Bovine respiratory syncytial virus in experimentally exposed and rechallenged calves; viral shedding related to clinical signs and the potential for transmission

Thea Blystad Klem
Siri Kulberg Sjurseth
Ståle Sviland
Britt Gjerset
Mette Myrmel
Maria Stokstad
Bovine respiratory syncytial virus in experimentally exposed and rechallenged calves; viral shedding related to clinical signs and the potential for transmission
10.1186/s12917-019-1911-zR E S E A R C H A R T I C L E Open Access
Background: Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen worldwide, detrimentally affecting the economy and animal welfare. To prevent and control BRSV infection, further knowledge on virus shedding and transmission potential in individual animals is required. This study aimed to detect viral RNA and infective virions during BRSV infection to evaluate duration of the transmission period and correlation with clinical signs of disease. The outcome of BRSV re-exposure on calves, their housing environment and effect of introduction of sentinel calves was also investigated. A live animal experiment including 10 calves was conducted over 61 days. Initially, two calves were inoculated with a non-passaged BRSV field isolate. Two days later, six naïve calves (EG: Exposed group) were introduced for commingling and four weeks later, another two naïve calves (SG: Sentinel group) were introduced. Seven weeks after commingling, EG animals were re-inoculated. Clinical examination was performed daily. Nasal swabs were collected regularly and analysed for viral RNA by RT-ddPCR, while virus isolation was performed in cell culture. BRSV serology was performed with ELISA.Results: All the EG calves seroconverted and showed clinical signs of respiratory disease. Viral RNA was detected from days 1-27 after exposure, while the infective virus was isolated on day 6 and 13. On day 19, all animals were seropositive and virus could not be isolated. Total clinical score for respiratory signs corresponded well with the shedding of viral RNA. The SG animals, introduced 27 days after exposure, remained negative for BRSV RNA and stayed seronegative throughout the study. Inoculation of the EG calves seven weeks after primary infection did not lead to new shedding of viral RNA or clinical signs of disease.Conclusion: Viral RNA was detected in nasal swabs from the calves up to four weeks after exposure. The detection and amount of viral RNA corresponded well with the degree of respiratory signs. The calves were shedding infective virions for a considerable shorter period, and naïve calves introduced after four weeks were not infected. Infected calves were protected from reinfection for at least seven weeks. This knowledge is useful to prevent spread of BRSV.

Background

Bovine respiratory syncytial virus (BRSV) is an important respiratory pathogen in cattle, detrimentally affecting the economy and animal welfare. The virus is distributed worldwide and is a major pathogen of the bovine respiratory disease complex [1,2]. Viral respiratory infections are also of concern with regards to antibiotic resistance, as they predispose cattle to secondary bacterial infections that are commonly treated with antibiotics [3]. Bovine respiratory disease is traditionally handled with management measures, vaccination and metaphylactic antibiotic treatment [4]. Another possible strategy is to prevent inter-herd transmission of the main pathogens by increasing biosecurity measures at herd level. Because live animal transport is considered one of the main modes of BRSV transmission between herds [5,6], proper mitigation must ensure that live animal transport be performed without compromising biosecurity. This requires knowledge on transmission risk associated with animal contact at different stages of infection. Knowledge of BRSV shedding related to clinical features would also be useful in order to assess the transmission risk of an infected herd without the use of viral diagnostic assays. For both of these areas, several knowledge gaps exists. Although way of infection may affect both viral shedding and clinical signs compared to naturally exposed animals, challenge studies are superior in the sense that aetiology and time of exposure is known and clinical features and virus excretion can be followed closely. Challenge studies, many of them aiming to evaluate the efficacy of vaccines [7][8][9][10][11] seldom last longer than one to two weeks. Grissett et al. [12] and Gershwin [13] concluded that shedding of BRSV begins on day three or four post-infection (p.i.) and usually lasts until day nine or ten. Grissett et al. [12] summarized that the median time to appearance, peak and resolution of clinical signs was 3, 6 and 12 days, respectively, based on information from 22 inoculation studies [7][8][9][10][11][14][15][16][17][18][19][20][21][22]. As studies outlasting the acute phase of infection are lacking, it is not known how long an animal can transmit infectious viruses to other animals. Appearance of clinical signs is usually the only information available in the field, and finding a clinical parameter that indicates shedding of infectious BRSV would be valuable. The existence of chronic or persistent infections in individuals is likewise still unclear [23][24][25][26].

During the acute phase of a BRSV infection, immunological protection develops, but it is assumed to be short-lived [27]. This might enable early reinfection and new shedding of the infective virus, which complicates the risk assessment. A few BRSV studies have been performed to shed light on this. In a study by Kimman et al. [23] they reported a strong local IgA response in the respiratory tract, but no virus shedding, when calves were re-exposed 3-4 months after primary BRSV infection. Stott et al. [28] indicated, referring to their own unpublished results, that reinfection in calves and heifers may occur as early as three weeks post-infection. However, early reinfection with BRSV is not well-documented, and more precise knowledge of the occurrence is needed.

The existing literature on BRSV shedding and transmission is based on various laboratory methods, such as detection of viral RNA and culturing of the virus. Although resource-demanding, virus transmission studies are preferably performed using live animals in sentinel trials.

The aim of the present study was, therefore, to study basic features of BRSV infection in calves infected by exposure to BRSV-shedding calves. This was performed by:

Methods

Study design

A descriptive live animal experiment with a total of 10 calves was performed. The experimental units were groups of calves, and the total experimental period was 61 days. All the calves were euthanized by the end of the experiment. The groups were designated as Inoculated group (IG: calf I 1 and I 2 ), Exposed group (EG: calf E 1 -E 6 ), and Sentinel group (SG: calf S 1 and S 2 ). The intervention for IG was inoculation with BRSV, while for EG it was exposure to IG from their acute stage until 26 days after challenge (D26) and inoculation with BRSV on D49. The intervention for SG was exposure to EG and their environment 27 days after first exposure to BRSV (D27). The read-out was presence of BRSV RNA, infective BRSV, anti-BRSV antibodies, clinical signs, weight gain and pathological lesions. Each single animal was used as its own control, mainly to detect whether the calves were clinically healthy, had an average weight gain and did not have antibodies against relevant respiratory viruses (BRSV, BCoV and BPIV3) before exposure to BRSV. The experiment was performed as an open-label trial, as blinding was not practically achievable. A timeline of the experimental period with an overview of interventions is provided in Fig. 1.

