within micelles . The effhthalene . Such chBurkholderia sp., Lanzon and Brown [Burkholderia multivorans [However, enhancement in solubilization in the presence of surfactants does not always lead to enhanced biodegradation. Some surfactants trap oil/hydrocarbons in the hydrophobic core of the micelles and make them unavailable to the microorganism , 42. Thend Brown  illustrativorans , althougtivorans . Enterobacter sp., Pseudomonas sp., and Stenotrophomonas sp., where the nonionic surfactants used were Tween 80, Triton X-100, and Tergitol NP-10. The increased degradation rates were attributed to micellar solubilization. Surfactant dose impacts the mode of action of surfactants and affects biodegradation. At high dose, surfactants may adversely affect the microorganisms responsible for the degradation of petroleum hydrocarbons and oil due to their inherent toxicity. Moreover, higher dose also translates to higher cost. The administration of appropriate surfactant dose is essential for effective bioremediation. Surfactants with lower CMC value are preferred for bioremediation since solubilization is effective only for surfactant concentration above the CMC. Surfactant concentration beyond the CMC is linearly related to increase in degradation up to a concentration that is toxic to the microorganism. Enhanced degradation with increasing surfactant dose above CMC due to micellar solubilization was demonstrated during the degradation of phenanthrene and pyrene using nonionic and anionic surfactants, Tween 20, Tween 80, Triton X-100, and SDS . Tween 8Burkholderia multivorans (NG1) as illustrated in Burkholderia cepacia (ES1) in the presence of Triton X-100. They reported that the rate of degradation of a model NAPL (NAPL A2) was faster as the surfactant concentration was increased from 0 to 5 CMC. Irrespective of the mechanism of action, increase in surfactant dose was found to increase the degradation rate of model NAPL A1 (0.1%) by Pseudomonas putida ATCC strain 17514. In general, low surfactant concentrations stimulated the degradation of PAHs even for surfactants such as Tween 20 that could be degraded by the culture. However, SDS had different impacts on fluoranthene and anthracene degradation such that it decreased fluoranthene degradation but increased anthracene degradation. CTAB had a detrimental effect on the degradation of both of the PAHs and also hindered culture growth. While micellar solubilization is the predominant phenomenon at the surfactant concentration above CMC, low surfactant concentration may also impact SEB by affecting the microorganism-substrate interaction. The role of surfactant type on such interactions was investigated by Rodrigues et al.  using TwMycobacterium spp. KR2 in the presence of low surfactant concentration less than 40 mg/L. The nonionic surfactants used were Tween 80, polyoxyethylene  sorbitan monooleate (Brij 30), POE (10) sorbitan monooleate (10LE), and POE (23) sorbitan monooleate (Brij 35). The anionic and cationic surfactants, linear alkyl benzene sulphonate (LAS), and tetradecyl trimethyl ammonium bromide (TDTMA) were also used. The presence of surfactants decreased the mineralization of phenanthrene and caused the release of phenanthrene intermediates in the form of volatile organic compounds (VOCs), although, culture growth was slightly enhanced due to preferential utilization of the surfactants as growth substrate when present at low concentration. In contrast, Jin et al.  reportedIn subsurface contamination scenarios, surfactant sorption also needs to be considered. Surfactants that depict lower sorption would be more effective in enhancing the mobility of the oil trapped in soil pores and in enhancing the bioavailability of oil. Anionic and nonionic surfactants typically depict lower sorption to mineral surfaces compared to cationic surfactants. Recent studies have also illustrated good potential for SEB using mixed surfactants, where the effectiveness can be enhanced at lower surfactant concentration. Zhu and Feng  found syBurkholderia multivorans (NG1) in the presence of the three synthetic surfactants Triton X-100, Igepal CA-630, and Tween 80 and exogenously added biosurfacatant JBR-515 during the degradation of model NAPL A1 and model NAPL A2 [Burkholderia multivorans (NG1), was enhanced in the presence of Triton X-100 but not in the presence of the biosurfactant during the degradation of model NAPL A1 [Burkholderia cepacia [It is imperative to conduct studies to decipher the surfactant- microorganism interaction which is unique in every scenario. The surfactant-microorganism interaction may directly impact the substrate uptake mechanism. As discussed in preceding sections, the direct uptake of hydrocarbons is favored by increase in cell surface hydrophobicity of the microorganism. Surfactants are reported to alter the cell surface hydrophobicity of microorganisms and affect the direct uptake of substrate from a NAPL or a solid phase.  NAPL A2 , 12, 44. cepacia . NAPL coBurkholderia multivorans (NG1) was less negative in the presence of surfactant Triton X-100 during the degradation of both NAPL A1 and NAPL A2  surfactants, Lutensol GD 70, and Glucopon 215, derived from natural and renewable sources, have been reported to facilitate the biodegradation of diesel oil in Achromobacter denitrificans by facilitating greater adherence through cell surface changes as measured in the BATH assay [Stenotrophomonas maltophilia. In another study by Rodrigues et al. [Pseudomonas putida so as to alter the cell surface charge and the tendency to adhere to n-hexadecane in BATH assay. SDS and CTAB both lowered the cell surface charge significantly such that agglomeration of cells was observed, whereas Tween 20 enhanced the cell surface charge. None of the surfactants caused adherence of the cells to n-hexadecane water interface in the BATH assay. However, most of the surfactants enhanced the degradation of PAHs possibly by inducing cell surface hydrophobicity. It may be emphasized that the %adherence measured in the BATH assay is not a true measure of hydrophobicity, since this assay is strongly affected by the solution phase interactions and the choice of NAPL used in the BATH assay [The influence of surfactant-microorganism interaction is also manifested through changes in zeta potential which reflects the cell surface charge. Mohanty and Mukherji  observed NAPL A2 . The decsurfaces . In a stTH assay . Enhances et al. , Tween 2TH assay , 77. AdhRhodococcus sp. F9-D79 and Pseudomonas sp. JA5-B45, degrading crude oil by direct uptake mechanism and uptake after solubilization, respectively, the Rhodococcus sp. primarily contributed to crude oil degradation in the absence of surfactants. In contrast, in presence of the surfactant, Igepal CO-630,the Pseudomonas sp. played a greater role in the degradation of aromatics in crude oil as Igepal CO-630 hindered the attachment of Rhodococcus sp. to the oil water interface. The change in hydrophobicity/surface charge that occurs in the presence of surfactants may thus depend on the inherent cell surface characteristics of the bacteria and its uptake mechanism, nature of the surfactant, and the manner in which it predominantly sorbs, that is, as monomers or as hemimicelles. Such changes have a strong influence on substrate uptake and the success of SEB. Some studies have shown that cultures with hydrophobic cell surfaces that typically tend to adhere to oil/hydrocarbons are adversely affected by the addition of surfactants , 78. TheAs previously discussed, the addition of surfactants to a contaminated site adds to pollution due to the surfactant itself. Thus, based on environmental sustainability considerations use of biodegradable surfactants for SEB may be preferable over those that are recalcitrant and persistent. However, the solubilization capacity and effects of surfactants on the biodegradation of hydrocarbons need to be a key consideration governing their choice. Surfactant biodegradation is associated with various positive and negative implications. As