The effect of the video game Mindlight on anxiety symptoms in children with an Autism Spectrum Disorder

Lieke A M W Wijnhoven
H M Daan
Creemers
Rutger C M E Engels
Isabela Granic
The effect of the video game Mindlight on anxiety symptoms in children with an Autism Spectrum Disorder
10.1186/s12888-015-0522-xS T U D Y P R O T O C O L Open Access
Background: In the clinical setting, a large proportion of children with an autism spectrum disorder (ASD) experience anxiety symptoms. Because anxiety is an important cause of impairment for children with an ASD, it is necessary that effective anxiety interventions are implemented for these children. Recently, a serious game called Mindlight has been developed that is focused on decreasing anxiety in children. This approach is based on recent research suggesting that video games might be suitable as an intervention vehicle to enhance mental health in children. In the present study it will be investigated whether Mindlight is effective in decreasing (sub) clinical anxiety symptoms in children who are diagnosed with an ASD.Methods/Design: The present study involves a randomized controlled trial (RCT) with two conditions (experimental versus control), in which it is investigated whether Mindlight is effective in decreasing (sub) clinical anxiety symptoms in children with an ASD. For this study, children of 8-16 years old with a diagnosis of an ASD and (sub) clinical anxiety symptoms will be randomly assigned to the experimental (N = 60) or the control (N = 60) condition. Children in the experimental condition will play Mindlight for one hour per week, for six consecutive weeks. Children in the control condition will play the puzzle game Triple Town, also for one hour per week and for six consecutive weeks. All children will complete assessments at baseline, post-intervention and 3-months follow-up. Furthermore, parents and teachers will also complete assessments at the same time points. The primary outcome will be child report of anxiety symptoms. Secondary outcomes will be parent report of child anxiety, child/parent report of depressive symptoms, and parent/teacher report of social functioning and behavior problems. Discussion: This paper aims to describe a study that will examine the effect of the serious game Mindlight on (sub) clinical anxiety symptoms of children with an ASD in the age of 8-16 years old. It is expected that children in the experimental condition will show lower levels of anxiety symptoms at 3-months follow-up, compared to children in the control condition. If Mindlight turns out to be effective, it could be an important contribution to the already existing interventions for anxiety in children with an ASD. Mindlight could then be implemented as an evidence-based treatment for anxiety symptoms in children with an ASD in mental health institutes and special education schools.

Background

In the clinical setting, a large proportion of children with an autism spectrum disorder (ASD) experience anxiety symptoms. Between 11 % and 84 % of all children with an ASD experience some degree of impairing anxiety [1]. More specifically, 21 % of the children with an ASD suffer from subclinical anxiety [2] and approximately 40 % of the children with an ASD meet the criteria of at least one anxiety disorder [3]. Some of the most frequently reported anxiety disorders and symptoms seen in children with an ASD are simple phobias, generalized anxiety disorder, separation anxiety disorder, obsessivecompulsive disorder and social phobia [1].

Moreover, anxiety is an underlying cause of several symptoms of ASD. For example, anxiety underlies or affects the stereotype and rigid behavior [4] and the problems in social functioning [5] that children with an ASD often show. Anxiety also underlies comorbid symptoms of children with an ASD, for example oppositional and aggressive behavior [6] and depressive symptoms [7]. Furthermore, anxiety in children with an ASD has a negative impact on adaptive functioning, daily living skills and relationships with peers, teachers and family [8][9][10]. Therefore, it is important that anxiety in children with an ASD is treated and prevented from further escalation.

Recognition of anxiety symptoms in children with an ASD is not new. In the original description of children with an ASD, Kanner [11] stated that a number of these children had "substantial anxiety problems". Yet, the evaluation and treatment of anxiety in children with an ASD has only recently received empirical attention [1]. Many studies showed the effectiveness of adapted versions of cognitive behavioral therapy (CBT; e.g. [12]) or new interventions especially developed for children with an ASD (e.g. [13]), reasoning that the traditional form of CBT is not suitable for children with an ASD. On the other hand, a recent study of Van Steensel and Bögels [14] has shown that CBT is effective in reducing anxiety symptoms in children with an ASD, and that CBT is as effective for children with an ASD as for children without an ASD.

However, there are some important limitations to CBT for anxiety, both in general and specifically for children with an ASD. First, CBT largely consists of teaching children to become conscious of their negative thoughts, to evaluate these thoughts, and eventually to challenge them and formulate thoughts that are more accurate. These sessions have a face-to-face, verbal and cognitively complex character. Because children with an ASD have a cognitive and social impairment, they have difficulties with learning skills in these CBT-sessions and as a result they are often not intrinsically motivated for CBT [15]. Therefore, a greater focus on visual aids and structured sensory information is an important requirement in anxiety treatment for children with an ASD [15,16]. Second, there is a large gap between the knowledge that children gain in CBT and the implementation and practice of this knowledge in daily life. Especially for children with ASD, frequent practice and exposure opportunities are important in anxiety interventions [1]. However, the exercises in CBT that do exist are mostly decontextualized and do not fully represent the situations in which children experience their anxiety. A third limitation of CBT is limited access to care and long waiting lists to care that is accessible [17]. Finally, the low cost effectiveness is a limitation of CBT, which many mental health institutions experience as a barrier to treatment delivery. Therefore, it is important that new anxiety interventions are developed that can provide a solution for the above mentioned limitations.

Recently, it has been shown that video games have the potential to enhance mental health and well-being in children and adolescents [18,17]. For example, Merry et al. [19] found that the video game 'SPARX' was effective in reducing depressive symptoms among adolescents in the age between 12-19 years old. They concluded that it was a potential alternative to usual care for adolescents with depressive symptoms in primary care settings and that it could be used to address some of the unmet demands for treatment. More recently, the serious game Mindlight (Playnice Institute) has been developed for the treatment of anxiety disorders in children. A recent study has tested the effect of Mindlight on anxiety symptoms in school children [20]. This study showed that both the anxiety of the children who played Mindlight as the anxiety of the children who played the control game significantly decreased over time. However, no studies to date have investigated the effect of a serious game on anxiety symptoms of children with an ASD.

Mindlight has the aim to tackle anxiety in children by using several treatment mechanisms. First of all, it uses exposure techniques, one of the most empirically-validated treatment components of CBT for anxious individuals [21]. During exposure, individuals are gradually exposed to the threatening cues. In this way, they are getting habituated to these cues and eventually they are getting more comfortable and less anxious when being exposed to them. Moreover, Mindlight uses neurofeedback mechanisms, which is effective in decreasing anxiety symptoms of children [22]. These mechanisms are focused on regulating arousal levels associated with anxiety through relaxation and concentration. Finally, attention bias modification is incorporated in Mindlight, which is a therapy mechanism in which children learn to (a) disattend to threatening cues and shifting attention away from those cues and to (b) focus on positive aspects of the environment in the service of relevant goals [23].

It is hypothesized that Mindlight has the potential to serve as an effective new intervention for children with ASD and comorbid (sub) clinical anxiety symptoms, and that it can overcome the limitations of CBT. First, it is known that children with ASD often feel a close affinity for technology and games, which means that the participating children are probably intrinsically motivated to play a game like Mindlight in therapy [24]. Moreover, it has been reported that computer based training could be an effective tool in treatment for children with ASD, due to its visual and structured character [15]. Mindlight uses visual aids and structured sensory information to a great extent, both for creating a 'scary' exposure environment and for teaching important treatment concepts. Furthermore, Mindlight includes frequent practice and exposure opportunities. Because Mindlight can be played repeatedly, with the difficulty level increasing as children become better players, there is a great deal of practice and exposure involved in playing this game. As a result, the gap between knowledge and behavior may be substantially decreased and effective cognitive and emotional coping skills can be automatized and possibly generalized with practice. Finally, therapy skills can be practiced at home, which means that children have an easier access to mental health care. In this way, the waiting lists can become shorter and the therapy costs can be decreased when implementing a game like Mindlight as therapy tool.

In the present study, the primary aim is to investigate whether Mindlight is effective in reducing (sub) clinical anxiety symptoms in children with an ASD. The secondary aim is to examine whether Mindlight is effective in reducing parent report of child anxiety, and the anxiety-related depressive symptoms, social functioning and behavior problems of the participating children. To investigate these aims, a multi method symptom assessment is used, including parent, teacher and child reports [1]. If Mindlight turns out to be effective for anxious children with an ASD, it could be considered as a new therapeutic intervention next to the already existing approaches for anxiety in children with an ASD.

Methods

The study design will be reported in line with the CON-SORT 2010 Statement for reporting parallel group randomized trials [25]. The medical ethics committee CMO Arnhem-Nijmegen in the Netherlands has given approval for the conduction of this study (NL50023.091.14). Moreover, the study is registered in the Dutch Trial Register for RCT's (NTR5069).

Design

The present study involves a randomized controlled trial (RCT) with two conditions (experimental versus control), in which it is investigated whether the new video game Mindlight is effective in treating (sub) clinical anxiety symptoms in children with an ASD. For this study, children in the age of 8-16 years old with a diagnosis of an ASD according to the Diagnostic and Statistical Manual of Mental Disorders 4th Edition -Text Revision (DSM-IV-TR; [26]) will be screened for anxiety symptoms. The children with (sub) clinical anxiety symptoms will be selected and approached for participation.

After the selection and recruitment, children will be randomly assigned to the experimental or control condition. At baseline (T0), children, parents and teachers will fill in questionnaires. Moreover, parents will undergo a semi-structured interview (ADIS-P; [27]) to determine whether their child meets the criteria of one or more anxiety disorders. At post-intervention (T1) and at 3months follow-up (T2), children, parents and teachers will fill in questionnaires again to evaluate the effect of Mindlight. At 3-months follow-up, parents will undergo the semi-structured interview again to test whether Mindlight also had an effect on the present anxiety disorder (s) in the participating children. Fig. 1 shows a schematic overview of the design in the present study.

Participants' eligibility

Children with an ASD (DSM-IV-TR: Autistic disorder, Asperger disorder, PDD-NOS; [26]) in the age between 8 to 16 years old will be assessed for eligibility by a screening. This screening consists of filling in an anxiety questionnaire by both children (SCAS-C; [28]) and parents (SCAS-P; [29]). When children have at least subclinical levels of anxiety, they are eligible for participation in the study. Moreover, they have to have sufficient knowledge of the Dutch language. Exclusion criteria are absence of parental permission and presence of prominent suicidal ideation or other severe psychiatric problems that need immediate treatment (e.g. severe trauma's).