Housing and husbandry

The experiment was conducted in the Laboratory Animal Unit at the Norwegian Veterinary Institute in Oslo, in a BSL2 facility consisting of three housing units with separate ventilation systems, and separate entrances with sluice rooms. In the sluice, the clothes and boots of the personnel were changed and hands were washed routinely, both before and after entrance to the calves. Each unit was provided with separate equipment for care and sampling and had one free-range housing pen with ad libitum water supply. The pens were bedded with sawdust and cleaned manually by a technician once a day. The pen where the IG was inoculated was 7 m 2 and the two other pens were 8 m 2 each. Roughage was fed ad libitum and a commercial concentrate formula was allotted twice a day. The allotment of concentrate started at 1 kg per day, gradually increasing to 2 kg within the first three weeks. From three weeks and throughout the study period, the ration was stable at 2 kg per day.

Experimental animal arrangement

Ten BRSV seronegative, conventionally reared and weaned Norwegian Red calves aged 2-4 months were included. The two heifer and eight bull calves were obtained from three private Norwegian dairy farms situated in the southeastern part of Norway, in proximity to the BSL2 facility. The allocation of animals to experimental groups was random with the exception that calves of different age were mixed to avoid age bias. The first eight calves were initially housed in three different rooms. After the 11-day acclimatization period, the two IG calves (room 3), were inoculated with BRSV (D-2 of the experiment). Two days later (D0), I 1 and I 2 were moved to room 1 and 2, respectively, each holding three EG calves. The EG calves were housed together with the IG calves until D26, when I 1 and I 2 were euthanized. On D27, S 1 and S 2 were brought in to rooms 1 and 2, respectively, where the EG calves were housed. On D48, the two SG calves were moved to a separate room (room 3). At the same time, the EG calves were rearranged to induce mild stress: Two of three calves in each room were moved to the other room, and rechallenged by inoculation with BRSV. On D50, S 1 and S 2 were moved back to room 1 and 2, respectively. On D61, the remaining eight calves were euthanized.

Inoculum and inoculation procedure

Prior to inoculation, a BRSV field isolate O4-4B/N/11 earlier described by Klem et al. [29] was centrifuged at 1200 x g and filtered through a 0.45 μm filter. To exclude the possibility of bacterial contamination the filtrate was seeded out on blood agar plates and cultured for 24 h at 37°C. The filtrate was also tested by RT-PCR for the presence of BRSV, bovine parainfluenza-3 virus (BPIV-3) [29] and bovine corona virus (BCoV) (in-house method, Norwegian Veterinary Institute, Oslo, Norway). The filtrate was positive for BRSV with a Ct-value of 21.4 and negative for BCoV, BPIV-3 and bacteria. The inoculum was prepared by diluting the filtrate 1:5 with sterile NaCl.

The inoculation procedure was performed on non-sedated calves via manual restraint. The head was kept in a horizontal position while 5 ml inoculum was nebulized intranasally by use of a perforated plastic tube with a diameter of 1.57 mm and an inner channel of 1.14 mm. The tube was inserted 15 cm into the left nostril. After nebulization, the left nostril was closed by hand to avoid outflow of the inoculum.

Treatment procedures

Moderately to severely depressed calves with a high respiratory rate (≥ 65/min) and temperature ≥ 40.0°C were treated with 0.5 mg/kg meloxicam for pain relief and anti-inflammatory effect. When bacterial pneumonia was suspected, 20 mg/kg procain benzylpenicillin was administered IM once daily for five days. Euthanasia was performed by stunning with a captive bolt, followed by bleed-out.

Clinical observations

Qualified personnel monitored the calves morning and evening, and more frequently if deemed necessary. A veterinarian examined all calves on a daily basis until D10, and three to four times a week thereafter throughout the experimental period. When the general health condition of the calves was negatively affected, the frequency of examination was increased. A clinical scoring system was developed, modified after Hägglund et al. [30], and is presented in Table 1. An overall clinical score was calculated by summing up the separate scores from the clinical registrations. The peak outbreak was defined as the day with the highest number of clinically affected calves.

Collection of biomaterial

Collection of blood samples and nasal swabs was performed three to four times a week from each animal. Nasal swabs (ESwab™ Copan, Brescia, Italy) for virus detection were placed approximately five cm into the right and the left nostril, alternately. The swab was pressed gently against the nasal septum and rolled up and down four times.

All swabs were kept on ice and stored at − 80°C within one hour, until analysis.

Blood was collected from the external jugular vein in plain tubes. Sera were stored at − 20°C until analysis.

Quantification of BRSV RNA (RT-ddPCR)

All the nasal swabs collected during the experimental period were analysed. RNA was extracted from 200 μl of the swab transport medium using the automated Nucli-Sens easyMAG protocol (Biomérieux, Marcy l'Etoile, France), according to the manufacturer's instructions. Quantification of BRSV genomes was conducted in duplicate, with Bio-Rad's QX200 ddPCR System (droplet digital PCR). Each run included a positive (RNA from the nostril of a BRSV-positive trial calf ) and negative control (water). Droplet generation and transfer of droplets were as described by the manufacturer. The One-Step RT-ddPCR Advanced Kit for Probes (BioRad, CA, USA) and 2 μl RNA were used. The sequence of primers and probe (5′ FAM and BHQ1 as quencher) was as described by M. Boxus et al., targeting a 123 bp region of the BRSV N gene [31]. Primers and probe concentrations were as recommended by the kit manufacturer and with the following cycling conditions: 50°C for 60 min, 95°C for 10 min and 40 cycles of 95°C for 30 s and 60°C for 1 min. The ramp rate was set to 2°C/second. Data processing and absolute quantification of BRSV genomes per μl RNA was performed with Quanta-Soft Version 1.7 (BioRad, CA, USA).

Positive nasal swabs from the calves were subjected to Kaplan-Meier survival analysis in Stata (Stata SE/ 14, Stata Corp., College Station, TX, USA). The function shows the cumulative survival, i.e. shedding of BRSV RNA over time, which decreases as nasal swabs from the calves turns BRSV RNA negative. As the exact time-point the nasal swab of a calf turned negative was unknown, the mid-point between the last positive and the following negative sample was used in the analysis.

BRSV isolation

Based on RT-ddPCR results, nasal swabs were selected for virus isolation at three different time points from all calves in group EG: 1) Early-D6, the second day that all calves tested positive for viral RNA; 2) Middle-D13, all calves were still positive; and 3) Late-D19, the first day where viral RNA was no longer detected in all EG calves (five of the six were positive).