discussed in the preceding sections, surfactants facilitate the utilization of petroleum hydrocarbons at higher rates by enhancing their bioavailability. Thus, degradation of the surfactants may hinder the uptake of petroleum hydrocarbons. In terms of their biodegradability and toxicity, surfactants may be classified as follows: readily degradable ; hardly degradable, yet not inhibiting the degradation of other carbon sources; and posing toxicity to the microorganism and inhibiting its growth. The various key factors that influence the degradability are surfactant properties, degradation capability of the microorganisms, and environmental conditions. Surfactant properties include the charge , structural complexity (simple chain or with polymeric structures), and source  . The biodegradation of surfactants results from the ability of the microorganisms to catabolically assimilate the surfactant as carbon and energy source. As the structural stability of the micelles is destroyed, the hydrocarbon present in the micellar core may be released. Lee et al.  reportedPositive effects of surfactant degradability include the removal of the surfactants from the polluted site and enhancement in the uptake of hydrocarbons. Sometimes, degradable surfactants serve as a primary substrate while the pollutant is degraded cometabolically . In yet Sphingomonas paucimobilis, which was unable to utilize the phenanthrene solubilized within surfactant micelles. Destabilization of the micelles caused release of phenanthrene into the aqueous phase which could subsequently be utilized by the culture. Sometimes, bioavailability of the primary substrate can be improved with biodegradation of the surfactant due to greater release of hydrocarbons from the micellar phase into the aqueous phase, making the substrates more readily available to microorganisms. The release of hydrocarbons from the micellar phase is often found to be a limiting factor when nonbiodegradable surfactants are used for SEB. A biodegradable surfactant was found to stimulate the direct uptake of n-decane and n-tetradecane along with the uptake of micelle solubilized hydrocarbons . Kim andAlthough surfactant biodegradation has been reported, they are often only partially transformed and these transformation products tend to accumulate in the environment. Chemical surfactants are more resistant to degradation compared to biosurfactants that are readily biodegradable . NumerouPseudomonas aeruginosa, Bacillus subtilis, and a microbial consortium obtained from municipal solid waste compost. It was observed that the cationic surfactant CTAB was toxic to the microorganisms, so neither glucose nor the surfactant was degraded. Triton X-100 was nontoxic; however, it was not degraded due to its structural complexity. The anionic surfactant SDS was easily degraded by all three microbial strains. The degradation of SDS is also reported in various other studies [Bacillus subtilis culture but not by the Pseudomonas strain producing it. The compost microorganisms showed much higher degradation efficiency due to the diversity of microbial species compared to Bacillus sp., which showed low degradation efficiency and a long lag phase.Zeng et al.  compared studies . The bioVibrio cyclotrophicus sp. Under aerobic condition, rhamnolipid and Triton X-100 depicted soluble COD removal efficiency of 74% and 47.1%, respectively, after 10 days. Under anaerobic, sulphate reducing and nitrate reducing conditions, COD removal efficiencies after 6 days were 47.2, 34.2, and 24.6% for rhamnolipids. The degradation of Triton X-100 was inhibited under these conditions. Mohan et al.  conducteVibrio fischeri. The latter studies have attempted to quantify the acute toxicity of surfactants based on effective concentration causing 50% inhibition in light output (EC50). This test provides a measure of nonspecific toxicity. Toxicity is found to increase with the increase in hydrophobicity which is commonly indicated by the octanol water partition coefficient, Kow [Surfactant toxicity is an important aspect which may adversely affect the SEB of oil and petroleum hydrocarbons. Surfactants applied at high concentration are often found to adversely affect the microbial growth on rich media or in the presence of easily degradable substrate. Surfactants added to resting cells at a high concentration have also been reported to cause a decrease in oxygen uptake rate. The concentration at which these adverse effects are manifested depends on the structure of the surfactant used and the nature of the microorganism. In contrast, growth inhibition and reduced substrate and oxygen uptake rate in the presence of oil and petroleum hydrocarbons are not necessarily a manifestation of toxicity since they may result from reduced bioavailability of the substrate. The toxicity of any surfactant is related to its capacity to adsorb and penetrate through the bacterial cell membrane . Surfactnt, Kow . HoweverThe ionic/nonionic nature of the hydrophilic head group on a surfactant affects its toxicity. Typically, nonionic surfactants are found to be less toxic to bacteria than ionic surfactants , 90–92. In studies with branched and linear ethoxylated alkylphenols (such as NPEOs and OPEOs), Hall et al.  reportedFor nonionic surfactants with the same head group and similar structure, the alkyl chain length of the hydrophobic moiety is reported to affect toxicity. For alkylpolyglucosides (APG), the surfactant having the longest alkyl chain length was found to exhibit the highest toxicity –97. In c50 was found to be dependent on the hydrophobic alkyl chain length, HLB and CMC of the surfactant such that toxicity increased as the alkyl chain length increased and HLB decreased. The toxicity of APGs increased as the CMC decreased. For fatty-alcohol ethoxylates, increasing the alkyl chain length lowered EC50 and increased toxicity, whereas increasing ethoxylation increased the HLB and lowered the toxicity. Surfactants with similar chemical structure varying in terms of their HLB value are reported to differ in terms of their toxicity to bacteria. It may be noted that HLB may increase due to the increase in hydrophilic chain length or due to the decrease in the hydrophobic chain length. The increase in HLB is thus expected to reduce nonspecific toxicity due to the reduction in hydrophobicity which inhibits its entrance into the lipid bilayer of the cell membrane , 98. DurKow, hydrophobic chain length, HLB, and CMC) using a freshwater shrimp and rotifer species. However, tests based on bacteria only reveal nonspecific toxicity. Specific toxicity of surfactants may be observed through toxicity tests based on organisms, such as algae, rotifers, fish, shrimp, and snails. Toxicity of surfactants, when tested on different aquatic organisms show variable LC50 values depending on the sensitivity of the test species [β-estradiol by interacting with the estrogen receptor. These transformation products are quite recalcitrant and tend to accumulate in aquatic sediments where they exert toxic effects towards plants and animals. Most of the relationships between toxicity and structural characteristics of the nonionic surfactants revealed through tests based on bacteria are also found to be valid for higher organisms. Uppgård et al.  confirme species , 94, 101 species . NonylphThe introduction of surfactants into oil contaminated soil and aquatic environments may add to pollution through the accumulation of petroleum hydrocarbon degradation intermediates and partial biotransformation products of the surfactants. This may pose a threat to aquatic and terrestrial plants and animals. These ecotoxicological implications need to be considered for successful application of SEB. Complete removal of oil rarely occurs. Some components in oil are only partially transformed such that accumulation of intermediates may occur. As surfactants enhance the bioavailability, some components that are not inherently degradable by microorganisms may get partially transformed. Such partial