Procedure

Contexts of recruitment are mental health institutes (e.g. GGZ Oost Brabant) and special education schools in the Netherlands. First, parents will receive a letter with information about the screening and the study. Moreover, children and parents will be asked to fill in the SCAS-C/ P [28,29]. When children have at least subclinical levels of anxiety and meet the other inclusion criteria, children and parents will be approached to participate in the study. If children and parents agree with participation, active written informed consent of the parents and the children who are above the age of 12 will be obtained.

After obtaining active written informed consent, children will be randomly allocated to the experimental or control condition. Children in the experimental condition will play Mindlight individually for one hour per week during 6 consecutive weeks at the recruitment location. Moreover, children may receive treatment as usual (TAU) parallel with Mindlight. TAU will mainly be offered by mental health institutes and might for example consist of psycho-education on ASD, play/drama therapy, parent guidance and/or medication. These types of TAU will be monitored and reported during the course of the study, and will be tested as potential confounders in the analyses. Children in the control condition will play the computer game Triple Town individually for one hour per week during 6 consecutive weeks at the recruitment location. Again, children may receive TAU parallel with the game. Moreover, they will have the opportunity to play Mindlight after the 3-months follow-up if this game turns out to be effective.

Sample size

A priori power analysis was performed in G*Power 3.1 [30] to calculate the sample size that is required in the present study. It is expected that Mindlight is significantly more effective in reducing anxiety symptoms of the participating children than Triple Town. Previous studies on the effect of already existing modified versions of CBT on anxiety among children with an ASD reported moderate to large effect sizes on anxiety symptoms (e.g. [31,12,32]). It is assumed that the implementation of Mindlight in clinical practice is worth consideration, when the effect of Mindlight is at least equal to the effect of the present anxiety treatments for children with an ASD. Therefore, the power calculation is conducted with a moderate expected effect size (f = .25) of condition (experimental or control condition) at 3-months follow-up. Moreover, it was expected that the Type I error was .05 and that the Type II error was .20 (power is .80). When computing these assumptions, it was calculated that a sample of 86 participants would be satisfactory in the present study. Eventually, the sample size was increased by 40 % to compensate for loss to follow-up (estimated at 20 %) and possible loss of power due to potential clustering of data in case of group formation (estimated at 20 %), resulting in 120 participants (60 in experimental condition and 60 in control condition).

Intervention

The intervention that will be investigated is called Mindlight. This is a video game aimed at children of 8-16 years old and is based on principles of cognitive-behavioural therapy (CBT) and neurofeedback, which are evidencebased interventions for anxiety-disordered children and adults. Briefly, the premise of the game is that Little Arthur is left on the doorstep of a scary mansion by his parents. Arthur must learn to use his own inner strength to overcome his greatest fears so the shadows in the house can hold no power over him. He can accomplish this by using his Mindlight, a light bubble that can shine on the surroundings and that can be controlled by his own inner strength. This 'inner strength' will be measured by a neurofeedback headset (the 'MindWave'; Neurosky USA; [33]), which children will put on when they are going to play Mindlight. This headset records EEG using dry sensor technology, which consists of an active and reference electrode. The signals that are measured will be filtered on Delta, Theta, Alpha and Beta waves. In Mindlight, especially the Alpha and Beta waves will be used for real time feedback. Research has shown that the Mindwave headset has good reliability and validity [33], and that it can be used in research with children who have a developmental disorder (e.g. ADHD; [34]).

In Mindlight, the Alpha and Beta waves will be used in several ways. First of all, the recorded Alpha waves reflect the degree of relaxation of the child. This feature is used in the exposure techniques (CBT) that are embedded in the game (see also Fig. 2): when the child sees threatening stimuli (e.g. monsters) several times during the game and learns to maintain calm when facing them, the child eventually gets habituated to them and can gain points more easily.

Furthermore, the recorded Beta waves reflect the degree of concentration and the allocation of attention of the player. Focused concentration allows the player to solve attention bias modification (ABM) puzzles. ABM is a training protocol that has its roots in CBT and that is based on the idea that distorted cognitions, particularly attentional biases characterized by hyper attention towards potential threats, play a role in the pathogenesis of childhood anxiety [22]. ABM has been shown to reliably reduce anxiety by retraining the attentional system to focus on positive stimuli [35]. Mindlight uses this principle in the ABM-puzzles, by rewarding children for focusing on positive aspects of the environment (measured by the neurofeedback device). More specifically, they learn to move towards, and quickly respond to, positive stimuli (e.g., portraits of happy faces) and disattend to, or shift attention away from, negative stimuli (e.g., mean faces, threatening animals).

To minimize the chance of finding placebo-effects of Mindlight, children in the control condition will also receive a computer game. In this way, the amount of attention that children in the experimental condition and children in the control condition receive is equal, and the effects that may be found can uniquely be ascribed to the game itself. The computer game that the children in the control condition are going to play is called 'Triple Town'. In this puzzle game, the player has to build a city. The bigger the city you build, the more points you can receive. To accomplish this, you have to combine elements (e.g. trees and houses) in a strategical way. Moreover, you have to block bears that try to hinder you in building the city. In this way, the child learns to think strategically in order to overcome challenges. Moreover, children learn to keep a goal and to persevere in order to reach this goal. However, the game is not specifically focused at reducing anxiety levels, which makes 'Triple Town' a suitable game for the control condition.

The gaming sessions will be led by qualified therapists, or by master students who are supervised by qualified therapists. In session 1, the therapist starts with psychoeducation on anxiety. After that, the anxiety of the child will be discussed, the therapist will explain the game and eventually the therapist will clarify that this game is focused on decreasing the anxiety of the child. Then, the child will play the game for approximately 40 min. After playing the game, the therapist will ask the following standardized questions: 1) How did the gaming go What did you find difficult/What did you find easy? 3) What did you learn in the game? 4) Could you apply and practice the skills you have learned in scary or difficult situations in daily life? In session 2-6, the therapist will start the session with discussing the previous week and the skills the child has practiced at home. When the child mentions that he has practiced the skills in a scary or difficult situation, this will be reinforced by the therapist. In this way, the therapist does not add explicit therapeutic elements to the gaming sessions, but children do get stimulated to think about their anxiety and the way they can apply and practice the skills they have learned in the game in daily life. Table 1 shows an overview of the different time points, the questionnaires that were filled in on each time point and the informants that were involved.

Study outcome measures

Screening measures

To test their eligibility, children will be screened on anxiety symptoms using the SCAS-C for child report and the SCAS-P for parent report [28,29]. Children are eligible for participation when the child and/or the parent report the presence of subclinical child anxiety. Moreover, demographical questions (e.g. sex, age, educational level) will be asked to both children and parents. Finally, some questions about the child's gaming behavior (e.g. 'How many hours per week do you game?') will be asked.

Primary outcome measure

Anxiety symptoms will be measured with the Dutch translation of the Spence Children's Anxiety Scale (SCAS; [28]).

The SCAS consists of 44 items (e.g. 'I am afraid when I have to sleep alone' , 'I worry about things') on a 4-point scale, ranging from 'never' to 'always'. Scores on items ranged from 0 to 3, with higher scores indicating more anxiety symptoms. Moreover, the scale consists of six subscales that are in line with the different anxiety disorders that are described in the DSM-IV: panic/agora phobia, separation anxiety, social phobia, generalized anxiety, obsessive compulsive anxiety and anxiety for physical injury. The SCAS has a high validity and reliability [36,37].

Secondary outcome measures

Anxiety of the child according to the parents will be measured with the Dutch translation of the Spence Child Anxiety Scale for Parents (SCAS-P; [29]). The SCAS-P consists of 38 items on a 4-point scale ranging from 0 (never) to 3 (always). The items of the SCAS-P were formulated as closely as possible to the corresponding item of the child version of the SCAS. Only items referring to an internal state (e.g. item 4: 'I feel afraid') were rephrased into observable behaviour for parents (e.g. 'My child complains of feeling afraid'). The SCAS-P consists of the same six subscales as the child version. The SCAS-P has a good reliability and validity [38].

The presence of anxiety disorders according to the parents will be assessed with the Dutch translation of the Anxiety Disorders Interview Schedule for DSM-IV, Parent version (ADIS-P; [27]). This is a semi-structured diagnostic parent interview that can be used to diagnose anxiety disorders in children of 7-17 years old. The interview will be administered by a qualified therapist or by a master student under supervision of a qualified therapist. In this study, the presence of the following DSM-IV anxiety disorders will be examined: separation anxiety disorder, social phobia, specific phobia, panic disorder, agora phobia and generalised anxiety disorder. The interview consists of standardized questions, with 'yes' , 'no' and 'different' as possible answers. At the end of the interview, the interviewer has to give his/her clinical judgement about the severity of every disorder. On basis of this judgement, the interviewer will make a definitive decision about the presence (yes/no) of the different anxiety disorders. The ADIS-P has a strong reliability and validity [27].

Depressive symptoms will be measured using the Dutch translation of the Child Depression Inventory 2 (CDI 2; [39]). The CDI 2 consists of 28 items measured on a 3-point scale ranging from 0 (depressive symptom is absent) to 2 (depressive symptom is always present) (e.g., 'I don't feel alone' = 0, 'I often feel alone' = 1, 'I always feel alone' = 2; 'Sometimes I'm sad' = 0, 'I'm often sad' = 1, 'I'm always sad' = 2). The children have to choose the answer that is most in accordance with their own thoughts and feelings. The validity and reliability of the Dutch CDI 2 (:P) is still being investigated, but it Table 1 Overview of assessments   Screening  T0  T1  Behavior problems (SDQ) χ χ χ *The primary caregiver of the child will fill in the questionnaires. The ADIS-P will be conducted with both parents (if possible) already has been shown that the original (American) version of the CDI 2 (:P) [40] has a good reliability, internal consistency and convergent validity [41]. Depression according to the parents will be measured with the Dutch translation of the Child Depression Inventory 2 for parents (CDI 2:P; [39]) will be used to measure parental assessment of depressive symptoms of their child. The CDI 2:P consists of 17 items measured on a 4-point scale ranging from 0 (not at all), 1 (some of the time), 2 (often), or 3 (most of the time) (e.g. 'My child looks sad'; 'My child seems lonely'). The parent has to assess to which extent the items are in accordance with their child's thoughts and feelings.