Virus isolation was performed by inoculating foetal bovine turbinate (FBT) cells propagated in Eagle's minimal essential medium (EMEM, Belgium) in 96 well plates in duplicate samples of 50 μl of 0,45 μm filtered nasal swab medium for 30 min. After adding medium with 2% FCS, negative replication control cells were harvested, while the parallel samples were incubated at 37°C , in 5% CO 2 . The cells were observed for cytopathogenic effect (CPE) and passaged after 7 days of incubation. Presence of CPE was further confirmed by a direct immunofluorescence test using FITC Moab a-BRSV (Bio-X Diagnostics, Rochefort, Belgium) and supernatants were tested by the BRSV RT-ddPCR described.

Antibody detection

Indirect ELISAs for the detection of serum antibodies specific to BRSV (SVANOVIR® BRSV-Ab), BPIV-3 (SVA-NOVIR® PIV3-Ab) and BCoV (SVANOVIR® BCV-Ab) (all from Svanova Biotech AB, Uppsala, Sweden) were performed according to the manufacturer's instructions. Sera collected from all calves every second day from D-2 to D60 were analysed for BRSV antibodies. In addition, sera were analysed for detection of antibodies against BCoV and BPIV-3 at specific time points: For IG on D-2 and D26, for EG on D26, D47 and D60, and for SG on D27, D47 and D60.

Pathological examination

Standard post-mortem examinations were performed on infected animals (IG and EG), including macro-and micro-pathological examinations of the respiratory tissue. Lung tissue was prepared and cultured for the presence of bacteria and sections were stained with haematoxylin-eosin for histological investigation.

Weight gain

The calves were weighed two to three times per week throughout the experimental period. Average daily weight gain was calculated for the infected animals (IG and EG) by dividing total weight gain by the number of days between each weighing. The median of the average daily weight gain (all calves) were calculated and the minimum (min) and maximum (max) median values were registered for the acclimatization period (D-11-D0), the peak infection period (D1-D15) and the restitution period (D16-D57).

Results

Clinical outcome

All IG and EG calves showed signs of respiratory disease, varying from mild to severe (Table 2). In IG, the first clinical sign was observed three days after inoculation (D1), when one calf had sparse serous nasal discharge. Four days later, both inoculated animals showed mild respiratory signs with mild depression, normal to seromucous nasal discharge and a sporadic cough. Nine days after inoculation (D7), the total clinical score was at its highest for the IG calves, and the median of the total clinical score value was 4.0. On D1, the first clinical sign appeared in three of the six EG calves as they showed mild signs of respiratory disease, including a sporadic cough, mild depression and/or sparse serous to mildly opaque nasal discharge. Calves E 5 and E 6 started to show severe signs of respiratory disease on D10 and D15, respectively, which qualified for medical treatment with penicillin and anti-inflammatory drugs. The association of the BRSV RT-ddPCR results versus scores of clinical parameters and total clinical score of the EG calves is shown in Fig. 2.

Viral RNA in nasal swabs

BRSV RNA was detected in nasal swabs from all IG and EG calves after the first challenge, and quantitative RNA values are given in Table 3. In IG, the first positive sample was detected two days after inoculation (D0). In EG, RNA was first detected on D1, and all animals were positive by D5. The peak was on D9. One IG calf was positive until D17, while two EG calves were still positive on D27. From D29, nasal swabs from all calves were negative. The respective log 10 numbers of genome copies are found in Table 3. The results of the Kaplan-Meier-survival analysis of viral RNA in nasal swabs from the calves are provided in Fig. 3. According to the analysis, the estimated longest persistence of viral RNA was until 28 days after exposure. The SG calves were negative for viral RNA throughout the study period and none of the EG calves were positive following the rechallenge.

Detection of infective viruses in nasal swabs

Cytopathogenic effect and positive immunostaining were observed in cells incubated with swab material from all six EG calves collected on D6 and D13. No clear CPE or immunostaining were caused by samples collected on D19. A high difference in supernatant viral RNA titer was found by RT-ddPCR between negative replication control and incubated samples from D6 and D13 (range: 2.9-7.6 and 2.5-5.3 log 10 GC per swab, respectively). Samples collected on D19 did not show any rise in supernatant viral RNA titer after two passages in FBT cells, which indicates no infective BRSV.

Serology

All calves tested negative for antibodies against BRSV before inoculation/exposure. In IG, one calf seroconverted on D11, and both calves were positive on D13. In EG calves, seroconversion was first detected on D13,  Table 3 Log 10 numbers of genome copies of BRSV per swab in calves of inoculated group (IG, calf I 1 and I 2 ) and exposed group (EG, calf E 1 -E 6 ) from first day of exposure of IG to EG to the last day where nasal swabs positive for viral RNA were detected with ddPCR (D27) Day postexposure EG Log 10 genome copies BRSV/swab in IG and EG calves
I 1 I 2 E 1 E 2 E 3 E 4 E 5 E 6 0 3.0 - - - - - - - 1 5.2 - - - - - 2.5 - 2 7.5 7.5 - - - 2.8 - 3.3 3 7.9 - 6.0 - 2.17 - 3.1 - 4.6 - - 4.8 4.2 19 - - - - - 3.2 3.8 - 21 - - - - - 4.6 - 5.0 25 - - - - 3.3 - - - 27 - - - - - 3.3 2.5 -
and all calves had seroconverted by D19. SG calves remained seronegative to BRSV throughout the study period. In Fig. 4, the corresponding median values of EG BRSV antibody titer and RNA quantities are presented.

No animals seroconverted towards BCoV or BPIV-3.

Pathological examination

All IG and EG animals had mild pathological lesions in the respiratory tract, with bronchointerstitial pneumonia, ranging from mild to moderate. Five out of eight calves had a mucopurulent bronchitis and/or bronchiolitis, six had a mild bronchiolitis obliterans, seven had mild to pronounced atelectasis and one calf had pleuritis. All the calves had mild to pronounced bronchus-associated lymphoid tissue hyperplasia and mild to moderate reactive hyperplasia of the lymphatic tissue of nasopharynx and the regional lymph nodes of the lungs. Bacterial culture from lung tissue showed no growth, except from one calf, where sparse occurrence of Trueperella pyogenes was identified.