transformation products that tend to accumulate are often more toxic than the parent compound. The intermediates are often acidic in nature and tend to cause pH drop in the system . This maThe application of surfactants in oil contamination scenarios may selectively facilitate the growth of specific microorganisms which may eventually hinder oil degradation . The addSince surfactants pose a secondary source of pollution, there is much concern over the fate of surfactants in the environment once the site is remediated with respect to petroleum hydrocarbons. If the surfactants are eventually completely degraded by naturally occurring microorganisms, the surfactant aided remediation technology may be considered more sustainable. The fate of surfactants in the environment is dependent on their chemical structure and is controlled by the sorption and biodegradation processes. Cationic and nonionic surfactants have much higher sorption on soil and sediment than anionic surfactants. Various factors such as physiochemical properties of the surfactants, surfactant concentration, the nature of the sediments, and environmental parameters influence the sorption of surfactants onto sediment/soil . SurfactBioconcentration of the surfactants and their degraded products in organisms pose another environmental risk. Bioconcentration increases with increase in hydrophobicity of the surfactants. For the highly hydrophobic long chain LAS homologues, the bioconcentration factors (BCFs) in rainbow trouts and fathead minnows are found to be in the range of 1.4–372 L/kg and 6–990 L/kg, respectively. Bioconcentration is more evident in aquatic ecosystems rather than in terrestrial ecosystems. Alkylphenols, such as NP and OP, which are common degradation products of alkylphenol ethoxylates, also tend to bioaccumulate in the aquatic environment due to their hydrophobic nature. Due to bioaccumulation, chronic toxic effects are eventually manifested in various organisms. Chronic toxicity of surfactants may be manifested at concentrations greater than 0.1 mg/L in the aquatic environment . Based on the above observations reported in the literature, it is evident that surfactant-enhanced degradation of oil is a complex process. The success/failure of SEB depends on numerous factors including the choice of surfactant and its dose to be applied at the contaminated site, the hydrocarbon degrading microorganisms present in the environment and their response to oil/hydrocarbons, the interaction of the hydrocarbon degraders with the surfactants, and surfactant biodegradability and toxicity considerations. The structure of the surfactant, its HLB value, and its dose will affect the distribution of oil in the system through emulsification or micellar solubilization. The surfactant dose selected is expected to play a pivotal role in the success of the whole process. The administration of high dose may have a detrimental effect on hydrocarbon mineralization due to the toxicity of the surfactant to the hydrocarbon degrading microorganisms or due to the reduced bioavailability of the hydrocarbons solubilized within surfactant micelles. In spite of micellar solubilization and emulsification, the biodegradation of oil may be adversely affected if the microbial cell surface changes in the presence of surfactants are unfavorable. Although the choice of a biodegradable surfactant is not necessarily beneficial for petroleum hydrocarbon degradation, a surfactant that is eventually completely biodegraded will cause less ecotoxicological concerns. A surfactant that degrades only partially may not only have adverse effect on petroleum hydrocarbon degradation due to reduction in surface activity, but may also cause accumulation of recalcitrant intermediates in the environment. The adverse ecotoxicological implications of such a choice may make the surfactant aided biodegradation unsustainable. Moreover, the application of surfactants should be economically affordable. The successful implementation of SEB is thus very challenging and depends on all of the above parameters discussed. Due to the multiple challenges associated with this technology, field trials are strongly recommended before the direct application of the process on a large scale.


Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes.Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont.The zoonotic Bartonella species between animals and might also transmit Bartonella spp. to humans.We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated  Bartonella species are facultative intracellular Gram-negative bacteria which can infect humans and a wide range of animal species. Cat-scratch disease (CSD) is probably the most common Bartonella infection in the northern hemisphere [Bartonella spp. are an important cause for blood-culture negative endocarditis [Bartonella henselae, with a confirmed link to disease in humans [B. henselae bacteremia [Ctenocephalides felis, are involved in transmission between cats and may be also able to transmit B. henselae to humans: Approximately 30% of patients with CSD do not recall traumatic cat contact [Bartonella species, including Bartonella clarridgeiae and Bartonella grahamii, have also been linked to human disease. However, there are only few reports of disease cases linked to these species [misphere ,2. The hmisphere . Regionamisphere . Thirty misphere ,5. Extramisphere ,5-8. Furcarditis -11. Disecarditis . The domn humans ,14. Infen humans . In a Ducteremia . Bites o contact -19. Soli species -23.Bartonella species [B. henselae DNA in various Ixodid tick species [B. henselae can be transmitted across the developmental stages of Ixodes ricinus [Bartonellaceae, particularly B. henselae, are present in I. ricinus ticks in the Netherlands and form a risk to public health.The number of cases with tick-borne diseases in the Netherlands is on the rise : This is species -27. Seve species ,28-32. A ricinus . Altoget ricinus . One of Rickettsiae. They are fastidious, mostly obligate intracellular alpha-proteobacteria. Hard ticks (Ixodidae) have been identified as vectors of the spotted fever syndrome in humans, which is caused by at least 15 different Rickettsia species [Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, and Rickettsia conorii, the causative agent of Mediterranean spotted fever [Rickettsia helvetica is the most prevalent rickettsial species found in I. ricinus ticks in the Netherlands [R. helvetica is unclear but infection with R. helvetica has been suspected in acute perimyocarditis, unexplained febrile illness, sarcoidosis and recently also meningitis [R. rickettsii and/or R. conorii, and serological cross-reaction with other rickettsial pathogens are common. Notably, some patients suspected to have (suffered from) a (mild) rickettsiosis do not recall a tick bite. In these cases, other potential sources of infection, for example cat fleas, might be involved. Cat fleas may maintain and transmit Rickettsia felis which is the causative agent of flea-borne spotted fever, also called cat flea typhus or summer flu [R. felis appears to have a global distribution [R. felis is also present in the Netherlands. Nevertheless, evidence for the presence of R. felis in Dutch cat fleas is lacking and no autochthonous clinical cases of flea-borne rickettsisos have been reported. In this study, we investigated whether R. felis is present in Dutch cat fleas.Another class of pathogens that can be transmitted by both Ixodid ticks and cat fleas is  species . Two infed fever . Rickettherlands ,38. To dningitis -47. Labommer flu . Clinicammer flu . Patientmmer flu -54. R. fribution , and it Nymphs and adult ticks were collected for recent studies between 2006 and 2010 by flagging vegetation at 16 different locations in The Netherland (Table Ticks from cats were collected by veterinarians between 2006 and 2009 as described . More thLipoptena cervi) and attached ticks were collected from red deer by hunters.Deer keds  were collected from wood mice caught in Duin and Kruidberg area .Myodes glareolus) and