Social functioning according to the parents and teacher will be measured with the 'Vragenlijst voor Inventarisatie van Sociaal gedrag van Kinderen' (VISK), a Dutch translation of the Children's Social Behaviour Questionnaire (CSBQ; Luteijn [42]). The VISK consists of 49 items measured on a 3-point scale (0 = not applicable, 1 = sometimes applicable, 2 = often applicable). The items are divided over six problem scales: being not well tailored to social situations; limited tendency to engage in social interactions; orientation problems in time, space and place; not understanding social information; stereotype behavior; and anxiety for and resistance against changes. Hartman and colleagues [43] reported that the VISK has a good validity and reliability.

Internalizing and externalizing behaviour problems according to the parents and teacher will be measured with the Dutch translation of the Strengths and Difficulties Questionnaire (SDQ; [44]). The SDQ consists of 25 items, measured on a 3-point scale (0 = not true, 1 = a little bit true, 2 = certainly true). Moreover, an impact supplement can be completed, by which the impact of the behavior problems on daily functioning of the child and its family can be assessed. The items of the SDQ are divided over the following scales: emotional problems, behavior problems, hyperactivity/ attention problems, problems with peers and social behavior. There are separate forms for parents and teachers. The SDQ has a sufficient reliability and validity [45]. Treatment expectancies according to child and parents will be measured using the Dutch translation of the Parent Expectancies for Therapy Scale (PETS; [46]). The PETS has a parent and child version, and each version consists of seven items measured on a 6-point scale ranging from 1 (I totally disagree) to 6 (I totally agree). The items of the PETS are divided over the following subscales: credibility, child improvement and parent involvement. The PETS has a good validity and reliability [47].

Data analysis/statistical analysis

Following the intention-to-treat principle [48], all children randomized to a condition will be included in the analyses to test the study objectives. Multiple imputations will be used for missing observations at post-intervention and 3-month follow-up. Reporting of the results of the study will be in accordance with the CONSORT 2010 Statement [25].

To investigate the differences in the development of anxiety symptoms between children in the experimental condition and children in the control condition, a 3 (within-subjects: pre, post, follow-up) by 2 (condition: experimental vs. control) ANOVA (repeated measures) will be conducted, with anxiety symptoms (child report) as the dependent variable. The direct effect of Mindlight on parent report of child anxiety, parent/child report of depressive symptoms, and parent/teacher report of social functioning and behavior problems will be investigated in the same way. Furthermore, remission rates of the anxiety disorders that were diagnosed at baseline (with ADIS-P) will be calculated at 3-months follow-up, and Chi-square (χ 2 ) tests will be conducted to compare remission rates between the experimental and control group. In the above mentioned analysis plan it is assumed that the children will play the game individually and that therefore the data will not be clustered. However, in case of clustered data due to the formation of groups in which several children play the game in the same room, the analyses will be conducted in MPLUS 6.11 [49].

Finally, possible baseline differences between the two conditions in demographic variables (e.g. age, sex, educational level), anxiety symptoms, gaming behaviour and treatment expectancies will be checked. Moreover, possible differences in TAU between the experimental and control condition will be checked at all time points. Variables that show different distributions between the two groups will be entered as confounders in all models testing the effectiveness of the intervention.

Discussion

The present study protocol gives an overview of a study design for a randomized controlled trial testing the effect of the serious game Mindlight in decreasing anxiety symptoms of children with an ASD. The primary aim is to investigate whether Mindlight is effective in reducing (sub) clinical anxiety symptoms of children with an ASD in the age of 8-16 years old. The secondary aims are to investigate whether Mindlight is effective in reducing parent report of child anxiety and the anxiety-related problems in social functioning, depressive symptoms and behavior problems of children with an ASD. It is expected that Mindlight is effective in reducing anxiety symptoms of the children in the experimental condition, compared to the children in the control condition that play Triple Town. Furthermore, it is expected that Mindlight is more effective than Triple Town in reducing parent report of child anxiety, parent/child report of depressive symptoms and parent/teacher report of social functioning and behavior problems.

Strengths and limitations

The present study design has a few strengths and limitations. A strength is that it is the first study investigating the effect of a serious game in a clinical context with children who are diagnosed with an ASD and who have comorbid (sub) clinical anxiety symptoms, which could lead to a new way of treating anxiety in children with an ASD. An additional strength is that children with an ASD often feel a close affinity for technology and games [24,15], and that the participating children are probably intrinsically motivated to play a game like Mindlight in therapy. Furthermore, Mindlight includes frequent practice, exposure opportunities, visual aids and structured sensory information, which all stimulate the automatization and the generalization of skills to daily life in the participating children. Another strength is that this study may lead to the implementation of Mindlight in mental health institutes, which may result in an easier access to mental health care, shorter waiting lists and lower therapy costs. Finally, this study has multiple outcome measures, which makes it possible to investigate other direct effects of Mindlight on anxiety-related symptoms (e.g. depressive symptoms) of the participating children.

A limitation of the present study design is that the children in the control condition might have played Triple Town already before the start of the study. This may have a positive (e.g. more practice) or negative (r boredom) influence on the effect of the game. Moreover, only short-term effects (3-months follow-up) of Mindlight will be investigated. In this way, no conclusions can be drawn about the long-term effects of Mindlight on anxiety symptoms of the participating children. Finally, there are no standardized protocols for offering and implementing a video game in a clinical therapy session. This implies that the best form of implementation still needs to be discovered and improved by experience.

Implications for practice

Anxiety symptoms are highly common in children with an ASD. Still, treatment on anxiety in children with an ASD only recently has received some empirical attention. This may be caused by the fact that anxiety is often underdiagnosed in children with an ASD [1]. In this way, anxiety treatment for children with an ASD is not common and the development of evidence-based anxiety treatments has not been focused upon until recently. By developing and investigating new anxiety treatments for children with an ASD, these may be more frequently offered in mental health institutes in the future. Moreover, if Mindlight turns out to be effective for anxious children with an ASD, it could be considered as a good and suitable therapeutic alternative to the already existing interventions for anxiety in children with an ASD. Mindlight could then be implemented as an evidence-based treatment for children with an ASD in mental health institutes and special education schools.

Conclusion

This paper aimed to describe a study that will investigate the effect of the serious game Mindlight on (sub) clinical anxiety symptoms of children with an autism spectrum disorder in the age of 8-16 years old. It is expected that children in the experimental condition will show lower levels of anxiety symptoms at 3-months follow-up, compared to children in the control condition. If Mindlight turns out to be effective, this could provide a significant contribution to the evidence-based treatment of anxiety in children with an ASD.

Fig. 1
1Flow diagram of recruitment, randomization and assessments. FU = Follow-up

Fig. 2
2Relaxation mechanic Mindlight: remain calm to enlighten environment today? 2)
AcknowledgementsFunding for this study was provided by grants from the Research & Innovation Foundation of GGZ Oost Brabant and the Behavioral Science Institute of the Radboud University in Nijmegen. They both conducted peer review of the study protocol and provided approval to the study.Competing interestsMindlight has been developed by the Playnice Institute. Prof. dr. Isabela Granic and Prof. dr. Rutger Engels are the founders of this institute. However, Mindlight is not yet commercialized. The chance of competing interests is only present in case of commercialization of this game. Dr. Daan Creemers and Lieke Wijnhoven (MSc) declare that they have no competing interests.Authors' contributionsLieke Wijnhoven (MSc) is responsible for the data collection, the data analysis and for reporting the study results. Dr. Daan Creemers, Prof. dr. Rutger Engels and Prof. dr. Isabela Granic are supervisors, grant applicators, and principal investigators. All authors have contributed to the writing of this manuscript. Moreover, all authors have read and approved the final manuscript.
Anxiety in children and adolescents with autism spectrum disorders. S W White, D Oswald, T Ollendick, L Scahill, 10.1016/j.cpr.2009.01.003Clin Psych Rev. 29White SW, Oswald D, Ollendick T, Scahill L. Anxiety in children and adolescents with autism spectrum disorders. Clin Psych Rev. 2009;29:216-29. doi:10.1016/j.cpr.2009.01.003.

Depression and anxiety symptoms in children and adolescents with autism spectrum disorders without intellectual disability. J F Strang, L Kenworthy, P Daniolos, L Case, M C Wills, A Martin, 10.1016/j.rasd.2011.06.015Res Autism Spectr Disord. 6Strang JF, Kenworthy L, Daniolos P, Case L, Wills MC, Martin A, et al. Depression and anxiety symptoms in children and adolescents with autism spectrum disorders without intellectual disability. Res Autism Spectr Disord. 2012;6:406-12. doi:10.1016/j.rasd.2011.06.015.

Anxiety disorders in children and adolescents with autistic spectrum disorders: a meta-analysis. Fja Van Steensel, S M Bögels, S Perrin, 10.1007/s10567-011-0097-0Clin Child Fam Psychol Rev. 14Van Steensel FJA, Bögels SM, Perrin S. Anxiety disorders in children and adolescents with autistic spectrum disorders: a meta-analysis. Clin Child Fam Psychol Rev. 2011;14:302-17. doi:10.1007/s10567-011-0097-0.

The relationship between anxiety and repetitive behaviours in autism spectrum disorder. J Rodgers, M Glod, B Connolly, H Mcconachie, 10.1007/s10803-012-1531-yJ Autism Dev Disord. 42Rodgers J, Glod M, Connolly B, McConachie H. The relationship between anxiety and repetitive behaviours in autism spectrum disorder. J Autism Dev Disord. 2012;42:2404-9. doi:10.1007/s10803-012-1531-y.

Effects of anxiety disorder severity on social functioning in children with autism spectrum disorder. Y Chang, J Quan, J J Wood, 10.1007/s10882-012-9268-2J Dev Phys Disabil. 24Chang Y, Quan J, Wood JJ. Effects of anxiety disorder severity on social functioning in children with autism spectrum disorder. J Dev Phys Disabil. 2012;24:235-45. doi:10.1007/s10882-012-9268-2.

The relationship of comorbid anxiety symptom severity and challenging behaviors in infants and toddlers with autism spectrum disorder. P Cervantes, J L Matson, K Tureck, H L Adams, 10.1016/j.rasd.2013.09.005Res Autism Spectr Disord. 7Cervantes P, Matson JL, Tureck K, Adams HL. The relationship of comorbid anxiety symptom severity and challenging behaviors in infants and toddlers with autism spectrum disorder. Res Autism Spectr Disord. 2013;7:1528-34. doi:10.1016/j.rasd.2013.09.005.