Average daily weight gain

During the acclimatization period (D − 11 to D0), the median of the average daily weight gain (all calves) was 991 g (min 878 g, max 1500 g). In the peak infection period (D1-D15), it was reduced to 583 g (min 117 g, max 1000 g), while increasing to 901 g (min -1350, max 1850 g) in the restitution period (D16-D57).

Discussion

The present study aimed to assess the transmission potential of BRSV from infected calves and their housing environment in an experimental setting. Only ten animals were included in the study. Although not ideal, the chosen study design is common in experimental infection studies in large animals [12]. This in contrast to other type of studies with traditional laboratory animals or more epidemical studies, e.g. natural infections in the field. There are several challenges related to the large size of the animals like the accessibility to experimental facilities of acceptable size and biosecurity level. It is also an economical aspect due to the high costs of housing in experimental facilities and to the cost of purchasing calves. The ethical aspect according to number of animals used is always of concern. Consequently, the present study is performed as a descriptive experimental infections study. The experimental setting allowed controlled conditions and control of time of exposure to BRSV, which is difficult to achieve under field conditions. Natural conditions were mimicked concerning host species, age, breed, feeding and social interaction with other calves. Several environmental factors for the calves are identified as risk factors of respiratory disease. Among these are low temperatures combined with wetness of the animals, dust, herd size, transport, shared housing between calves and adult cattle, large differences in age and commingling of many calves in the same pen [32,33]. In the present experiment, most of these factors were not advisable or possible to implement, but a few mild stressors were introduced, such as moving animals and commingling with other calves (i.e., rearrangement during the experiment). The bedding was dry, leaving the animal rooms dustier than in an average herd. Although severe respiratory disease was observed, clinical symptoms might have been even more severe if the virus had been introduced to a regular cattle herd. A non-passaged BRSV strain obtained from a field outbreak of respiratory disease was used for inoculation, in order to avoid changes in virulence due to cultivation in cell lines. For the main calf group (EG), natural exposure to virus-shedding calves was used to mimic field conditions. Experimental studies usually include control groups where naïve animals are kept in the same environment. However, due to biosecurity measures and room capacity, the present housing facilities did not allow for this. Therefore, each calf functioned as its own control, by using the period prior to the challenge as a control period.

The long experimental period allowed the study of the presence of nasal mucosa-associated viral RNA for an extended time period, which revealed that BRSV was present for up to four weeks, starting on D1-D5.

Compared to results of the group of calves investigated in the present study, previous studies have concluded that viral shedding usually begins later, and lasts for a shorter period [7][8][9][10][11][13][14][15][16][17][18][19][20][21][22]. Other studies have claimed that BRSV replication in the upper respiratory tract only occurs in an early stage of infection [34,35], which has led to the conclusion that sampling of bronchoalveolar lavage and lung tissue is necessary for diagnostic purposes in later stages of the disease. The present results indicate that diagnostic sampling using nasal swabs is suitable for up to one month after exposure to BRSV. This is in line with another study by Klem et al. [29], and enables easier, cheaper and more frequent testing of animals at risk.

Although viral RNA was found in nasal swabs for up to four weeks, cell culture propagation of BRSV from nasal swabs indicated that the shedding period of infectious virus amounts is considerably shorter, approx. 13-18 days, indicating a shorter period of actual transmission risk. The two SG calves remained uninfected after commingling with EG calves and their environment four weeks after EG calves were exposed to BRSV. The number of calves was unfortunately too low to conclude if this would be valid in a real herd with a larger number of animals, Nevertheless, it would be interesting to investigate this further in a study with more animals included and a sequential study design. In a recent experimental infection study on BCoV [36], a similar pattern was observed; RNA from BCoV was detected for approximately a month after infection, but the infectivity of the virus was found to be considerably shorter, lasting for up to 18 days. These results suggest that the same strategies can be applied to prevent inter-herd transmission for both bovine respiratory disease complex pathogens.

Some differences were found in the viral shedding patterns of EG and IG calves, which might reflect differences between infection caused by natural exposure to the virus and the more artificial inoculation proceduree.g., the time from exposure until the detection of viral RNA in the nasal swabs was shorter in EG calves than the time from inoculation to viral shedding in IG calves. The virus in the nasal swabs might reflect the presence of the inhaled virus, but no active BRSV infection. Bovine respiratory syncytial virus is a labile, enveloped virus and freezing and thawing can detrimentally affect infectivity [2,37,38]. As the amount of infective virus was unknown in the inoculate, it may have contained less infective virus than the amount the EG calves were exposed to. This might explain why inoculation led to a longer incubation period and less severe infection in IG calves. In addition, viral RNA was detected for a longer period of time in the EG calves. The late outbreak peak of clinical signs of the EG compared to earlier reports from experimental infections [12] might have been caused by a more prolonged process in a group of animals naturally exposed to BRSV. This adds to the view that studies based on natural exposure are more valid than inoculation studies.

Clinically, the infection resembled a naturally occurring BRSV infection, with all infected calves showing respiratory signs but with individual differences in severity. Comparison of the median values of the total clinical score and the BRSV RNA titer showed a clear conformity between the two parameters during the course of the trial. In general, the detection of replicating viral RNA occurred a few days before the onset of clinical signs. Rise in temperature and coughing is commonly used as a measure to detect respiratory disease in the field. In the present experiment, these parameters increased during the same time period as the number of BRSV genome copies raised. However, the coughing continued intermittently for a much longer period than viral RNA was detected. The median values of the rectal temperature score was only slightly elevated. Increased respiratory rate and nasal discharge appeared to be the clinical parameter corresponding best with the detection of viral RNA for the included calves. The clinical signs also corresponded well with the periods when an active BRSV infection was detected. After approximately three weeks, no infective virus could be isolated and most clinical signs were very mild or no longer present.

The pathological examination showed that both IG and EG calves had mild pathological lesions in the respiratory tract. This was unexpected, as the calves were no longer clinically ill at the time of euthanasia. Nevertheless, the low average daily weight gain in the peak infection period and the failure to regain normal growth during the recovery period are in agreement with previous findings indicating a negative effect on growth and production, months after apparent recovery [39].