wood mouse (Apodemus sylvaticus) were derived from a previous study [Fleas from bank voles . Briefly, the gltA gene was amplified using 5'-GGGGACCAGCTCAtGGTGG and 5'-AATGCAAAAAGAACAGTAAACA as primers, yielding amplification products of approximately 380 base pairs. A serial dilution of the cultivated B. henselae ATCC 49882 strain was used as positive control [Nymphal and adult vegetation ticks, larvae from Vrouwenpolder, ticks collected from cats and deer, deer keds, rodent tissue samples, fleas collected from rodents and pet animals were tested for the presence of  control . The hig control .Rickettsia spp. DNA by PCR followed by reverse line blotting (RLB) as previously described [R. conorii), 5'-GCTAATACCATATATTCTCTATG (R. helvetica), 5'-GTATATTCTCTACGGAAAAAAG (Rickettsia sp. IRS3), and 5'-TATATTCTCTACAGAGGAAAGATT (R. felis). In order to detect potential double infections of ticks with R. helvetica and other rickettsial species, two RLB probes which were able to hybridize to DNA of most Rickettsia species except for R. helvetica: 5'-AATACCGTATATTCTCTACGGA (NonHelv1) and 5'-AATACCGTATATTCTCTGCGGA (NonHelv2). Plasmids containing a 16S rRNA sequence from R. helvetica, R.conorii, R. typhi or R. prowazekii were used as positive controls.Tickets collected from cats as well as fleas from dogs and cats were tested for the presence of escribed  but withAmplification of parts of the citrate synthase gene was done as described by De Sousa and colleagues using cs409d/rp1258n and cs535d/rp1258n as primer pairs .Ca. Midichloria mitochondrii" was amplified from tick lysates of larvae collected in Vrouwenpolder as described [Part of the 16S rRNA gene of "escribed , using 5Rickettsia sp., which were analyzed by RLB, all PCR products were separated and analyzed by TAE agarose gel-electrophoresis. To minimize cross contamination and false-positive results, positive and negative controls were included in each batch tested for Bartonella or Rickettsia by the PCR. In addition, DNA extraction, PCR mix preparation, sample addition, and PCR analysis were performed in separated, dedicated labs.With the exception of the 16S rRNA PCR done for Bartonella henselae in the Dutch tick population 1527 Ixodes ricinus nymphal and adult ticks from 16 geographically different locations were collected from recent field studies  to an uncultured Bartonella-related sp. isolate from I. scapularis (Genbank accession number EF662054). Neither B. henselae nor any other Bartonella species were detected in any of the lysates of these questing ticks.Vegetation types of most sampling areas have been described previously with the exception of Vrouwenpolder. Vrouwenpolder is a vegetation rich dune area with several species of deciduous trees and shrubs and approximately 70% of the soil was covered with vegetation litter. Only very scarce and incomplete information is available about the fauna of all locations. Nymphs (98%) and adults (2%) were tested for I. ricinus larvae from Vrouwenpolder were subjected to the same PCR against the gltA-gene. 41 larvae were positive and their sequences were identical to the Bartonella-related sp. from nymphs and adults. Under normal conditions, questing larvae have not yet had a blood meal. Transstadial but not transovarial  transmission of Bartonella has been described in ticks [Bartonella-related sp. in larvae is directly derived from a vertebrate host. The sequences of the Bartonella-related sp. might be derived from another microorganism present in ticks, which is capable of vertical transmission. This is supported by the fact that gltA-gene of this Bartonella-related sp. does not cluster with other Bartonella species . An additional, independent PCR using generic primers on the 16S rRNA of Bartonella species was performed [Bartonella isolated from the gut of Apis mellifera mellifera (EU055544), Phyllobacterium myrsinacearum (HQ380017) to a presumed Bartonella grahamii isolated from Apodemus agrarius (AB529498). As so many different microorganisms had similar homology, we concluded that this part of 16S rRNA gene is not suitable for detection and identification of Bartonella species.Next, lysates from 192 questing in ticks . Therefohondrii" . 56 tickerformed  on the sB. henselae, adult ticks may acquire this pathogen while feeding on cats. 265 adult I. ricinus ticks removed from domestic cats were tested by PCR. B. henselae could not be detected in any of these tick lysates. In 56 lysates a fragment of approximately 380 bp was observed. Sequencing revealed that these samples contained the same Bartonella-related sp. as found in questing ticks  yielded a 380 bp on the agarose gels. Sequencing revealed that at least 26 ticks contained DNA identical to B. schoenbuchensis (AJ564632), and 13 ticks contained the Bartonella-related sp. Sequencing was not successful in all cases, probably also because of mixed sequences of both Bartonella-related sp and B. schoenbuchensis (not shown). As B. schoenbuchensis was never detected in questing ticks  have been suggested to transmit B. schoenbuchensis between red deer as they have shown to be infected with B. schoenbuchensis [B. schoenbuchensis (AJ564632).The absence of herlands , where mensis AJ5632, and Bartonella-related sp. from questing ticks was further investigated. One of the most prominent vertebrate hosts of I. ricinus are probably rodents. To determine whether mice are a potential reservoir of this Bartonella-related sp., 96 DNA samples from ear tissue of small rodents, Myodes glareolus and A. sylvaticus, from the Duin en Kruidberg area were tested by PCR and sequencing a 380 bp part of the gltA gene. Twelve rodent samples were positive, but sequencing revealed that these sequences (n = 5) were 98-99% similar to Bartonella isolates from A. flavicollis (yellow-necked mouse) from Slovenia  and Greece . The Bartonella sequences from these rodents were only 70% similar to the Bartonella-related sp. from questing ticks  were positive for B. henselae. Three pools were positive for B. clarridgeiae and 43 pools for R. felis  DNA of R. felis was detected. No Bartonella sp. was detected in dog fleas. One third of the sampled dogs (n = 44) and cats (n = 109) carried fleas positive for R. felis, and one sixth of the animals had fleas positive for B. henselae . Therefore the risk of contracting bartonellosis from I. ricinus ticks in the Netherlands seems to be very low.In this study, we have identified several henselae . None ofI. ricinus larvae were subjected to PCR using generic primers on the 16S rRNA of Bartonella species [Bartonella isolated from the gut of Apis mellifera mellifera, Phyllobacterium myrsinacearum to a presumed Bartonella grahamii. This high variety of hits indicates that the 16S rRNA gene has too little resolution power to positively identify a Bartonella species or to distinguish Bartonella from other, closely related genera. Furthermore, results of a BLAST depend on the quality of sequences in the database. In an earlier publication we discussed the pitfalls of using a database with only verified sequence types which neglects large amounts of less well verified but valuable data [Bartonella sp., Rickettsia sp. and "Ca. Midichloria mitochondrii" are very difficult to culture and some species have not yet been cultured at all. For uncultured microorganisms often only a limited number of genes are known and based on the limited data available it can be difficult to identify a microorganism to genus level. This can lead to misnaming of database entries which in turn can lead to further misidentifications [Questing  species . Five ofble data . Using aications .Bartonella transmission, other ectoparasites, especially fleas, might be involved in the enzootic cycle of Bartonella in animals. B. henselae and B. clarridgeiae were found in cat fleas, and B. grahamii and another rodent-related Bartonella species were found in fleas collected from rodents, and B. schoenbuchensis in deer keds. All these ectoparasites could transmit Bartonella between animals and maybe also from animals to