Age-related differences in the prevalence and correlates of anxiety in youth with autism spectrum disorders. R A Vasa, L Kalb, M Mazurek, S Kanne, B Freedman, A Keefer, 10.1016/j.rasd.2013.07.005Res Autism Spectr Disord. 7Vasa RA, Kalb L, Mazurek M, Kanne S, Freedman B, Keefer A, et al. Age-related differences in the prevalence and correlates of anxiety in youth with autism spectrum disorders. Res Autism Spectr Disord. 2013;7:1358-69. doi:10.1016/j.rasd.2013.07.005.

Exploring the manifestations of anxiety in children with autism spectrum disorders. V Hallett, L Lecavalier, D G Sukhodolsky, 10.1007/s10803-013-1775-1J Autism Dev Disord. Hallett V, Lecavalier L, Sukhodolsky DG, et al. Exploring the manifestations of anxiety in children with autism spectrum disorders. J Autism Dev Disord. 2013. doi:10.1007/s10803-013-1775-1.

Effects of cognitive behavioral therapy on daily living skills in children with high functioning autism and concurrent anxiety disorders. A Drahota, J J Wood, K M Sze, M Van Dyke, 10.1007/s10803-010-1037-4J Autism Dev Disord. 413Drahota A, Wood JJ, Sze KM, van Dyke M. Effects of cognitive behavioral therapy on daily living skills in children with high functioning autism and concurrent anxiety disorders. J Autism Dev Disord. 2011;41(3):257-65. doi:10.1007/s10803-010-1037-4.

The prevalence of anxiety and mood problems among children with autism and Asperger syndrome. J A Kim, P Szatmari, S E Bryson, D L Streiner, F J Wilson, Autism. 42Kim JA, Szatmari P, Bryson SE, Streiner DL, Wilson FJ. The prevalence of anxiety and mood problems among children with autism and Asperger syndrome. Autism. 2000;4(2):117-32.

Autistic disturbances in affective contact. L Kanner, Nervous Child. 2Kanner L. Autistic disturbances in affective contact. Nervous Child. 1943;2:217-50.

The Coping Cat program for children with anxiety and autism spectrum disorder: a pilot randomized controlled trial. R H Mcnally Keehn, A J Lincoln, M Z Brown, D A Chavira, 10.1007/s10803-012-1541-9J Autism Dev Disord. 43McNally Keehn RH, Lincoln AJ, Brown MZ, Chavira DA. The Coping Cat program for children with anxiety and autism spectrum disorder: a pilot randomized controlled trial. J Autism Dev Disord. 2013;43:57-67. doi:10.1007/s10803-012-1541-9.

Randomized controlled trial: multimodal anxiety and social skill intervention for adolescents with autism spectrum disorder. S W White, T Ollendick, A M Albano, D Oswald, C Johnson, M A Southam-Gerow, 10.1007/s10803-012-1577-xJ Autism Dev Disord. 43White SW, Ollendick T, Albano AM, Oswald D, Johnson C, Southam-Gerow MA, et al. Randomized controlled trial: multimodal anxiety and social skill intervention for adolescents with autism spectrum disorder. J Autism Dev Disord. 2013;43:382-94. doi:10.1007/s10803-012-1577-x.

Cognitive-behavioral therapy for anxiety disorders in children with and without ASD. Fja Van Steensel, S M Bögels, 10.1037/a0039108J Consult Clin Psychol. 833submittedVan Steensel FJA, Bögels SM. Cognitive-behavioral therapy for anxiety disorders in children with and without ASD. J Consult Clin Psychol. 2015;83(3):512-23. doi:10.1037/a0039108. Epub 2015 Apr 20. submitted.

Evaluation of a new computer intervention to teach people with autism or Asperger syndrome to recognize and predict emotions in others. M Silver, P Oaks, 10.1177/1362361301005003007doi:10.1177/ 1362361301005003007Autism. 5Silver M, Oaks P. Evaluation of a new computer intervention to teach people with autism or Asperger syndrome to recognize and predict emotions in others. Autism. 2001;5:299-316. doi:10.1177/ 1362361301005003007.

Treating anxiety disorders in children with high functioning autism spectrum disorders: a controlled trial. A Chalfant, R Rapee, L Carroll, 10.1007/s10803-006-0318-4J Autism Dev Disord. 37Chalfant A, Rapee R, Carroll L. Treating anxiety disorders in children with high functioning autism spectrum disorders: a controlled trial. J Autism Dev Disord. 2007;37:1842-57. doi:10.1007/s10803-006-0318-4.

The benefits of playing video games. I Granic, A Lobel, Rcme Engels, 10.1089/cyber.2013.0296Am Psychol. Granic I, Lobel A, Engels RCME. The benefits of playing video games. Am Psychol. 2014. doi:10.1089/cyber.2013.0296.

Friends, fun, frustration and fantasy: child motivations for video game play. C J Ferguson, C K Olson, 10.1007/s11031-012-9284-7Motiv Emot. 37Ferguson CJ, Olson CK. Friends, fun, frustration and fantasy: child motivations for video game play. Motiv Emot. 2013;37:154-64. doi:10.1007/s11031-012-9284-7.

The effectiveness of SPARX, a computerised self help intervention for adolescents seeking help for depression: randomised controlled non-inferiority trial. S N Merry, K Stasiak, M Shepherd, C Frampton, T Fleming, Mfg Lucassen, 10.1136/bmj.e2598Br Med J. 344Merry SN, Stasiak K, Shepherd M, Frampton C, Fleming T, Lucassen MFG. The effectiveness of SPARX, a computerised self help intervention for adolescents seeking help for depression: randomised controlled non-inferiority trial. Br Med J. 2012;344:1-16. doi:10.1136/bmj.e2598.

A randomized controlled trial testing the effectiveness of the video game Mindlight in preventing anxiety in children. E A Schoneveld, M Malmberg, A Lichtwarck-Aschoff, G P Verheijen, Rcme Engels, I Granic, Schoneveld EA, Malmberg M, Lichtwarck-Aschoff A, Verheijen GP, Engels RCME, Granic I. A randomized controlled trial testing the effectiveness of the video game Mindlight in preventing anxiety in children. 2015;

Exposure therapy for anxiety: principles and practice. J S Abramowitz, B T Deacon, Sph Whiteside, Guilford PressNew YorkAbramowitz JS, Deacon BT, Whiteside SPH. Exposure therapy for anxiety: principles and practice. New York: Guilford Press; 2011.

An integrative, multi-factor conceptualization of hypnosis. D C Hammond, 10.1080/00029157.2005.10401508Am J Clin Hypnosis. 48Hammond DC. An integrative, multi-factor conceptualization of hypnosis. Am J Clin Hypnosis. 2005;48:131-5. doi:10.1080/00029157.2005.10401508.

Distorted cognition and pathological anxiety in children and adolescents. P Muris, A P Field, 10.1080/02699930701843450doi:10.1080/ 02699930701843450Cogn Emotion. 22Muris P, Field AP. Distorted cognition and pathological anxiety in children and adolescents. Cogn Emotion. 2008;22:395-421. doi:10.1080/ 02699930701843450.

Designing affective video games to support the social-emotional development of teenagers with autism spectrum disorders. M Khandaker, 10.3233/978-1-60750-017-9-37Ann Rev Cyber. Wiederhold WK, Riva GTelem. AmsterdamIos PressKhandaker M. Designing affective video games to support the social-emotional development of teenagers with autism spectrum disorders. In: Wiederhold WK, Riva G, editors. Ann Rev Cyber Telem. Amsterdam: Ios Press; 2009. doi:10.3233/978-1-60750-017-9-37.

Statement: updated guidelines for reporting parallel group randomized trials. K F Schulz, D G Altman, D Moher, C Group, Consort, 10.1186/1745-6215-11-32Trials. 113232Schulz KF, Altman DG, Moher D, Group C. CONSORT 2010 Statement: updated guidelines for reporting parallel group randomized trials. Trials. 2010;11(32):32. doi:10.1186/1745-6215-11-32.

American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders 4th Edition Text Revision (DSM-IV-TR). American Psychiatric AssociationWashington, DCAmerican Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders 4th Edition Text Revision (DSM-IV-TR). Washington, DC: American Psychiatric Association; 2000.

Anxiety disorders interview schedule for DSM-IVchild version. ADIS-C handleiding. B M Siebelink, Pda Treffers, Harcourt Test PublishersAmsterdamSiebelink BM, Treffers PDA. Anxiety disorders interview schedule for DSM-IV- child version. ADIS-C handleiding. Amsterdam: Harcourt Test Publishers; 2001.

Spence children's anxiety scale (Dutch translation of child version). A Scholing, M H Nauta, S H Spence, AmsterdamNL: University of AmsterdamScholing A, Nauta MH, Spence SH. Spence children's anxiety scale (Dutch translation of child version). Amsterdam, NL: University of Amsterdam; 1999.

Spence children's anxiety scale (Dutch translation of parent version). A Scholing, M H Nauta, S H Spence, AmsterdamNL: University of AmsterdamScholing A, Nauta MH, Spence SH. Spence children's anxiety scale (Dutch translation of parent version). Amsterdam, NL: University of Amsterdam; 1999.

Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. F Faul, E Erdfelder, A G Lang, A Buchner, 10.3758/bf03193146Behav Res Meth. 39Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Meth. 2007;39:175-91. doi:10.3758/bf03193146.

Group cognitive behavior therapy for children with high-functioning autism spectrum disorders and anxiety: a randomized trial. J Reaven, A Blakeley-Smith, K Culhane-Shelburne, S Hepburn, 10.1111/j.1469-7610.2011.02486.xJ Child Psychol Psychiatry. 53Reaven J, Blakeley-Smith A, Culhane-Shelburne K, Hepburn S. Group cognitive behavior therapy for children with high-functioning autism spectrum disorders and anxiety: a randomized trial. J Child Psychol Psychiatry. 2012;53:410-9. doi:10.1111/j.1469-7610.2011.02486.x.

Cognitive behavioral therapy for children with autism: review and considerations for future research. J T Danial, J J Wood, 10.1097/DBP.0b013e31829f676cJ Dev Behav Pediatr. 34Danial JT, Wood JJ. Cognitive behavioral therapy for children with autism: review and considerations for future research. J Dev Behav Pediatr. 2013;34:702-15. doi:10.1097/DBP.0b013e31829f676c.

EEG from a single-channel dry-sensor recording device. S J Johnstone, R Blackman, J Bruggemann, 10.1177/1550059411435857Clin EEG Neurosci. 43Johnstone SJ, Blackman R, Bruggemann J. EEG from a single-channel dry-sensor recording device. Clin EEG Neurosci. 2012;43:112-20. doi:10.1177/1550059411435857.