During infection, the calves showed clinical signs and pathological changes normally associated with BRSV infection in calves without co-infections. However, infections with BRSV are known to predispose cattle to secondary bacterial infections, and despite the fact that bacterial examinations of lung tissue post mortem were negative in all calves but one (who had sparse numbers of Trueperella pyogenes), it is not possible to completely rule out the possibility of co-infections-especially considering the fact that, during the experiment, two of the calves were medically treated with penicillin. BRSV is also known to occur in co-infection with other viruses or with Mycoplasma spp. In the present study, none of the animals were infected with BCoV or BPIV-3, and the Norwegian cattle population is currently free from many of the internationally recognized agents of bovine respiratory disease; for example, bovine herpes virus type 1 [40], bovine viral diarrhoea virus [41] and Mycoplasma bovis have never been detected [32].

Calves in the present study were not reinfected by inoculation seven weeks after the primary infection, indicating that protective immunity is longer than what has previously been suggested [23,28]. The sentinel animals remained seronegative after contact with the EG animals one month after the onset of infection, and the seroconversion in the EG calves after about two to three weeks, coincided with the period when the active infection had been cleared and the virus no longer seemed to be infective in the cell culture.

Taken together, protective immunity seemed to correspond with the detection of serum antibodies, and to last for at least 7 weeks.

The median clinical score, mean viral RNA detection and virus infectivity coincided and were strongly reduced about three weeks after exposure to BRSV. The negative results in the sentinel study further support the argument that BRSV infected animals and their housing environment pose a lower risk after three to four weeks. Combining this knowledge with the registration of clinical signs of respiratory disease, with an emphasis on the occurrence of increased respiratory rate, could be useful information when implementing preventive biosecurity measuressuch as periods of quarantine-to avoid spreading the virus. However, an experimental setting with relatively few animals of the same age will differ from the transmission dynamic in a regular herd. One of the challenges in a conventional herd setting is to know at which time point the virus has circulated throughout the whole herd. Since animals may develop subclinical infection, the time point when the last animals clear the infection will often be unknown. Despite the efforts in this experiment to mimic natural conditions, these factors must be considered when the results are applied in a herd.

Conclusions

In this study, viral BRSV RNA was detected in nasal swabs from calves up to 27 days after exposure to the virus. The period when infective viruses were found was shorter: between 13 and 19 days. The total clinical score, the increase in the respiratory rate and nasal discharge corresponded best with the shedding of viral RNA. Contact between EG calves and naïve calves four weeks after exposure did not result in infection. EG calves were protected from reinfection seven weeks after initial exposure. This adds to our basic understanding of transmission patterns of BRSV, within-and between-herds, and can provide support for preventive action when used to design inter-herd biosecurity measures.

Fig. 1
1Timeline of interventions during the experimental period. Three groups of animals were included in the experiment: the Inoculated group (IG: calf I 1 and I 2 ), Exposed group (EG: calf E 1 -E 6 ), and Sentinel group (SG: calf S 1 and S 2 )

Fig. 2
2Mean number of genome copies of bovine respiratory syncytial virus (BRSV) related to median clinical score of infection. Day = days post-exposure to BRSV, GC = genome copies

Fig. 3
3Kaplan-Meier survival function for BRSV RNA in nasal swabs from infected calvesFig. 4 Mean number of genome copies of bovine respiratory syncytial virus (BRSV) related to median antibody titre. Day = days post-exposure to BRSV, GC = genome copies, PP = percent positivity

Abbreviations
BCoV: Bovine corona virus; BPIV-3: Bovine parainfluenza-3 virus; BRSV: Bovine respiratory syncytial virus; CPE: Cytopathogenic effect; D: Days after challenge; EG: Exposed group; FBT cells: Foetal bovine turbinate cells; GC: Genome copies; IG: Inoculated group; P.i.: Post infection; PP: Percent positivity; RT-ddPCR: Reverse transcription droplet digital polymerase chain reaction; SD: Standard deviation; SG: Sentinel group

Investigating whether the calves and their environment are not infectious to naïve in-contact calves four to nine weeks post-infection despite rechallenge with BRSV and mild stress induction.1) Investigating the shedding of viral RNA and
infective virions:
a. related to clinical outcome during the experimental
period, lasting for two months
b. in calves rechallenged by inoculation seven
weeks p.i.
2)

Table 1
1Clinical scoring system. The score from each category was added to give a daily clinical score for each of the calves in the experimentScore
Respiratory rate
(breaths/min)

Rectal temperature (°C)
Cough
Nasal discharge
Lung auscultation
Demeanor

0
≤ 49
≤ 39,5
No cough observed
Normal
No abnormal
sounds

Bright, alert

1
5 0 -54
39,6-39,9
Sporadic cough
Serous or mucous
Wheezing
sounds

Mildly depressed

2
5 5 -64
40-40,4
More than one sporadic
cough every 10 min of
observation

Mucopurulent or
purulent

-
Moderately depressed

3
6 5 -74
40,5-40,9
-
-
-
Severely depressed

4
7 5 -85
> 40,9
-
-
-
-

Mild score: clinical score > 2 on 3 consecutive days
Moderate score: clinical score ≥ 4 on 3 days or more
Severe score: clinical score ≥ 9

Table 2
2Key clinical signs of respiratory disease after experimental infection with BRSVAnimal
group

Calf
no.

Peak
rt.°C

Number of days with

AcknowledgementsThe authors would like to acknowledge Tormod Mørk at the Norwegian Veterinary Institute for his contribution to the sharing of ideas and his practical assistance with carrying out the animal experiment.FundingThis study was funded by the Norwegian Veterinary Institute, the Norwegian University of Life Sciences and Geno SA. The funders had no role in the design of the study, data collection, analyses, decision to publish or preparation of the manuscript.Availability of data and materials Thea Blystad Klem can be contacted for additional data and material.Authors' contributions TBK, SKS, SS, BG, MM and MS contributed to the designing and performance of the BRSV experiment and wrote, read and approved the manuscript.Ethics approval and consent to participateThe animal experiment was conducted in line with national and international guidelines for the care and use of experimental animals. The experiment was approved by the Norwegian Animal Research Authority (Approval/Ref no. 7468). Written informed consent to use the animals in the study was obtained from the owners of the animals.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.
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Continuing education for the prevention of mild cognitive impairment and Alzheimer's- type dementia: a systematic review protocol