humans. B. henselae is well known to be associated to human cases of bartonellosis in the form of cat scratch disease [B. henselae to humans via ticks has not been proven and the risk of transmission by tick is probably negligible [B. clarridgeiae has also been frequently reported from cats and their ectoparasites [B. clarridgeiae transmission but the route of transmission has not yet been established.While ticks seem to play a negligible role in  disease ,8. It is disease . To dategligible . B. clararasites ,72 but harasites ,22. Cat B. schoenbuchensis has been hypothesized to be involved in the development of deer ked dermatitis, a sometimes long-lasting skin condition following bites of deer keds [B. schoenbuchensis. In the general population exposure to B. henselae is much more likely than exposure to any of the other discussed Bartonella species. It is therefore also likely to cause most bartonellosis cases. Further research is necessary to determine the risk of zoonotic transmission of different Bartonella species upon a bite of cat fleas and other arthropods.eer keds . While tI. ricinus ticks is negligible while other arthropods, including deer keds and fleas, can potentially transmit Bartonella sp. to humans. Earlier reports of high infection rates of questing ticks with a Bartonella species might in fact be due to the misidentification of "Ca. Midichloria mitochondrii" as Bartonella sp..Risk of human bartonellosis transmitted by The authors declare that they have no competing interests.FJ, WT, JRR, HS and PAMO were involved in study design. FG, EKEH, AMN and PO collected ticks and fleas, contributed to laboratory analyses and analyzed data. ETK and MF developed new methodology, performed laboratory analyses and analyzed data. HS acquired funding, was involved in data analyses and wrote the initial draft. ETK performed phylogenetic analyses and wrote the final draft. All authors were involved in completing the manuscript and approved the final version


Bartonella species comprise a group of zoonotic pathogens that are usually acquired by vector transmission or by animal bites or scratches.Bartonella 16S-23S intergenic spacer (ITS) region was used in conjunction with BAPGM  enrichment blood culture to determine the infection status of the family members and to amplify DNA from spiders and woodlice. Antibody titers to B. vinsonii subsp. berkhoffii (Bvb) genotypes I-III, B. henselae (Bh) and B. koehlerae (Bk) were determined using an IFA test. Management of the medical problems reported by these patients was provided by their respective physicians.PCR targeting the Bartonella henselae seroreactive and B. henselae DNA was amplified and sequenced from blood, serum or Bartonella alpha-proteobacteria (BAPGM) enrichment blood cultures from the mother and oldest son. Also, B. henselae DNA was PCR amplified and sequenced from a woodlouse and from woodlouse hunter spiders collected adjacent to the family’s home.In this investigation, immediately prior to the onset of symptoms two children in a family experienced puncture-like skin lesions after exposure to and presumptive bites from woodlouse hunter spiders. Shortly thereafter, the mother and both children developed hive-like lesions. Over the ensuing months, the youngest son was diagnosed with Guillain-Barre (GBS) syndrome followed by Chronic Inflammatory Demyelinating Polyradiculoneuropathy (CIDP). The older son developed intermittent disorientation and irritability, and the mother experienced fatigue, headaches, joint pain and memory loss. When tested approximately three years after the woodlouse hunter spider infestation, all three family members were B. henselae infections were acquired by spider bites or whether the spiders and woodlice were merely accidental hosts, physicians should consider the possibility that B. henselae represents an antecedent infection for GBS, CIDP, and non-specific neurocognitive abnormalities.Although it was not possible to determine whether the family’s  Bartonella is composed of fastidious, Gram-negative and aerobic bacilli belonging to the Alpha proteobacteria group. Bartonella species (spp.) are hemotropic, arthropod-borne bacteria that cause long-term bacteremia in mammalian reservoir hosts [Bartonella species that have been discovered among diverse animal reservoir hosts in geographical regions throughout the world. Since 1990, over thirty Bartonella species and sub-species have been characterized and named, with many other putative species yet to be described. Globally, these bacteria reside in diverse ecological niches; many cause persistent intravascular infection in reservoir hosts and 17 Bartonella spp. have been associated with an expanding spectrum of human and animal diseases, ranging from acute febrile illnesses to more severe disease manifestations, including encephalopathy, endocarditis, myocarditis, sensory and motor neuropathies, pleural and pericardial effusion, pneumonia, granulomatous hepatitis and hemolytic anemia [The genus ir hosts ,2. Durinc anemia -7.Bartonella spp. consists of one or more reservoir hosts and one or more transmission competent arthropod vectors. A vertebrate, generally a mammal, sustains a chronic intravascular infection, which in some instances is associated with a relapsing pattern of bacteremia. The persistently infected host serves as the blood reservoir for perpetuation of the transmission cycle, with an arthropod vector transferring the bacteria from the reservoir host to a susceptible uninfected host [Bartonella spp. are arthropods. The vector for B. quintana is the body louse  and for B. bacilliformis is the sandfly (Lutzomyia verrucarum). Fleas  play a major role in the natural transmission cycle for many bartonellae, especially B. henselae among pets and wildlife [Bartonella species. Genetic diversity and bacterial strain variability appear to enhance the ability of Bartonella spp. to infect not only specific reservoir hosts, but also accidental hosts, as has been shown for B. henselae[The natural history for seemingly all ted host . Most vewildlife ,10. Ther henselae.Bartonella research study. Three family members were B. henselae seroreactive and B. henselae DNA was amplified and sequenced from the mother and older son’s blood, and from a woodlouse and woodlouse hunter spiders.Because neurological abnormalities developed in both children after woodlouse hunter spider bites were suspected by their parents, a family from Kentucky was directed to our laboratory for inclusion in a Prior to moving to a new apartment housing location in suburban Louisville, Kentucky on May 1, 2008, all four family members were healthy and had normal sleep patterns. Two months earlier, while in a previous apartment, a bat was removed by an exterminator. Although the bat was flying free within the apartment when the family awakened, there was no indication of bite wounds. The family’s dog had also experienced a flea infestation prior to moving to the new apartment. The family dog was the only pet, there was no history of family members experiencing bites or scratches and no flea infestations were reported after moving into the new apartment or subsequently to a new house in the same neighborhood.Dysdera crocata) in the apartment, including in the children’s beds and on the children. During August 2008, the parents suspected that both sons  were bitten by woodlice hunter spiders. The mother (41 years-old) did not knowingly experience any spider bites. After the apartment was treated by an exterminator, no woodlice or woodlice hunter spiders were observed in or around the apartment. Subsequently, the mother and both sons developed recurrent rash-like skin lesions, disruptive sleep patterns and both boys developed anxiety accompanied by episodes of inconsolable crying, irritability, and panic attacks. In July 2009, the oldest son was examined by a surgeon because of enlarged lymph nodes in the neck. Over the ensuing months, the mother developed symptoms, including fatigue, headaches, joint pain, eye pain, insomnia, memory