Neurocognitive training for children with and without AD/HD. ADHD atten def hyp disord. S J Johnstone, S Roodenrys, R Blackmann, E Johnstone, K Loveday, S Mantz, 10.1007/s12402-011-0069-84Johnstone SJ, Roodenrys S, Blackmann R, Johnstone E, Loveday K, Mantz S, et al. Neurocognitive training for children with and without AD/HD. ADHD atten def hyp disord. 2012;4:11-23. doi:10.1007/s12402-011-0069-8.

Review: attention bias modification (ABM): a novel treatment for anxiety disorders. Y Bar-Haim, Research, 10.1111/j.1469-7610.2010.02251.xJ Child Psychol Psychiatry. 51Bar-Haim Y. Research Review: attention bias modification (ABM): a novel treatment for anxiety disorders. J Child Psychol Psychiatry. 2010;51:859-70. doi:10.1111/j.1469-7610.2010.02251.x.

Correlations among two self-report questionnaires for measuring DSM-defined anxiety disorder symptoms in children: the screen for child anxiety related emotional disorders and the Spence Children's anxiety scale. P Muris, H Schmidt, H Merckelbach, 10.1016/s0191-8869(99)00102-6Pers Individ Dif. 28Muris P, Schmidt H, Merckelbach H. Correlations among two self-report questionnaires for measuring DSM-defined anxiety disorder symptoms in children: the screen for child anxiety related emotional disorders and the Spence Children's anxiety scale. Pers Individ Dif. 2000;28:333-46. doi:10.1016/s0191-8869(99)00102-6.

Psychometric properties of the Spence Children's anxiety scale with young adolescents. S H Spence, P M Barrett, C M Turner, 10.1016/s0887-6185(02)00236-0J Anxiety Disord. 17Spence SH, Barrett PM, Turner CM. Psychometric properties of the Spence Children's anxiety scale with young adolescents. J Anxiety Disord. 2003;17:605-25. doi:10.1016/s0887-6185(02)00236-0.

A parent-report measure of children's anxiety: psychometric properties and comparison with child-report in a clinic and normal sample. M H Nauta, A Scholing, R Rapee, M Abbott, S H Spence, A Waters, 10.1016/S0005-7967(03)00200-6Behav Res Ther. 42Nauta MH, Scholing A, Rapee R, Abbott M, Spence SH, Waters A. A parent-report measure of children's anxiety: psychometric properties and comparison with child-report in a clinic and normal sample. Behav Res Ther. 2004;42:813-39. doi:10.1016/S0005-7967(03)00200-6.

Handleiding Children's depression inventory (herziene versie). B Timbremont, C Braet, J Roelofs, Amsterdam: Pearson Assessment and Information BTimbremont B, Braet C, Roelofs J. Handleiding Children's depression inventory (herziene versie). Amsterdam: Pearson Assessment and Information B.V; 2008.

Children's Depression Inventory 2 (CDI 2). M Kovacs, Multi-Health Systems IncNorth Tonawanda, NY2nd edKovacs M. Children's Depression Inventory 2 (CDI 2). 2nd ed. North Tonawanda, NY: Multi-Health Systems Inc; 2011.

Children's Depression Inventory 2 (CDI 2). Y Bae, Test Review, M Kovacs, 10.1177/0734282911426407J Psychoed Asses. 302nd ed.Bae Y, Test Review, Kovacs M. "Children's Depression Inventory 2 (CDI 2)" (2nd ed.). J Psychoed Asses. 2012;30:304-8. doi:10.1177/0734282911426407.

The development of the Children's social behavior questionnaire: preliminary data. E F Luteijn, A E Jackson, F R Volkmar, R B Minderaa, 10.1023/a:1026060330122J Autism Dev Disord. 28Luteijn EF, Jackson AE, Volkmar FR, Minderaa RB. The development of the Children's social behavior questionnaire: preliminary data. J Autism Dev Disord. 1998;28:559-65. doi:10.1023/a:1026060330122.

Vragenlijst voor inventarisatie van sociaal gedrag van Kinderen. Herziene handleiding. C A Hartman, Amsterdam: HarcourtHartman CA et al. Vragenlijst voor inventarisatie van sociaal gedrag van Kinderen. Herziene handleiding 2007. Amsterdam: Harcourt; 2007.

Dutch version of the Strengths and Difficulties Questionnaire (SDQ). B M Van Widenfelt, A W Goedhart, Pda Treffers, R Goodman, 10.1007/s00787-003-0341-312Eur Child Adolesc PsychiatryVan Widenfelt BM, Goedhart AW, Treffers PDA, Goodman R. Dutch version of the Strengths and Difficulties Questionnaire (SDQ). Eur Child Adolesc Psychiatry. 2003;12:281-9. doi:10.1007/s00787-003-0341-3.

Vragen naar psychische problemen bij kinderen en adolescenten: de strengths and difficulties questionnaire. Maandblad Geestelijke Volksgezondheid. A Goedhart, F Treffers, B Widenfelt, 10.1007/s00787-003-0341-358Goedhart A, Treffers F, Widenfelt B. Vragen naar psychische problemen bij kinderen en adolescenten: de strengths and difficulties questionnaire. Maandblad Geestelijke Volksgezondheid. 2003;58:1018-35. doi:10.1007/s00787-003-0341-3.

Parent expectancies for therapy scale. A E Kazdin, L Holland, Child Conduct Clinic. Kazdin AE, Holland L. Parent expectancies for therapy scale. New Haven, CT: Yale university, Child Conduct Clinic; 1991.

Parent expectancies for child therapy: assessment and relation to participation in treatment. M K Nock, A E Kazdin, 10.1023/a:1016699424731J Child Fam Stud. 10Nock MK, Kazdin AE. Parent expectancies for child therapy: assessment and relation to participation in treatment. J Child Fam Stud. 2001;10:155-80. doi:10.1023/a:1016699424731.

The application of the principle of intention-to-treat to the analysis of clinical trials. D Gillings, G Koch, 10.1177/009286159102500311Drug Inform J. 25Gillings D, Koch G. The application of the principle of intention-to-treat to the analysis of clinical trials. Drug Inform J. 1991;25:411-24. doi:10.1177/009286159102500311.

Mplus user's guide. L A Muthén, B O Muthén, Muthén and MuthénLos Angeles, CA5th edMuthén LA, Muthén BO. Mplus user's guide. 5th ed. Los Angeles, CA: Muthén and Muthén; 1998-2007.


Diversity of Clostridium perfringens toxin- genotypes from dairy farms

Svenja Fohler
Guenter Klein
Martina Hoedemaker
Theresa Scheu
Christian Seyboldt
Amely Campe
Katharina Charlotte Jensen
Amir Abdulmawjood
Diversity of Clostridium perfringens toxin- genotypes from dairy farms
10.1186/s12866-016-0812-6R E S E A R C H A R T I C L E Open Access
Background: Clostridium (C.) perfringens is the causative agent of several diseases in animals and humans, including histotoxic and enteric infections. To gain more insight into the occurrence of its different toxin-genotypes in dairy herds, including those toxin genes previously associated with diseases in cattle or humans, 662 isolates cultivated from feces, rumen content and feed collected from 139 dairy farms were characterized by PCR (detecting cpa, cpb, iap, etx, cpe, and both allelic variants of cpb2).Results: Isolates from feces were assigned to type A (cpa positive, n = 442) and D (cpa and etx positive, n = 2). Those from rumen content (n = 207) and feed (n = 13) were all assigned to type A. The consensus and atypical variants of the cpb2 gene were detected in 64 (14.5 %) and 138 (31.22 %) of all isolates from feces, and 30 (14.5 %) and 54 (26.1 %) of all isolates from rumen content, respectively. Conclusion: Both allelic variants of cpb2 occurred frequently in animals without signs of acute enteric disease, whereby the atypical variant dominated. Five (0.8 %) of all type A isolates were positive for the cpe gene. Therefore, the present study indicates that dairy cows are no primary source for potentially human pathogenic enterotoxin gene positive strains.

Background Clostridium (C.) perfringens is the primary cause of a range of diseases in animals and humans [1]. Most important are gas gangrene, which occurs mainly after contamination of wounds with cells or spores [2], and enteric diseases that either solely affect the gut or in addition induce generalized symptoms, e.g. pulpy kidney disease in ruminants [3]. It is classified into five types, A to E, depending on the production of four major toxins (alpha, beta, epsilon, and iota) [3,4]. Distinct types of these are associated with certain diseases. In addition, clinical importance is attributed to the beta-2 and the enterotoxin, two of up to 12 potentially occurring minor toxins [5].

The alpha toxin is the major pathogenicity factor in gas gangrene. The coding gene (cpa) is chromosomallylocated, highly-conserved, and therefore present in all C.

perfringens strains [6]. The second major toxin that is coded by cpb is the beta toxin. It is involved in necrotic enteritis in several species, including cattle, and enterotoxaemia like lamb dysentery [4]. The third major toxin, the epsilon toxin coded by etx, is important for enterotoxaemia in sheep, goats, and less frequently in cattle [4]. Type E that produces the iota toxin, which consists of two components coded by iap and ibp, was previously associated with enterotoxaemia in calves as well as in lambs and rabbits [7].

The role of the beta-2 toxin in intestinal diseases in different animal species is still not entirely clear [4]. Until now, it has been demonstrated to be a key factor for enteritis in piglets [8,9]. The reported frequencies for cattle and the conclusions drawn from different studies concerning its role, especially in enteric disease cases, differ widely [10]. It is coded by the plasmid-borne cpb2 that occurs in two variants (consensus and atypical) [11]. The most important minor toxin for human intestinal diseases is the enterotoxin. This toxin is coded by cpe that can be located on the chromosome or on a plasmid. It causes food poisoning, antibiotic-associated diarrhea, and sporadic diarrhea [12]. In animals it was previously suspected of being associated with enteritis in several species, but its importance still remains unclear [5,13]. C. perfringens was also suspected of being involved in the development of additional diseases, including an enterotoxaemia in calves as well as an acute necro-hemorrhagic enteritis in adult cattle [14,15].

Till now studies on the diversity of C. perfringens types in mature dairy cattle testing for the coding genes of all above mentioned toxins are rare. Thus, within this study C. perfringens isolates, cultured from samples collected from a large number of dairy farms, were characterized by testing with a multiplex PCR for the four major toxin genes (cpa, cpb, iap, and etx) as well as the presence of the cpb2 (consensus and atypical variant) and cpe.