Nina Matyas
Stefanie Auer
Christoph Gisinger
Monika Kil
Filiz Keser Aschenberger
Irma Klerings
Gerald Gartlehner
Continuing education for the prevention of mild cognitive impairment and Alzheimer's- type dementia: a systematic review protocol
10.1186/s13643-017-0553-0P R O T O C O L Open Access
Background: Because of the enormous social and economic burden of disease, the prevention of mild cognitive impairment and Alzheimer's-type dementia has become a major global public health priority. Studies show that cognitively stimulating activities during middle adulthood might have a protective effect on the brain by boosting the cognitive reserve. The aim of this review is to identify evidence investigating the effects of continuing education for the prevention of mild cognitive impairment and Alzheimer's-type dementia in late life. Methods: Our approach employs a two-stage design: First, we will conduct a systematic review to assess the preventive effects of continuing education on mild cognitive impairment and Alzheimer's-type dementia. Second, because we expect to find few studies, we will perform a review of systematic reviews on leisure activities that mimic formal continuing education to determine their effects on the prevention of mild cognitive impairment and Alzheimer's-type dementia. We will search electronic databases (e.g., MEDLINE, PsycINFO, EMBASE, CENTRAL, CINAHL, and Scopus) for published studies and gray literature databases (e.g., trial registries) for unpublished studies. Two authors will independently screen abstracts and full-texts using pre-defined eligibility criteria, select studies, extract data, and assess the quality of included studies or reviews. Outcomes of interest include the incidence of mild cognitive impairment or Alzheimer's-type dementia, quality of life, functional capacity, and psychological wellbeing. Intermediate outcomes are cognitive (test) performance, cognitive functioning, and social inclusion. The review team is a multidisciplinary group consisting of methodological experts and dementia, geriatrics, and continuing education researchers. Discussion: We anticipate that our review will highlight serious gaps in the current evidence. Results will build the basis for further research regarding the relation of continuing education and cognitive decline and dementia. Systematic review registration: PROSPERO CRD42017063944

Background

Rationale

As a consequence of the rapidly aging world population, neurocognitive disorders such as mild cognitive impairment or Alzheimer's-type dementia have become a major public health challenge. The World Health Organization (WHO) has designated the prevention and control of neurocognitive disorders as a public health priority [1].

Alzheimer's-type dementia is the most common form of dementing illnesses. In 2015, 47.5 million people globally lived with Alzheimer's disease or another closely related dementing illness [2]. The prevalence is projected to rise up to 135.5 million patients in 2050 [2].

Because of the progressing loss of independent functioning of patients with Alzheimer's-type dementia, the social and economic burden of the disease is enormous. In 2016, the US economic burden associated with dementia was estimated to be 236 billion U.S. Dollars [3], the total global costs for dementia were 818 billion in 2015 [4].

The exact risk factors of neurocognitive disorders are largely unknown but the risk increases substantially with age. For example, the prevalence rate for Alzheimer's disease surges from 3.5% in persons aged 75 or older to 46.3% in those aged 95 years or older [5].

Clinically, the onset of Alzheimer's-type dementia is a slow process of cognitive deterioration. When the decline of cognitive functioning reaches a level that can be measured objectively, it is often referred to as mild cognitive impairment or mild neurocognitive disorder [6]. Petersen describes this as a "transitional period" between normal aging and the diagnosis of Alzheimer's-type dementia. In this study, mild cognitive impairment refers to "amnestic" mild cognitive impairment (aMCI), which is defined as a condition where memory loss is predominant [7]. In approximately 32% of patients with aMCI, cognitive decline progresses to a degree that the ability of a person to perform everyday activities is significantly impaired. Such a state is called major neurocognitive disorder due to Alzheimer's-type dementia [6,8]. Based on the criteria of the Diagnostic and Statistical Manual (DSM-5), Alzheimer's disease is characterized by a significant decline of intellectual abilities in one or more cognitive domains (learning and memory, language, executive function, complex attention, perceptual motor function, social cognition) outside the context of delirium [8].

The underlying risks for Alzheimer's-type dementia are not yet thoroughly understood, and no curative treatment has been found [9]. Systematic reviews assessing risk factors indicate that low educational level, decreased physical activity, unhealthy diet, smoking, and alcohol abuse might be predictors of dementia [10][11][12][13]. Likewise, chronic medical conditions such as cardiovascular diseases, diabetes, obesity, cancers, depression, thyroid disorder, or genetic factors increase the risk of dementia [10]. Some studies, however, found a protective association of cognitively stimulating activities, such as learning a new language in middle age, with a slower cognitive decline during late life [14][15][16][17][18]. Such results underpin a theory called the "cognitive reserve hypothesis" [19,20]. According to this theory, through every activity that stimulates the brain, the cognitive reserve gets boosted and the resistance towards any dementia-related brain pathology gets stronger [21]. A larger cognitive reserve acquired by continuing education activities, thus, might protect against cognitive decline [21,22].

For this systematic review, we define continuing education as structured learning activities and programs provided by formal and non-formal educational institutions for persons beyond the age of compulsory schooling. These activities are designed to help individuals satisfy learning needs and interests, to enrich knowledge, to develop and improve abilities and skills, and to foster personality, social competences, families, networks, health, and professional life. Continuing education is voluntary and based on topics and courses that are not directly connected to any special job position or vocational training [23][24][25][26][27][28].

To date, the preventive effect of continuing education on cognitive impairment and Alzheimer's-type dementia has not been assessed in an objective and systematic way.

Objectives

The purpose of our review is to summarize the evidence investigating the effects of continuing education on the development of cognitive impairment and Alzheimer'stype dementia. Based on discussions with experts in the fields of neurocognitive disorder, aging, and continuing education, we designed the following five key questions that will guide our systematic review:
Key

Methods

This systematic review protocol has been designed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Protocol (PRISMA-P) statement [29] (see Additional file 1). Our protocol is registered on PROSPERO (international prospective register of systematic reviews) (registration number CRD42017063944). Figure 1 presents an analytic framework of the effects of continuing education on relevant health outcomes.

Study design

Because we expect few studies that assess formal continuing education, we will also include leisure activities that mimic continuing education regarding content, but are not organized educational activities (e.g., learning a new language privately versus learning a new language as an organized educational activity). Because leisure activities are not our primary focus of interest but can be considered as surrogate interventions in some circumstances, we will address our questions of interest with two different methodological approaches:

1. We will employ a systematic review of primary studies to assess the preventive effects and potential harms of continuing education provided by formal and non-formal institutions. 2. We will use a review of systematic reviews to determine the preventive effects and potential harms of related leisure activities.

A review of systematic reviews is a synthesis of evidence from multiple systematic reviews [30]. For both approaches, we will employ the same criteria for populations, control interventions, outcomes, timing, and settings as outlined below. Criteria for interventions and eligible study designs will be different.