loss, disorientation, irritability, weakness in the upper extremities and loss of sensation to both legs. In May 2010, as detailed in the case report below, the youngest son was diagnosed with Guillain-Barre syndrome (GBS), and subsequently with Chronic Inflammatory Demyelinating Polyneuropathy (CIDP).In July 2008, their new apartment flooded, after which there was a large influx of woodlice (order Isopoda). Subsequently, the mother reported seeing occasional wood louse hunter spiders . Beginning in August 2011, blood and serum samples from the mother, both sons and the dog were submitted for Bartonella testing. As the parent’s initial concerns related to the youngest son’s CIDP diagnosis, this child was tested in August 2011, followed by the mother and dog in November, and the oldest son in April 2012. The father did not recall being bitten by a spider himself, remained healthy during the course of this investigation and was never tested for evidence of Bartonella sp. infection. Over a one-year period (2011–2012), spiders, identified as wood louse hunter spiders (Dysdera crocata), and several woodlice (Isopoda order) collected from around the family’s new house (located three miles from the spider infested apartment) were submitted by express mail for manual DNA extraction and Bartonella PCR.After contacting the corresponding author and describing the family's medical history, the mother elected to enter her sons and herself into an ongoing study regarding Bartonella testing. Collection and analyses of these data were conducted in conjunction with North Carolina State University Institutional Review Board approval (IRB no. 1960-11).Aseptically obtained EDTA (ethylene diaminetetraacetic acid)-anti-coagulated blood and serum samples from the family, their dog, whole spiders and woodlice collected from around the apartment in Kentucky, were submitted to the North Carolina State University College of Veterinary Medicine Intracellular Pathogens Research Laboratory (NCSU-CVM-IPRL) for Bartonella vinsonii subsp. berkhoffii genotypes I, II, III, B. henselae (Houston 1strain), B. henselae (San Antonio 2 strain), and Bartonella koehlerae antibodies were determined in the Intracellular Pathogens Research Laboratory (IPRL) following traditional immunofluorescence antibody assay (IFA) practices with fluorescein conjugated goat anti-human IgG (Pierce Biotechnology Rockford IL), as described in previous studies from our laboratory [Bartonella organisms of feline isolates of B.koehlerae (NCSU 09FO-01) and B.henselae H-1 (NCSU 93FO-23), B. henselae SA2 (NCSU 95FO-099) and canine isolates of B.vinsonii berkhoffii genotype I (NCSU 93CO-01), II (NCSU 95CO-08) and III (NCSU 06CO-01) were passed from agar grown cultures into cell cultures to obtain antigens for IFA testing Heavily infected cell cultures were spotted onto 30-well Teflon coated slides (Cel-Line/Thermo Scientific), air dried, acetone fixed and stored frozen. Serum samples were diluted in phosphate buffered saline (PBS) solution containing normal goat serum, Tween-20 and powdered nonfat dry milk to block non-specific antigen binding sites. Sera were screened at dilutions of 1:16 to 1:8192. To avoid confusion with possible non-specific binding found at low dilutions and to standardize with other laboratories such as the CDC, a cutoff titer of 1:64 was used to define a seroreactive titer.boratory -14. BartBartonella spp. DNA from blood, serum and enrichment BAPGM  enrichment blood culture was used to test EDTA-anti-coagulated whole blood and centrifuged serum samples [2O followed by a single wash with 95% ethanol. For DNA extraction, the entire body of each arthropod was pulverized to a fine powder by bead-beating using stainless steel beads. DNA from spiders and woodlice were manually extracted using DNeasy blood and tissue mini kit following manufacturer’s instructions . Bartonella DNA was amplified using conventional Bartonella genus PCR primers targeting the 16S-23S intergenic spacer region (ITS) as previously described [Bartonella genus PCR was performed using oligonucleotides 425 s (5′CCGGGGAAGGTTTTCCGGTTTATCC 3′), 325 s (5′ CCTCAGATGATGATCCCAAGCCTTTTGGCG 3′) and 1000as (5′ CTGAGCTACGGCCCCTAAATCAGG 3′) as forward and reverse primers, respectively. Amplification was performed in a 25-ul final volume reaction containing 12.5 uL of MyTaq Premix (Bioline), 0.2 uL of 100 umol/L of each forward and reverse primer , 7.3 uL of molecular grade water, and 5 uL of DNA from each sample tested. Conventional PCR was performed in an Eppendorf Mastercycler EPgradient®  under the following conditions: a single cycle at 95°C for 2 s, followed by 55 cycles with DNA denaturing at 94°C for 15 s, annealing at 66°C for 15 s, and extension at 72°C for 18 s. The PCR reaction was completed by a final cycle at 72°C for 30s. All PCR reactions were analyzed by 2% agarose gel electrophoresis. Amplicons obtained from arthropod and human samples were sequenced to identify the Bartonella sp. and ITS strain type. Bacterial species and strains were defined by comparing similarities with other sequences deposited in the GenBank database using the Basic Local Alignment Search Tool (Blast version 2.0).A previously described approach that combines PCR amplification of  samples ,13,15-17escribed ,19. BartIn August 2008, the youngest son, a developmentally normal child with precocious motor skills, sustained puncture-like bite lesions in the skin overlying the mid humerus and proximal femur Figure . Based uth thoracic vertebrae through the remainder of the spinal cord. Neurological examination was significant for areflexia and weakness in the lower extremities. Guillain-Barre Syndrome was diagnosed and he was treated with 2 grams per kilogram of intravenous gammaglobulin (IVIG) over 4 days. He improved rapidly and was discharged following the infusions.In May 2010, about a month after an upper respiratory infection, the boy was unable to climb stairs. His parents brought him to an emergency room, where it was noted that he was unable to stand from a seated position on the floor. A lumbar puncture revealed an elevated CSF protein of 110  with 4 white blood cells. An MRI of the spine demonstrated enhancement of the ventral nerve roots and pial enhancement from the 11Bartonella infection. Due to this concern, treatment with azithromycin was initiated for 10 days, and it was felt that this was associated with some improvement. Bartonella henselae IgM and IgG antibodies were not detectable at a 1:16 dilution . By July 2011, despite medical therapy, pain and the right foot drop had worsened.When re-examined one month later, his leg strength was judged to be improved to 85% of normal and no further treatment was given. By July 2010, he was less able to walk without stumbling and was unable to stand from a seated position. He also complained of tingling and discomfort around his mouth and pain in the legs. IVIG was administered at a dose of 1 gram/kilogram on each of 2 days. Electromyelogram (EMG) findings were consistent with chronic sensory motor demyelinating polyneuropathy with secondary axonal features and conduction block. CIDP was diagnosed and IVIG treatments were re-instituted every 4 weeks, along with a 4 week course of prednisone and gabapentin for pain. By May 2011, following internet searches, his mother became concerned that the boy’s symptoms might be related to Bartonella testing at the NCSU-CVM-IPRL. While awaiting test results, treatment with azithromycin for 30 days was reinitiated. The family felt that they noted almost immediate improvement of the patient’s symptoms and requested that the next IVIG treatment be delayed. The boy was seroreactive to multiple Bartonella spp. antigens  was confirmed serologically and by PCR amplification and DNA sequencing. Between February and July 2012, she was treated with doxycycline 200 mg once daily and rifampin 300 mg twice a day. Following this antibiotic course, the mother reported substantial overall improvement and was almost symptom free. However, she continues to experience