Methods

Sample collection

Fecal samples (n = 1388) from the rectum and rumen content (n = 1389) were collected from each of ten lactating cows from 139 dairy farms in Germany within a study examining the occurrence of Clostridium species in dairy herds. The study population was previously described by Seyboldt et al. [16]. Feces and rumen content were transferred into 120 and 50 ml screw cap tubes, respectively. The texture was documented for each fecal sample. Additionally, 410 feed samples were collected (up to five per farm depending on the fed ration). All samples were immediately cooled at < 8°C.

Culture conditions

Anaerobic cultivation of Clostridium isolates was performed at the Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover. Fecal and ruminal fluid samples were directly spread on Schaedler agar plates (Becton Dickinson, Heidelberg, Germany) supplemented with 10 mg/l Vit K1 (Roth, Karlsruhe, Germany) and 5 % bovine blood (WDT, Memsen, Germany), and incubated at 37°C for 48 h under anaerobic conditions. 25 g of each feed sample were transferred into 225 ml peptone water (Oxoid, Wesel, Germany) and suspended for 20 min in a shaking water bath at room temperature. Afterwards, 1 ml was transferred into liver-bouillon (Oxoid; Wesel, Germany). Additionally, 0.3-0.5 g of each feces and ruminal fluid was transferred into 8 ml liver-bouillon. Spores were selected by heating the bouillon at 80°C for 10 min in a water bath, followed by incubation at 37°C for 48 h. Afterwards, one loopfull was spread by dilution-streaking on Reinforced Clostridial Agar (Oxoid, Wesel, Germany) with 12 mg/l Kanamycin (AppliChem, Darmstadt, Germany), 75 mg/l Neomycin (Serva, Heidelberg, Germany) and 10 % bovine blood, as well as on Schaedler agar plates. These plates were then incubated anaerobically at 37°C for 48 h. Colonies suspected of being Clostridium species were transferred to pure cultures. Simultaneously, all isolates were tested for growth under aerobic conditions. Only Gram-positive or -intermediate isolates showing no growth under aerobic conditions were transferred as pure cultures on Columbia agar plates containing sheep blood (Oxoid, Wesel, Germany) to the Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover for further investigation.

DNA isolation and amplification of 16S rRNA gene

Colonies from pure cultures were transferred into 180 μl of lysis buffer (lysozyme 20 mg/ml), and incubated for 30 min at 37°C. DNA was isolated with the DNeasy Blood and Tissue Kit (Qiagen, Darmstadt, Germany) according to the manufacturer's instructions for Gram-positive bacteria. Elution was carried out with 100 μl of elution buffer. All primers applied in this study were added to the reaction mixture at a concentration of 10 pmol/μl. PCR reaction mixtures for amplification of the 16S rRNA gene consisted of 15 μl mastermix (RedˈyˈGold Mix, Eurogentec, Cologne, Germany), 1 μl primer 16SUNI-L (Table 1), 1 μl primer 16SUNI-R, 0.5 μl MgCl 2 (50 mM), 7.5 μl nuclease free water, and 5 μl sample DNA. The thermocycling program started with an initial denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 45 s. Amplification products (5 μl) were loaded onto a 1.5 % agarose gel (Pequlab, Erlangen, Germany), and electrophoresis was performed for 45 min at 10 volt/cm. The remaining 25 μl of amplification product were used for sequencing in both directions (Eurofins genomics, Ebersberg, Germany) with sequencing primers 533 F and 907R (Table 1). All sequences were aligned against two gene databases, NCBI-BLAST (database for 16S ribosomal RNA sequences (Bacteria and Archaea) using the Megablast Tool; http://blast.ncbi.nlm.nih.gov/Blast.cgi? PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_ LOC=blasthome) and SepsiTest™-BLAST (http://www.sep sitest-blast.de/de/index.html).

Toxin-genotyping

All isolates identified as C. perfringens were further genotyped using two multiplex PCR assays with primers developed and validated by Baums et al. [17]. Each 25-μl reaction mixture for detection of the four major toxin genes contained 12.5 μl of mastermix (RedˈyˈGold Mix); 0.5 μl of nuclease-free water; 0.5 μl of each primer CPA5L, CPA5R, CPIL, CPIR, ETXL, ETXR; 1 μl of each primer CPB1L, CPB1R; 2 μl MgCl 2 (50 mM); and 5 μl DNA. PCR assays for detection of the two minor toxin genes cpe and cpb2 were carried out with mixtures containing 12.5 μl of mastermix (Red'y'Gold Mix), 2.5 μl of nuclease-free water, 1 μl of primer CPEL, 1 μl primer CPER, 0.5 μl primer CPB2L, 0.5 μl primer CPB2R, 2 μl MgCl2, and 5 μl DNA. The amplification program of both assays started with an initial denaturation at 95°C for 2 min, followed by 35 cycles of 20 s at 94°C, 20 s at 55°C and 30 s at 72°C. For positive controls, in-house collection strains were used (type A: CCUG 1795 T; type B: CCUG 2035; type C: CCUG 2036; type D: CCUG 2037; type E: CCUG 44727). Isolates that were negative for all toxin genes were additionally tested with another primer set for the presence of the cpa gene (Table 2) [18]. Reaction mixtures contained 12.5 μl mastermix (RedˈyˈGold Mix), each 0.5 μl of forward and reverse primer, 0.6 μl MgCl 2 (50 mM), 10.9 μl nuclease-free water, and 5 μl of sample DNA. Amplification was carried out after denaturation for 5 min at 94°C; with 35 cycles comprising 94°C for 30 s, 55°C for 30 s and 72°C for 30 s; and a final elongation at 72°C for 10 min. As the cpb2 primers used in the duplex assay only target the consensus variant of the gene, all isolates were additionally tested with the PCR assay developed by Jost et al. [11], to target both allelic variants, consensus (cpb2con) and atypical (cpb2aty). Reaction mixtures contained of 12.5 μl mastermix (RedˈyˈGold Mix), 0.5 μl of each primer (CPB2ATYPF, CPB2CONF, CPB2R), 0.6 μl MgCl 2 (50 mM), 7.9 μl nuclease-free water, and 2.5 μl of sample DNA. Amplification was carried out after denaturation for 5 min at 94°C; with 35 cycles comprising 94°C for 30 s, 55°C for 1 min and 72°C for 1 min; and a final elongation at 72°C for 10 min. The positive control for the consensus gene was the above mentioned type A strain (CCUG 1795 T). A field isolate of the in- house collection (LMQS-BP-8160, bovine type A) served as positive control for the atypical cpb2 variant. Separation of PCR products was performed for all typing assays on 2.0 % agarose gels.

Results

In total, 662 isolates of C. perfringens were cultured from samples from 129 farms. Of these, 442 originated from feces (from 378 individuals without changes in fecal texture and from 16 cows with diarrhea) and 207 from ruminal fluid (from 192 individuals). The 13 feed isolates were obtained from one potato pulp, one pressed pulp silage, and all others from grass silages cut at different times during the year and stored for variable periods, ranging from 8 up to 72 weeks.

With the PCR developed by Baums et al. [17], 17 (2.57 %) of the C. perfringens isolates were negative for all six toxin genes, also with repetition. Additional testing with the primers developed by Schlapp et al. [18] lead to a cpa positive result for all these 17 isolates. A positive result for the consensus cpb2 gene was obtained with the duplex PCR for 31 of the 662 isolates [17]. In contrast, with the PCR that was additionally applied to target both allelic variants [11], 94 isolates (including all 31 positive using the duplex PCR assay) and 194 isolates were positive for the consensus and the atypical cpb2 gene, respectively.

Solely positive for one major toxin gene (cpa) and therefore assigned to type A of C. perfringens were 660 isolates. Two isolates were assigned to type D (positive for cpa and etx). Additionally, the consensus and atypical variants of the cpb2 gene were present in 64 (14.5 %) and 138 (31.2 %) of all isolates from feces, and 30 (14.5 %) and 54 (26.1 %) of all obtained from rumen content, respectively. Of the type A isolates, 5 (0.8 %) were positive for cpe. None carried cpb or iap. Table 3 shows the occurring types in individual samples. This study was not carried out to determine differences between cows with diarrhea and those without. However, after clinical examination when fecal samples were collected, diarrhea was observed in some (n = 16) animals. This was considered as potential sign of acute enteric disorder. According to the sampling scheme feces were collected and investigated also from these cows. To exclude study bias toxin typing results of fecal isolates from these animals are given separately in Table 3. No considerable differences were observed between typing results for fecal samples from cows showing diarrhea and those without. Nevertheless, the number of investigated isolates from animals with diarrhea was too small to draw conclusions on differences.

Discussion

Studies on the occurring types of C. perfringens in mature dairy cattle including all toxin genes investigated within this study are rare. For the presence of the cpb2 and the cpe gene broad differences were reported for cattle [19][20][21][22]. Therefore, 662 C. perfringens isolates from dairy cattle were toxin genotyped to gain further insight into the presence of its different types on dairy farms.

Mainly C. perfringens type A was detected. This is the most frequently isolated type in samples of human, animal or environmental origin [3]. Only two isolates (0.3 %) of all were designated to be C. perfringens type D (cpa and etx positive), which is known to cause enterotoxaemia in small ruminants. Additionally, reports exist which assume that it plays a role in disease in calves [5,23,24]. Both investigated type D positive cows showed no signs of acute disorders, similar to those described for disease in small ruminants affected by type D. Within this study, none of the investigated isolates carried genes for the beta or the iota toxin found in types B, C and/or E. Though, for example, iota toxin gene positive C. perfringens (type E) were previously detected in healthy calves as well as in those suffering from diarrhea [25].