Eligibility criteria

We specified our inclusion and exclusion criteria based on the population, intervention, comparators, outcomes, timing, and settings (PICOTS) identified through a topic refinement exercise with experts in the field (Table 1). We will exclude study designs without control groups to ensure that our pool of included studies can inform the causal link between the intervention and outcomes.

Types of participants

We will include studies with adults (45 years or older) who do not have a clinical diagnosis of cognitive impairment or Alzheimer's-type dementia. This population also includes adults with subjective cognitive impairment who do not fulfill the criteria for a clinical diagnosis of mild cognitive impairment.

Subgroups of interest are based on age, sex/gender, race or ethnicity, level of education, socio-economic status, and duration of intervention. We will not include patients younger than 45 years. Furthermore, we will exclude populations with other severe health conditions that are likely to be associated with loss of cognitive function such as HIV (human immunodeficiency virus) infections, multiple sclerosis, stroke, post traumatic brain injuries, and psychiatric conditions (e.g., alcohol abuse, drug abuse, major depressive disorder).

Types of interventions

The systematic review will examine any intervention that can be categorized as "continuing education". In accordance with our definition, we include all courses, classes, lectures, workshops, and trainings that are provided by institutions of formal and non-formal education. The eligible learning activities are based on topics that are not directly connected to any special occupation. Individuals engage in continuing education voluntarily and intentionally to satisfy their learning needs and interests and to develop or improve their abilities and skills [23][24][25][26][27][28]. Because the focus of this research is on the effects of cognitive activities, all continuing education involving a physical activity will be excluded. For the review of systematic reviews, we include leisure activities that mimic the content of continuing adult education but in an informal setting. For example, we would include playing chess as a leisure activity if no studies on playing chess in a formal continuing education setting are available. We will not include leisure activities for which formal continuing education programs are unlikely (e.g., going to the cinema), or for which physical exercise is the main goal (e.g., jogging, yoga).

Types of comparators

Studies with any type of control group will be included.

Types of outcome measures

Our primary outcome of interest will be the incidence of mild cognitive impairment or Alzheimer's-type dementia. Furthermore, we are interested in any health outcome such as psychological wellbeing, functional capacity, or quality of life. In the absence of evidence on health outcomes, we will include intermediate outcomes such as cognitive functioning, cognitive (test) performance, or social inclusion.

Timing

The minimum duration for any intervention is 3 months; the minimum follow-up time is 1 year.

Setting

We will include any communities and institutions that offer continuing education. We will exclude institutions such as retirement homes, hospitals, or nursing homes.

Types of studies

We will include studies with control groups only. Eligible for inclusion are randomized controlled trials, nonrandomized controlled trials, prospective controlled cohort studies, retrospective controlled cohort studies, and case-control studies.

The review of systematic reviews will include any systematic review or meta-analysis that addresses a question of interest. We define systematic reviews based on the Cochrane handbook as a literature review that attempts to collate all empirical evidence using (a) a clearly stated objective and pre-defined eligibility criteria, (b) an explicit reproducible methodology, (c) a systematic search, (d) an assessment of the validity of the findings of the included studies, and (e) a systematic presentation, and synthesis, of the characteristics and findings of the included studies [30].

Information sources and literature search

We will systematically search, review, and analyze the scientific evidence for each key question. To identify articles relevant to each key question, we will begin with a Ovid MEDLINE search for eligible interventions using a combination of medical subject headings (MeSH®) and title and abstract keywords, limiting the search to humanonly studies without applying any language limitations (see Additional file 2: Ovid MEDLINE search strategy). We will also search the Cochrane Library, Embase, PsycINFO, CINAHL (Cumulative Index to Nursing and Allied Health Literature), ALOIS (the Cochrane Dementia and Cognitive Improvement Group Specialized Register), and ERIC (Education Resources Information Center) using analogous search terms. We selected these databases based on preliminary searches and consultation with content experts. The search period will go back to January 1990. An experienced information specialist will perform all searches; the electronic Ovid Medline search strategy was peer-reviewed by another information specialist following the PRESS (peer review of the electronic search strategy) statement [31].

In addition, we will search for gray literature (i.e., unpublished studies) relevant to this review. Potential sources of gray literature include ClinicalTrials.gov, the World Health Organization's International Clinical Trials Registry Platform, websites of relevant organizations, and dissertation databases (e.g., DART-Europe).

In an attempt to avoid retrieval bias, we will manually search the reference lists of landmark studies and background articles on this topic to look for any relevant citations that our electronic searches might have missed.

We will include studies that meet all the inclusion criteria and contain enough methodological information to enable us to assess risk of bias.

Data abstraction and data management

Two authors will independently review all titles and abstracts identified through searches for eligibility against our inclusion/exclusion criteria. Studies marked for possible inclusion by either reviewer will undergo a full-text review. For studies without adequate information to determine inclusion or exclusion, we will retrieve the full text and then make the determination. All results will be tracked in an EndNote® X8 bibliographic database (Thomson Reuters, New York, NY).

We will retrieve and review the full text of all titles included during the abstract review phase. Two authors will independently review each full-text article for inclusion or exclusion based on the eligibility criteria described above. If both reviewers agree that a study does not meet the eligibility criteria, the study will be excluded. If the reviewers disagree, conflicts will be resolved by discussion and consensus or by consulting a third member of the review team. We will record the reason that each excluded full-text publication did not satisfy the eligibility criteria so that we can later compile a comprehensive list of such studies.

For studies that meet our inclusion criteria, we will abstract important information into evidence tables. We will design data abstraction forms to gather pertinent information from each article, including characteristics of study populations, settings, interventions, comparators, study designs, modifiable risk factors [32], methods, and results. Trained reviewers will extract the relevant data from each included article into the evidence tables. A second member of the team will review all data abstractions for completeness and accuracy.

Risk of bias assessment

To assess the risk of bias (internal validity) of studies, we will use the Cochrane Risk of Bias Tool for randomized controlled trials [33] and the Risk of Bias in Nonrandomized Studies of Interventions (ROBINS-I) tool for nonrandomized studies [34]. For the appraisal of systematic reviews of RCTs, we will use the AMSTAR (Assessing the Methodological Quality of Systematic Reviews) tool [35].

We will consider the risk of bias for each relevant outcome of a study.

Two independent reviewers will assess the risk of bias for each outcome in each study. Disagreements between the two reviewers will be resolved by discussion and consensus or by consulting a third member of the team.