occasional irritability, confusion, dizziness, nausea, and pain involving the shoulder, hip and the bottoms of her feet.The mother, who takes care of her children and home full-time, was healthy prior to August 2008. She reported limited exposure to cats, wildlife or production animals, but did allow the family dog to sleep in her bed. Prior vector exposure was infrequent, but included fleas, ticks and mosquitoes. She did not recall being bitten by a spider. Subsequent to the spider infestation of the apartment, she developed fatigue, memory difficulties, headaches, irritability, eye pain, insomnia, chest pain, blurred vision, shortness of breath, rash and skin lesions and anxiety attacks. She had also experienced a loss of sensation in her legs, joint pain involving the shoulders and ankles, ear pain, and she frequently had a sore throat. In July 2009, she was examined due to an abdominal rash and shingles was tentatively diagnosed. The mother reported that her symptoms persisted between 2008–2011, without notable improvement or deterioration, during which time she sought care from her family physician, an otolaryngologist and a neurologist. Using blood and serum samples submitted in November 2011, infection with B. henselae SA2 strain was confirmed by serology and BAPGM enrichment blood culture PCR in April 2012. Treatment with clarithromycin 250 mg twice daily was instituted in May 2012. On August 29, 2012, rifampin 300 mg twice daily was added to the treatment regimen, which has continued through November, without known adverse side effects. The sore throat, ear and eye pain resolved by October; however, during the fall 2012 school year, his parents reported increased irritability and rage episodes. In addition, the boy’s teacher indicated a lack of attention during class, and suggested that the child might have an Attention Deficit Hyperactivity Disorder (ADHD). After consultation with the attending physician and a psychiatrist, the parents declined therapy for ADHD.In August 2008, the older son, was examined by his pediatrician due to suspected spider bites and a rash. This child also had occasional exposure to cats and dogs and had exposure to fleas and mosquitoes. Subsequently, the boy complained of a sore throat, occasional ear pain and pain in the thigh region. In July 2009, the boy’s parents sought medical consultation with a dentist and surgeon for a swollen lymph node in the neck that had persisted for approximately 3 months. The lymph node regressed in size without therapy. During 2009–2012, the parents indicated that the boy experienced episodes of unexplained depression, irritability, and anxiety, but was otherwise healthy. Infection with Bartonella serology and PCR results for the three family members and the dog are summarized in Table B. henselae SA2, B. henselae HI and between August 29th and August 31st, there was a four-fold or greater increase in antibody titers to B. koehlerae and to B. vinsonii subsp. berkhoffii genotypes I, II, and III. Bartonella sp. DNA was not amplified from his blood, serum or BAPGM enrichment blood cultures. The mother was seroreactive to B. henselae HI, B. henselae SA2, B. vinsonii subsp. berkhoffii genotypes II and III and B. koehlerae. Bartonella henselae bacteremia was confirmed in the mother by PCR amplification from two BAPGM enrichment blood cultures. Based upon the amplified DNA sequences, the B. henselae strain in the mother’s samples were 99.8% and 100% similar, respectively to B. henselae SA2, (GenBank accession AF369529). The oldest son was seroreactive to B. henselae SA2 and B. vinsonii subsp. berkhoffii genotype II. Based upon the DNA sequences amplified from a blood sample and BAPGM enrichment blood culture sample, the oldest son was bacteremic with a B. henselae SA2 strain (99.2% and 99.6% homology with GenBank accession AF369529). The dog was seronegative to all Bartonella spp. antigens and no Bartonella DNA was amplified from blood or the BAPGM enrichment blood culture. Following BAPGM enrichment culture, no subculture isolates were obtained from any family member. When retested in November 2012, the youngest son was only seroreactive to the B. henselae SA2 strain and Bartonella sp. DNA was not amplified from blood, serum or the BAPGM enrichment blood culture.Bartonella henselae SA2 DNA (97.0% homology with GenBank accession AF369529) was amplified and sequenced from pooled woodlice  was amplified and sequenced from two spiders and B. vinsonii subsp. berkhoffii genotype III DNA  from one spider. As the family had moved from the apartment in which the bites occurred into a house, all of the spiders and woodlice were collected approximately 3 miles from the original suspected spider bite location.B. henselae SA2 DNA from two family members, woodlouse hunter spiders, and a woodlouse collected at least three years after family members were exposed to and the children were presumably bitten by similar spiders, was unexpected. To the best of our knowledge, this is the first report of the presence of Bartonella spp. DNA in spiders or in woodlice. Although B. henselae DNA was amplified from two spiders collected 13 months apart, a woodlouse, and from serum, blood and BAPGM enrichment culture samples from two family members, these results should be interpreted with caution, as it is not clear whether Bartonella was acquired at the time of the infestation and spider bites or whether the spiders and woodlice are accidental hosts for Bartonella spp. Since the woodlouse hunter spider is thought to feed exclusively on woodlice (a land-dwelling crustacean), the amplification of Bartonella DNA from spiders and woodlice suggests that the B. henselae-infected spiders fed on infected woodlice. Preliminary results (unpublished data) obtained in our laboratory indicates that washed woodlice can become PCR-positive for B. henselae after feeding on food contaminated with the bacteria. Although the length of time that B. henselae can remain viable within the environment has not been investigated to any degree, the bacteria remains viable in flea feces for several days. Whether bacteria ingested by woodlice remain viable, whether replication can occur, how long a Bartonella sp. can be retained within the isopod and whether a spider feeding on this crustacean can acquire or transmit Bartonella are subjects for future studies. Although the family experienced a flea infestation prior to moving into the new apartment, the family dog was not seroreactive to Bartonella sp. antigens and was PCR negative in blood and BAPGM enrichment blood culture, making the dog and potentially fleas a less likely source of B. henselae transmission to family members. To date, B. henselae has not been reported in bats to the author’s knowledge, no family member experienced a bat bite, and the bat exposure occurred several months before the onset of illness in the children and mother.PCR amplification and sequencing of Bartonella DNA has also been amplified from non-hematophagous arthropods, such as honey bees [Bartonella organisms through environmental contact. In a recent report, a patient with neuroretinitis, a well documented ocular pathology induced by B. henselae, was diagnosed with bartonellosis following the bite (sting) of a bull ant (genus Myrmecia) in Australia [B. henselae was probably transmitted to the patient via the stinger or mandibles, which provided a portal for bacterial entry into the skin. These recent publications indicate that physicians should routinely review a patient’s medical history for arthropod exposure. Based upon recent clinical and research observations, there appears to be a growing spectrum of arthropods that might serve as vectors for Bartonella species, thereby emphasizing the critical importance of and the need for additional experimentally controlled vector competence studies. In addition, localizaton of Bartonella sp. replication within arthropods, further documentation of other potential animal reservoirs, and the determination of trans-ovarian transmission in various