(100)

type A = cpa + ; type Aβ2c = cpa + + cpb2con + ; type Aβ2a = cpa + + cpb2aty + ; type Ae = cpa + + cpe + ; type D = cpa + + etx + ; type Dβ2c = cpa + + etx + + cpb2con + Isolation of more than one genotype from single samples is indicated by listing each genotype separated by a "+"

The beta-2 toxin is known to be associated with enteritis in piglets and a causative role of it in enteric disease of calves was assumed [8,9,26]. Of all isolates cultured in this study, 202 (45.7 %) and 82 (39.6 %) obtained from feces and rumen content harbored the cpb2 gene, respectively. A similar rate (46 %) was found by Lebrun et al. who investigated isolates obtained from 14 healthy calves [27]. A screening conducted in North America for cpb2 in field isolates obtained from clinical laboratories detected the gene in 12.8 % of all isolates from cattle (including isolates from animals of all ages with and without enteric disease) [19]. Within that study a much higher rate of 47.3 % in isolates from calves suffering from enteric disease was found. This was mainly due to the high detection rate in type E isolates (97.3 %). A previous study conducted in Belgium, detected this gene in around 30 % of all isolates that were obtained equally from around 60 % of all healthy and diarrheic calves [25]. Gurjar et al. investigated feces of cows from seven dairy farms and found the toxin gene in 68 % of all C. perfringens positive fecal samples [20]. Within the present study we distinguished between both allelic variants of cpb2. The so called atypical cpb2 was detected as well in feces as in rumen content about twice as often as the consensus variant. Previous studies found that in porcine isolates the consensus variant occurs more frequently [11]. In contrast, in isolates of non-porcine origin the atypical variant is more common, whereby variations exist between different animal species [11,28]. Until now mainly small numbers of isolates obtained from cattle have been investigated discriminating between the two allelic variants of this gene. Jost et al. tested 24 isolates from cattle and found the atypical cpb2 in seven of ten type A, both type C and 12 of 12 type E strains [11]. Kircanski et al. tested bovine isolates and found the consensus and the atypical variant in 1 and 5 of 11 isolates, respectively [28]. The study from Lebrun et al. focusing on calves suffering from diarrhea detected in these solely the consensus variant [27]. Also, in isolates obtained from eight healthy calves the consensus variant (67.5 %) was detected more frequently than the atypical allele (32.5 %). One larger study was carried out on 218 bovine C. perfringens isolates from healthy cattle and animals suffering from acute enteric disease [29]. Deviating from the present study, Schlegel et al. found in 13 % of all isolates the atypical variant, but in none the consensus cpb2. Despite the varying detection frequencies, our results and those of previous studies demonstrate that both allelic variants occur frequently in cattle, whereby the atypical cpb2 seems to dominate.

Human enteric diseases, due to C. perfringens, are mainly caused by strains producing the enterotoxin [4]. These food poisoning strains are among those pathogens most frequently associated with foodborne illnesses [12,30]. cpe was only detected in five (0.8 %) of all investigated isolates in the present study indicating that dairy cows are no primary source for these food poisoning strains. Kukier et al. reported similar findings of 0.34 % cpe positive isolates from bovine fecal samples [21]. A higher rate was reported by Gurjar et al. [20], who found the enterotoxin gene in 4.6 % of all PCR positive fecal samples from dairy cows in Pennsylvania, USA. Miwa et al. even reported a rate of 26 % cpe positive feces of healthy slaughter cattle in Japan using nested PCR [22]. Most studies on the epidemiology of enterotoxigenic C. perfringens in farm animals did not recover isolates, and therefore Lindstroem et al. presumed that maybe unspecific amplification lead to the finding of high rates [12,22]. In contrast, within the present study only cultured isolates confirmed to be C. perfringens were tested for the presence of cpe. Differences between studies may also display the reported divergence in detection of C. perfringens toxin types in sample material of different geographical locations, as suggested for cpb2 positive isolates [10].

C. perfringens was found not only ubiquitous in feces but also in rumen content of cows within this study. This is to the authors knowledge the first investigation, that toxinotyped such a large number of C. perfringens isolates (n = 207) obtained from rumen content of cows. From this matrix only type A was cultured. A few isolates were tested positive for cpe and a larger amount for consensus or atypical cpb2.

Due to the culture conditions, not specifically selective for C. perfringens, isolates were obtained from fewer samples than it could have been expected with a more selective method. Nonetheless, most previous studies tested field isolates (often from animals with acute enteric disease) or isolates from calves. In contrast, this study examined a large number of isolates obtained from mature dairy cattle. Diarrhea was considered within this study as a sign of acute enteric disease. Thus, typing results of fecal samples of these animals are presented separately (Table 3), although the study was not carried out to determine differences between cows with diarrhea and those without. No considerable differences were detected between fecal samples of animals showing diarrhea and those without.

With the PCR developed and validated by Baums et al. [17], 17 (2.57 %) of all C. perfringens, that had previously been identified on the basis of 16S rRNA gene sequencing, gave negative results for the cpa gene. As it is known to be chromosomally coded and highly conserved, all C. perfringens isolates are expected to carry this gene [1]. Therefore, an additional PCR was applied using previously published primers to test the negative isolates for the presence of the cpa gene. With the primers developed by Schlapp et al. [18] all isolates that were negative with the first primer set gave positive results. Also other authors found small numbers of presumed cpa negative isolates of animal origin [29,31]. When tested with another PCR, these isolates of those previous studies may also give positive results. A possible reason for these negative PCR outcomes may be nucleotide variations in the primer binding region [22]. C. perfringens confirmation is often done by detection of the cpa gene. Considering that around 2.6 % of the investigated isolates gave false negative results using one PCR assay, this point needs attention. The primer set for cpb2 detection used in the duplex assay [17] only detects the consensus variant of the cpb2 gene. Thus, we additionally applied a PCR that distinguishes between the consensus and the atypical variant. We found differing results between the two primer sets targeting the consensus variant of the cpb2 gene published by Baums et al. [17] and Jost et al. [11]. With the primers used in the duplex assay 31 isolates were tested positive for the consensus cpb2, whereas with the more recent primer set described by Jost et al. 94 isolates (including all 31 isolates positive with the first assay) gave a positive result. A previous study investigated the genetic diversity of cpb2 genes and found for the consensus variant sequence similarities between 93.3 and 100 % [32]. Thus, apart from the genetically low relatedness between the consensus and the atypical variant also within the consensus allele nucleotide variations exist. This could explain the differing results obtained with both assays.

Conclusion

The diversity of C. perfringens types was low as mainly type A was cultivated. Of all isolates from feces and rumen content 202 (45.7 %) and 82 (40.6 %) harbored one variant of the cpb2 gene, respectively. The atypical allele occurred about twice as often as the consensus variant. The enterotoxin gene was present in only 0.8 % of all isolates. Thus, the present study indicates that dairy cows are no primary source for potentially human pathogenic enterotoxin gene positive strains.

Table 1
1Oligonucleotide primer for 16S rRNA gene amplification and sequencingIndicating the position of each primer relative to the 16S rRNA gene sequence of E. coli J01859Primer
Sequence (5′-3′)
Location c

16S rRNA gene amplification primer a

16SUNI-L
AGAGTTTGATCATGGCTCAG
8-27

16SUNI-R
GTGTGACGGGCGGTGTGTAC
1410-1391

16S rRNA gene sequencing primer b

533F
GTGCCAGCAGCCGCGGTAA
514-532

907R
CCGTCAATTCMTTTGAGTTT
925-906

a

Kuhnert P, Capaul SE, Nicolet J, Frey J. Phylogenetic positions of Clostridium
chauvoei and Clostridium septicum based on 16S rRNA gene sequences. Int J
Syst Bacteriol. 1996;46:1174-1176

b

Petti CA. Detection and identification of microorganisms by gene
amplification and sequencing. Clin Infect Dis. 2007;44:1108-1114

c

Table 2
2Oligonucleotide primers for C. perfringens toxin gene detection Schlapp T, Blaha I, Bauerfeind R, Wieler LH, Schoepe H, Weiss R, Baljer G. Synthesis and evaluation of a nonradioactive gene probe for the detection of Clostridium-perfringens alpha-toxin. Mol Cell Probe. 1995;9:101-109 Jost BH, Billington SJ, Trinh HT, Bueschel DM, Songer JG. Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin.Primer
Sequence (5′-3′)
Amplificationproduct
size [bp]

Primer concentration
[mM]

Toxin
Coding toxin
gene

Multiplex PCR (major toxin genes) a

CPA5L
AGTCTACGCTTGGGATGGAA
900
200
alpha
cpa

CPA5R
TTTCCTGGGTTGTCCATTTC
200

CPBL
TCCTTTCTTGAGGGAGGATAAA
611
400
beta
cpb

CPBR
TGAACCTCCTATTTTGTATCCCA
400

CPIL
AAACGCATTAAAGCTCACACC
293
200
iota
iap

CPIR
CTGCATAACCTGGAATGGCT
200

CPETXL
TGGGAACTTCGATACAAGCA
396
200
epsilon
etx

CPETXR
TTAACTCATCTCCCATAACTGCAC
200

Duplex PCR (minor toxin genes) a

CPEL
GGGGAACCCTCAGTAGTTTCA
506
400
entero
cpe

CPER
ACCAGCTGGATTTGAGTTTAATG
400

CPB2L
CAAGCAATTGGGGGAGTTTA
200
200
beta-2
cpb2

CPB2R
GCAGAATCAGGATTTTGACCA
200

Singleplex PCR for cpa detection b

224
AGGAACTCATGCTATGATTGTAACTCAAGG
775
200
alpha
cpa

972inverse
ACCACTAGTTGATATGTAAGCTACTAG
200

PCR for cpb2 variants c

CPB2ATYPF
ATTATGTTTAGGAATACAGTTA
304
200
beta-2 atypical
cpb2aty

CPB2CONF
CAATTGGGGGAGTTTATCCACAA
741
200
beta-2 consensus
cpb2con

CPB2R
CAATACCCTTCACCAAATACTC
200

a

Baums CG, Schotte U, Amtsberg G, Goethe R. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet Microbiol. 2004;100:11-16

b

c

Infect
Immun. 2005;73(1):652-6

Table 3
3Genotypes of C. perfringens present in feces, rumen content and feedC. perfringens types

Sample material
A
Aβ2c
Aβ2a
Ae
A + Aβ2c
A + Aβ2a
A + Ae
Aβ2c + Aβ2a
D
Dβ2c
Total

AcknowledgementsThe authors wish to thank the veterinarians Annika Wichern, Frieder Gundling and Phuong Do Duc from the Clinic for Cattle, University of Veterinary Medicine Hannover for the elaborative animal examination and sampling. Thanks also go to Mike Schiwek and Kevin Schenke from the Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover for their excellent technical assistance.FundingThis work was financially supported by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE), grant numbers 2810HS005 and 2810HS036.Availability of data and materialThe manuscript includes all used materials and data that is of interest for potential readers.Authors' contributions MH, AC, GK, AA and CS were involved in the conception and design of the study. MH, TS, KJ visited the farms and collected sample material. SF and AA conducted the laboratory investigations (DNA isolation, PCR assays and data interpretation). SF and KJ did the analysis of results. SF, AA and CS were involved in drafting of the manuscript. MH, GK, AA and AC performed the critical revision of the manuscript and gave important intellectual advice for approval. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publication Not applicable.Ethics approval and consent to participate The investigations and examinations on animals were performed according to the German Animal Welfare Act with permission of the respective authorities (Lower Saxony, Lower Saxony State Office for Consumer Protection and Food Safety LAVES, reg. no. 13a308). All farmers participated voluntarily and gave their written consent to collect samples from their animals. Submit your next manuscript to BioMed Central and we will help you at every step:
Clostridium perfringens: toxinotype and genotype. L Petit, M Gibert, M R Popoff, Trends Microbiol. 7Petit L, Gibert M, Popoff MR. Clostridium perfringens: toxinotype and genotype. Trends Microbiol. 1999;7:104-10.