Data synthesis and statistical analysis

If we find three or more similar studies for a comparison of interest, we will consider quantitative analysis (i.e., meta-analysis) of the data from those studies. For all analyses, we will use random-effects models to estimate pooled effects.

To determine whether quantitative analyses are appropriate, we will assess the clinical and methodological heterogeneity of the studies under consideration following established guidance [36]. We will do this by qualitatively assessing the PICOTS of the included studies, looking for similarities and differences. For meta-analyses, we will assess statistical heterogeneity in effects between studies by calculating the chi-square statistic and the I 2 statistic (the proportion of variation in study estimates attributable to heterogeneity) [37,38]. The importance of the observed value of I 2 depends on the magnitude and direction of effects and on the strength of evidence for heterogeneity (e.g., p value from the chi-squared test or a confidence interval for I 2 ). If we include any meta-analyses with considerable statistical heterogeneity, we will provide an explanation for doing so, considering the magnitude and direction of effects. We will also examine potential sources of heterogeneity using sensitivity analyses or analysis of subgroups. We plan to stratify analyses and/or perform subgroup analyses when possible and appropriate to examine clinical heterogeneity.

For any quantitative analyses, we will conduct sensitivity analyses including high risk of bias studies. Planned stratifications or categories for subgroup analyses include the subgroups listed in the analytic framework. When quantitative analyses are not appropriate (e.g., because of heterogeneity, insufficient numbers of similar studies, or insufficiency or variation in outcome reporting), we will synthesize the data qualitatively.

To assess publication bias, we will use funnel plots and Kendall's tests.

We will assess the quality of evidence for individual comparisons and outcomes using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach [39].

Discussion

To the best of our knowledge, this will be the first review to assess the potential benefits and harms of continuing education for the prevention of mild cognitive impairment and Alzheimer's-type dementia. We anticipate that this review will identify serious gaps in the current evidence. Our results will build the basis for further research and highlight implications for practice. Through our multidisciplinary team, results can easily reach a variety of stakeholders and findings can be disseminated through many channels.

Additional files

Fig. 1
1Analytic framework for continuing education to prevent mild cognitive impairment and Alzheimer's-type dementia

Additional file 1 :
1PRISMA-P 2015 checklist. (DOCX 35 kb) Additional file 2: Ovid MEDLINE search strategy. (DOCX 16 kb) Abbreviations AD: Alzheimer's disease; aMCI: Amnestic Mild Cognitive Impairment; AMSTAR: Assessing the Methodological Quality of Systematic Reviews; CEDEFOP: European Centre for the Development of Vocational Training; CENTRAL: Cochrane Central Register of Controlled Trials; DSM: Diagnostic and Statistical Manual of Mental Disorders; GRADE: Grading of Recommendations Assessment, Development and Evaluation; ICD: International Classification of Diseases; MCI: Mild Cognitive Impairment; MMSE: Mini-Mental State Examination; OECD: Organisation for Economic Co-operation and Development; PRISMA-P: Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Protocols; UNESCO: United Nations Educational, Scientific and Cultural Organization; WHO: World Health Organization

Table 1
1Criteria for inclusion and exclusion of studies in the review Abbreviations: MCI mild cognitive impairment, MMSE Mini-Mental State Examination, HIV human immunodeficiency virus, RCT randomized controlled trialCriteria

Category
Inclusion
Exclusion

Population
-Adults (45 years or older) without a clinical
diagnosis of cognitive impairment; this includes
people with subjective cognitive impairment

-People younger than 45 years
-People with a clinical diagnosis of impaired cognition (e.g., MMSE < 24)
-Populations comprised exclusively of patients with primary diseases
with an increased risk for dementia such as Parkinson disease, HIV
infection, multiple sclerosis, stroke, post traumatic brain injuries,
infectious diseases, psychiatric conditions (e.g., alcohol abuse, drug
abuse, major depressive disorder)

Subgroups
-Age
-Sex/gender
-Race/ethnicity
-Level of education
-Duration of intervention

Geography
No limit
No limit

Date of search
Searches will go back until 1990

Settings
Community-dwelling adults
Institutionalized people, e.g., people in nursing homes

Interventions
For systematic review
-All cognitive activities that are provided by
formal and non-formal educational institutions
-Classes/courses/trainings that are based on
individual interests and that are attended
voluntarily
For review of systematic reviews
-Leisure activities that are cognitively stimulating
and mimic the content of continuing education
but in an informal setting.

Formal (vocational) education and training, physical activities,
topics and courses that are related to any special job position
and/or occupation

Control interventions
No continuing education
Any educational activities and physical activities

Outcomes
Health outcomes
-Incidence of dementia
-Incidence of MCI
-Psychological wellbeing
-Functional capacity
-Quality of life
-Other relevant health outcomes
Intermediate outcomes
-Cognitive functioning
-Cognitive (test) performance
-Social inclusion

Timing
Minimum duration of the intervention: 3 months
Minimum follow-up time: 1 year

Publication language
No language restrictions

Study design
For systematic reviews
-RCTs
-Nonrandomized controlled trials
-Prospective controlled cohort studies
-Retrospective controlled cohort studies
-Case-control studies
-Nonrandomized studies must have a minimum
sample size of 300 or more participants
For review of systematic reviews
-Systematic reviews and meta-analyses

-Case series
-Case reports
-Cross over trials
-Nonsystematic reviews
-Studies without a control group
-Nonrandomized studies with fewer than 300 participants

AcknowledgementsNot applicable.FundingThis work was funded by internal funds of the Danube-University, Krems, Austria.Availability of data and materials Not applicable.Authors' contributionsAll authors made substantial contribution to the conception and design of this protocol. As an information specialist, IK developed the search strategy. NM has drafted the protocol. Our dementia experts SA and CG revised the background critically. MK and FK who are working in continuing education research helped in finding the appropriate intervention. GG peer-reviewed the protocol. All authors have given approval for this version to be published. NM is the guarantor of the review.Authors' informationThe systematic review is a multidisciplinary project performed at the Danube-University, Krems. NM is a MD (medical doctor) working in the Department for Evidence-based Medicine (EbM) and Clinical Epidemiology, and IK with a MSc in cultural studies is an information specialist. SA is a professor of dementia research and clinical psychologist. CG is a professor of geriatrics working at the Center for Geriatric Medicine and Geriatric Nursing. Ethics approval and consent to participate Not applicable.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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