arthropod species represent other important issues that require scientific attention.ney bees . Those australia . These aBartonella sp. bacteremia in immunocompetent patients [Bartonella bacteremic children [B. henselae DNA was only amplified from the mother's extracted serum samples, whereas B. henselae DNA was amplified from both blood and a BAPGM enrichment blood culture from the oldest son [Bartonella DNA was never amplified from a negative control and DNA from a B. henselae H1 strain (not B. henselae SA2 as found in this study) was used as a positive control for all PCR testing, therefore laboratory contamination is an unlikely explanation for the PCR and DNA sequencing results reported in this study. Due to the fact that B. henselae induces a relapsing bacteremia in cats [B. birtlesii induces a relapsing bacteremia in experimentally infected rodents [B. henselae bacteremia in human patients. Also, as reported previously from our laboratory [Bartonella spp. antigens over a one week period. The youngest son had identical antibody titers to B. henselae strains H1 and SA2, but seemingly seroconverted to B. vinsonii subsp. berkhoffii genotypes I, II III and B. koehlerae. Administration of IVIG ten days prior to collection of the initial blood sample may well have influenced the youngest son’s serological results, particularly if IVIG has antibacterial properties [B. henselae SA2 strain, but not to a B.henselae H1 strain. All serum sample sets from each patient were tested at the same time, by the same experienced technician, using the same conjugate and IFA antigen slides. Whether these serological discrepancies are related to sample collection and storage issues, a prozone effect associated with excess antigen, IVIG or other unknown factors requires additional investigation.From a clinical perspective, the non-specific symptoms reported in the mother are consistent with previous reports of patients ,22. Althchildren ,23,24. Idest son . Bartone in cats  and B. b rodents , three bboratory , there woperties . In contB. henselae serology was requested. Her B. henselae IgG titer was 1:1024 and a specific IgM titer was “positive”, although a value was not reported. Her convalescent IgM titer was negative and the IgG antibody titer had decreased. To date, CIDP has not been associated with Bartonella infection. Although serology supported Bartonella exposure in the younger son, prior administration of IVIG complicates interpretation of his antibody titers and potentially his BAPGM enrichment culture PCR test results. It is possible that the source of Bartonella antibodies was the IVIG and that repeated immunoglobulin administration suppressed the level of bacteraemia below the level of successful PCR amplification. CIPD, also referred to as relapsing polyneuropathy, is a neurological disorder characterized by progressive weakness and impaired sensory function in the legs and arms. As was true in the boy in this report, CIPD is often diagnosed as the chronic counterpart of GBS. Prior infection or vaccination can precipitate GBS, and Campylobacter jejuni has become the most well recognized antecedent infection [B. henselae as an antecedent infection for GBS and CIPD. Physicians should pursue the medical history in these patients to determine if they have experienced animal bites or scratches or arthropod bites or stings.Similar to the initial diagnosis in the youngest son, GBS due to neurobartonellosis was diagnosed in a 10-year-old girl, who was hospitalized due to progressive leg weakness . Seven dnfection . ConsideBartonella, there has been enhanced focus on known and suspected arthropod vectors. Because of their ability to reside within erythrocytes of a diverse number of mammalian hosts in conjunction with their diverse ecological niches, there is the potential opportunity for various Bartonella spp. to be transmitted by a variety of arthropod vectors. Several blood-feeding arthropods, Lutzomyia verrucarum, Pediculus humanus humanus, Ctenocephalides felis and some rodent fleas  have been confirmed to be competent vectors for transmission of Bartonella species [Bartonella spp. has been a controversial subject in recent years [Ixodes ricinus) transmission of a Bartonella sp. was recently demonstrated experimentally, thus supporting the possibility that Ixodes sp. ticks are transmitting Bartonella spp. throughout the northern hemisphere [B. henselae DNA in Ixodes ricinus[Ixodes scapularis[Ixodes pacificus[Bartonella spp. DNA is amplified. The amplification of Bartonella spp. DNA in the woodlouse hunter spiders in this study does not provide definitive proof of vector competence and may merely represent an accidental infection associated with ingestion of Bartonella-infected blood from an infected host (isopod). Although B. henselae was amplified and sequenced from woodlouse hunter spiders and from their associated prey, the woodlouse, definitively establishing the source of bacterial transmission to this family was not possible.As scientists, physicians and veterinarians learn more about the medical importance of the genus  species . Tick trnt years ,34; howemisphere . Previous ricinusIxodes sccapularis and Ixodpacificus. In concBartonella species. The location of Bartonella replication within arthropods, the documentation of other potential reservoirs, and the determination of trans-ovarian transmission in various arthropod species represent important public health issues that need to be resolved. As B. henselae SA2 DNA was amplified from woodlouse spiders and from a woodlouse collected nearly three years after the reported bites, it is not clear if the B. henselae infections in this family were acquired by spider bites or whether spiders and woodlice were accidental hosts. Also, additional studies are needed to determine whether B. henselae bactermia can predispose patients to GBS, CIDP and neurocognitive abnormalities.There appears to be a growing spectrum of arthropods that might serve as vectors for various Written informed consent was obtained from the patient for publication of this report and any accompanying images. The parents contacted the investigators to be entered into an ongoing IRB approved research study and were full supportive of investigations described in this manuscript.GBS: Guillain-Barre syndrome; CIDP: Chronic inflammatory demyelinating polyneuropathy; BAPGM: Bartonella alpha proteobacteria growth medium; EDTA: Ethylene diaminetetraacetic acid; IPRL: Intracellular pathogens research laboratory; EMG: Electromyelogram; IVIG: Intravenous gammaglobulin; ADHD: Attention deficit hyperactivity disorder; IFA: Immunofluorescence antibody assay.Bartonella species infection in animals and human patients. Dr. Ricardo Maggi has lead research efforts to optimize the BAPGM platform and is the Scientific Technical Advisor for Galaxy Diagnostics. Dr. Robert Mozayeni was one of the attending physicians for the patients described in this study and has recently joined Galaxy Diagnostics as the chief medical officer. All other authors have no potential conflicts.In conjunction with Dr. Sushama Sontakke and North Carolina State University, Dr. Breitschwerdt holds U.S. Patent No. 7,115,385; Media and Methods for cultivation of microorganisms, which was issued October 3, 2006. He is the chief scientific officer for Galaxy Diagnostics, a company that provides diagnostic testing for the detection of PM and RM performed the BAPGM enrichment blood culture and PCR testing of the patient samples, woodlice and spiders, performed DNA sequencing and alignments, and generated the first draft of the manuscript. SH is a pediatric neurologist who cared for the youngest son and drafted his case report. BRM is a rheumatologist who cared for the mother and oldest son and drafted their case reports. CT performed PCR testing of the woodlice and spiders. JB and BH assisted in sample acquisition and serological testing. EB coordinated various aspects of the investigation and helped to draft the final manuscript. All authors read and approved the manuscript.