Life-threatening clostridial infections. D L Stevens, M J Aldape, A E Bryant, Anaerobe. 18Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe. 2012;18:254-9.

Clostridial enteric diseases of domestic animals. J G Songer, Clin Microbiol Rev. 3Songer JG. Clostridial enteric diseases of domestic animals. Clin Microbiol Rev. 1996;3:216-34.

Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. F A Uzal, J C Freedman, A Shrestha, J R Theoret, J Garcia, M M Awad, V Adams, R J Moore, J I Rood, B A Mcclane, Future Microbiol. 9Uzal FA, Freedman JC, Shrestha A, Theoret JR, Garcia J, Awad MM, Adams V, Moore RJ, Rood JI, McClane BA. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol. 2014;9:361-77.

Clostridium perfringens toxins involved in mammalian veterinary diseases. F A Uzal, J E Vidal, B A Mcclane, A A Gurjar, Open Toxinology J. 2Uzal FA, Vidal JE, McClane BA, Gurjar AA. Clostridium perfringens toxins involved in mammalian veterinary diseases. Open Toxinology J. 2010;2:24-42.

Genome organization of the anaerobic pathogen Clostridium perfringens. B Canard, Cole St, Proc Natl Acad Sci U S A. 86Canard B, Cole ST. Genome organization of the anaerobic pathogen Clostridium perfringens. Proc Natl Acad Sci U S A. 1989;86:6676-80.

Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature. J G Songer, D W Miskimmins, Anaerobe. 10Songer JG, Miskimmins DW. Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature. Anaerobe. 2004;10:239-42.

Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined using genotyping and subtyping PCR assays. H S Garmory, N Chanter, N P French, D Bueschel, J G Songer, R W Titball, Epidemiol Infect. 124Garmory HS, Chanter N, French NP, Bueschel D, Songer JG, Titball RW. Occurrence of Clostridium perfringens beta2-toxin amongst animals, determined using genotyping and subtyping PCR assays. Epidemiol Infect. 2000;124:61-7.

Beta2 toxin, a novel toxin produced by Clostridium perfringens. M Gibert, C Jolivet-Renaud, M R Popoff, Gene. 203Gibert M, Jolivet-Renaud C, Popoff MR. Beta2 toxin, a novel toxin produced by Clostridium perfringens. Gene. 1997;203:65-73.

The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the β2-toxin in enteric disease of domestic animals, wild animals and humans. A J Van Asten, G N Nikolaou, A Gröne, Vet J. 183van Asten AJ, Nikolaou GN, Gröne A. The occurrence of cpb2-toxigenic Clostridium perfringens and the possible role of the β2-toxin in enteric disease of domestic animals, wild animals and humans. Vet J. 2010;183:135-40.

Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin. B H Jost, S J Billington, H T Trinh, D M Bueschel, J G Songer, Infect Immun. 73Jost BH, Billington SJ, Trinh HT, Bueschel DM, Songer JG. Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin. Infect Immun. 2005;73:652-6.

Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. M Lindström, A Heikinheimo, P Lahti, H Korkeala, Food Microbiol. 28Lindström M, Heikinheimo A, Lahti P, Korkeala H. Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiol. 2011;28:192-8.

A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea. J S Weese, H R Staempfli, J F Prescott, Equine Vet J. 33Weese JS, Staempfli HR, Prescott JF. A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea. Equine Vet J. 2001;33:403-9.

Hemorrhagic bowel syndrome in dairy cattle: 22 cases. A C Dennison, D C Vanmetre, R J Callan, P Dinsmore, G L Mason, R P Ellis, J Am Vet Med Assoc. 221Dennison AC, VanMetre DC, Callan RJ, Dinsmore P, Mason GL, Ellis RP. Hemorrhagic bowel syndrome in dairy cattle: 22 cases (1997-2000). J Am Vet Med Assoc. 2002;221:686-9.

Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves. C Manteca, G Daube, V Pirson, B Limbourg, A Kaeckenbeeck, J G Mainil, Vet Microbiol. 81Manteca C, Daube G, Pirson V, Limbourg B, Kaeckenbeeck A, Mainil JG. Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves. Vet Microbiol. 2001;81:21-32.

Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows. C Seyboldt, S Discher, Jordan E Neubauer, H Jensen, K C Campe, A Kreienbrock, L Scheu, T Wichern, A Gundling, F Doduc, P Fohler, S Abdulmawjood, A Klein, G Hoedemaker, M , Vet Microbiol. 177Seyboldt C, Discher S, Jordan E, Neubauer H, Jensen KC, Campe A, Kreienbrock L, Scheu T, Wichern A, Gundling F, DoDuc P, Fohler S, Abdulmawjood A, Klein G, Hoedemaker M. Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows. Vet Microbiol. 2015;177:398-402.

Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. C G Baums, U Schotte, G Amtsberg, R Goethe, Vet Microbiol. 100Baums CG, Schotte U, Amtsberg G, Goethe R. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet Microbiol. 2004;100:11-6.

Synthesis and evaluation of a nonradioactive gene probe for the detection of Clostridium perfringens alpha-toxin. T Schlapp, I Blaha, R Bauerfeind, L H Wieler, H Schoepe, R Weiss, G Baljer, Mol Cell Probe. 9Schlapp T, Blaha I, Bauerfeind R, Wieler LH, Schoepe H, Weiss R, Baljer G. Synthesis and evaluation of a nonradioactive gene probe for the detection of Clostridium perfringens alpha-toxin. Mol Cell Probe. 1995;9:101-9.

Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype. D M Bueschel, B H Jost, S J Billington, H T Trinh, J G Songer, Vet Microbiol. 94Bueschel DM, Jost BH, Billington SJ, Trinh HT, Songer JG. Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype. Vet Microbiol. 2003;94:121-9.

Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle. A A Gurjar, N V Hegde, B C Love, B M Jayarao, Mol Cell Probes. 22Gurjar AA, Hegde NV, Love BC, Jayarao BM. Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle. Mol Cell Probes. 2008;22:90-5.

Occurrence of Clostridium Perfringens in Food Chain. E Kukier, K Kwiatek, Bull Vet Inst Pulawy. 54Kukier E, Kwiatek K. Occurrence of Clostridium Perfringens in Food Chain. Bull Vet Inst Pulawy. 2010;54:571-6.

Most probable numbers of enterotoxigenic Clostridium perfringens in intestinal contents of domestic livestock detected by nested PCR. N Miwa, T Nishina, S Kubo, H Honda, J Vet Med Sci. 59Miwa N, Nishina T, Kubo S, Honda H. Most probable numbers of enterotoxigenic Clostridium perfringens in intestinal contents of domestic livestock detected by nested PCR. J Vet Med Sci. 1997;59:557-60.

Clostridium perfringens type D epsilon intoxication in one-day-old calves. P J Watson, S F Scholes, Vet Rec. 164Watson PJ, Scholes SF. Clostridium perfringens type D epsilon intoxication in one-day-old calves. Vet Rec. 2009;164:816-7.

Enterotoxemia in newborn calves due to C. perfringens types A, C and D. R Lulov, A K Angelov, Vet Med Nauki. 23Lulov R, Angelov AK. Enterotoxemia in newborn calves due to C. perfringens types A, C and D. Vet Med Nauki. 1986;23:20-8.

Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea. M C Ferrarezi, T C Cardoso, I S Dutra, Anaerobe. 14Ferrarezi MC, Cardoso TC, Dutra IS. Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea. Anaerobe. 2008;14:328-31.

A role for the Clostridium perfringens beta 2 toxin in bovine enterotoxaemia?. C Manteca, G Daube, T Jauniaux, A Linden, V Pirson, J Detilleux, A Ginter, P Coppe, A Kaeckenbeeck, J G Mainil, Vet Microbiol. 86Manteca C, Daube G, Jauniaux T, Linden A, Pirson V, Detilleux J, Ginter A, Coppe P, Kaeckenbeeck A, Mainil JG. A role for the Clostridium perfringens beta 2 toxin in bovine enterotoxaemia? Vet Microbiol. 2002;86:191-202.

The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome. M Lebrun, P Filee, B Mousset, D Desmecht, M Galleni, J G Mainil, A Linden, Vet Microbiol. 120Lebrun M, Filee P, Mousset B, Desmecht D, Galleni M, Mainil JG, Linden A. The expression of Clostridium perfringens consensus beta2 toxin is associated with bovine enterotoxaemia syndrome. Vet Microbiol. 2007;120:151-7.

The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed. J Kircanski, V R Parreira, S Whiteside, Y Pei, J F Prescott, Vet Microbiol. 159Kircanski J, Parreira VR, Whiteside S, Pei Y, Prescott JF. The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed. Vet Microbiol. 2012;159:371-4.

Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome. B J Schlegel, V J Nowell, V R Parreira, G Soltes, J F Prescott, JHSSchlegel BJ, Nowell VJ, Parreira VR, Soltes G, Prescott JF. Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS).

. Can J Vet Res. 76Can J Vet Res. 2012;76:248-54.

Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens. J E Grass, L H Gould, B E Mahon, Foodborne Pathog Dis. 10Grass JE, Gould LH, Mahon BE. Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998-2010. Foodborne Pathog Dis. 2013;10:131-6.

The presence of toxin genes of Clostridium perfringens isolated from camels and humans in Egypt. M E Mohamed, I I Suelam, M A Saleh, Vet Arhiv. 80Mohamed ME, Suelam II, Saleh MA. The presence of toxin genes of Clostridium perfringens isolated from camels and humans in Egypt. Vet Arhiv. 2010;80:383-92.

Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge. A Johansson, A Aspan, E Bagge, V Baverud, B E Engstrom, K E Johansson, BMC Microbiol. 647Johansson A, Aspan A, Bagge E, Baverud V, Engstrom BE, Johansson KE. Genetic diversity of Clostridium perfringens type A isolates from animals, food poisoning outbreaks and sludge. BMC Microbiol. 2006;6:47.