Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds: the value of precise field data

Ynte H Schukken
Robert H Whitlock
Dave Wolfgang
Yrjo Grohn
Annabelle Beaver
Joann Vankessel
Mike Zurakowski
Rebecca Mitchell
Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds: the value of precise field data
10.1186/s13567-015-0187-yR E S E A R C H A R T I C L E Open Access
Longitudinal infection data on Mycobacterium avium subspecies paratuberculosis (MAP) was collected on three dairy farms in Northeastern United States during approximately 10 years. Precise data on animal characteristics and animal location within farm were collected on these farms. Cows were followed over time with regard to MAP status during biannual fecal and serum sampling and quarterly serum sampling. Approximately 13 000 serum samples, 6500 fecal samples and 2000 tissue samples were collected during these years. Prevalence of positive samples was 1.4% for serological samples, 2.2% in fecal samples and 16.7% in tissue samples. Infection dynamics of MAP was studied and resulted in a number of potential changes in our understanding of MAP infection dynamics. First, a high prevalence of MAP infection was observed in these herds due to lifetime follow up of cows, including slaughter. Second, two distinctly different infection patterns were observed, so called non-progressors and progressors. Non-progressors were characterized by intermittent and low shedding of MAP bacteria and a virtual absence of a humoral immune response. Progressors were characterized by continuous and progressive shedding and a clearly detectable and progressive humoral immune response. Strain typing of MAP isolates on the three farms identified on two of three farms a dominant strain type, indicating that some strains are more successful in terms of transmission and infection progression. Continuous high quality longitudinal data collection turned out to be an essential tool in our understanding of pathobiology and epidemiology of MAP infections in dairy herds.

Introduction

Johne's disease (JD), or paratuberculosis, is a chronic enteric disease of cattle and other ruminants due to an infection with Mycobacterium avium subsp. paratuberculosis (MAP) [1,2]. Herd-level MAP infection prevalence has gradually increased in the past decade; in a recent survey, it was found that 68% of US dairy herds have apparently at least one cow that is infected with MAP [3]. This estimate was obtained from a survey published by the USDA's National Animal Health Monitoring System in 2007 [3]. The economic impact of MAP infections to the dairy industry in the United States varies but the cost to dairy producers was estimated to be more than $200 million per year [4].

The epidemiology of MAP in dairy herds is difficult to study as the infection shows a very slow progression from initial infection to clinical disease [5]. Many infected animals never show clinical signs and, many infected animals are only detected using diagnostic tests a few years after initial infection or are actually never detected [1]. Even more, under commercial farm circumstances, diagnostic testing is infrequent and there is a low diagnostic test sensitivity for animals shedding either intermittently or low levels of MAP [6]. Hence, precise information on the infection status of animals is difficult to obtain. Still, the best data necessary to understand epidemiology and pathobiology of MAP will likely be obtained from animals that are studied intensively during their complete lifetime under field conditions on commercial dairy farms [7]. Particularly if the longitudinal study on-farm is followed by culture of tissues at slaughter with a known predilection of MAP infection [8].

An important issue in our understanding infection dynamics of MAP has been the relative low prevalence of animals that are apparently infected (test positive). In most studies, farm prevalences between 3 -10% are the dominant category [9][10][11]. With such a low prevalence, infection fade out would be expected in a large proportion of infected farms [12,13]. In reality very few farms, if any, have been reported that successfully eliminated infected. The combination of low prevalence with infection persistence provides for a MAP conundrum with so far not a reasonable rational explanation [14].

The use of field samples is also complementary to studies in animal models. Field studies, of course, do not control the environment, MAP exposure, host and bacterial genotype. Therefore such field data are often difficult to interpret, as the sources of variation are often not well understood. Through genome-wide association studies using high densities of single nucleotide polymorphism markers, joint analysis across animals and herds have become a reality [15,16]. Hence combining precise longitudinal data on infection status and detailed host genetic and bacterial strain type information may now be combined to evaluate the impact of genetic susceptibility to infectious disease such as MAP. In such studies, the choice of the disease phenotype is essential to identify a relevant genetic susceptibility that may be used for selection based disease control programs [17].

To be able to estimate infection incidence and prevalence, longitudinal data will be essential [18]. This is obvious for incidence of infection as animals susceptible to infection will need to be followed to determine when and whether they become MAP infected. However with infections such as MAP with a very slow progression and a long time delay between infection and the first measurable signs of infection, a single cross-sectional measurement will not provide an accurate estimate of infection prevalence [19]. Recent studies have provided initial evidence that transmission routes of MAP include calf-to-calf transmission [20] and adult-to-adult transmission [5]. These routes have typically not been taken into account in MAP control programs. Quantitative estimates of the importance of these transmission routes would be essential to decide on relevant control procedures.

Here we describe the collection and use of long-term longitudinal data on three commercial dairy herds in the North East of the United States. Data were collected for approximately 10 years on these farms. The objective of the paper is to show the particular value of longitudinal data on slow infections such as MAP. Specifically, we describe the long-term collection of data on commercial dairy farms and we try to answer specific questions using longitudinal data:

1. Is the true prevalence based on longitudinal data different from estimates based on cross-sectional data? 2. Based on strain typing of isolates, are there within a herd over time multiple infection dynamics concurrently, rather than a single infection dynamic? 3. Are new infections occurring throughout life, and not just in young animals?

Materials and methods

The longitudinal dataset that we will describe here was obtained from three commercial dairy farms in the northeastern United States: farm A in New York State, farm B in Pennsylvania, and farm C in Vermont [5]. All three farms participated in the Regional Dairy Quality Management Alliance (RDQMA) project, which is a multistate research program conducted under a cooperative research agreement between the USDA Agricultural Research Service (ARS) and four Universities, Cornell University, Pennsylvania State University, University of Pennsylvania, and University of Vermont. The project emphasized longitudinal data collection in areas in which infectious diseases of public and animal health concern in dairy herds are endemic. For a more complete description, including information on farms, samplings, and microbial analyses, see Pradhan et al. [5]. Briefly, the milking herds consisted of approximately 330, 105, and 145 cows on farms A, B, and C, respectively. Sampling commenced in February, March, and November 2004 on farms A, B, and C, respectively, and continued for approximately 10 years, for farm A even up until today and still continuing. The project design included biannual collection of individual fecal samples and quarterly serology from all milking and non-lactating cows. Additionally, culled cows were tracked as much as possible from the farm to the slaughterhouse, and at the slaughterhouse four gastrointestinal tissues and a fecal sample were collected with the cooperation of USDA Food Safety and Inspection Service personnel. A summary of the sampling scheme is shown in Figure 1. During the study, the farms remained closed and did not purchase animals. Farm B was constituted from several herds just prior to the start of the study. Farm C included a number of cows from a neighboring dairy for a while due to a barn fire at this neighboring dairy. Farm A was a closed farm for years before the start of the study and remained a closed farm throughout the study. Throughout the study, the farm owners received all results from all tests and were advised with regard to optimal MAP management practices that would result in lower MAP prevalences. The harvested tissues included two lymph nodes located at the ileocecal junction and two pieces of ileum, one taken from 20 cm proximal to the ileocecal valve and the other taken from very near the ileo-cecal valve. In addition to the sampling of animals, farm environment was sampled in approximately 20 locations on a biannual basis [21]. On each of the farms, demographic data, production data and herd management information was collected. Precise demographic data included birth date, birth location, calving dates, fertility data, animal location data (pen status at any point in time), dry-off dates and eventually culling information and cull dates. This demographic data was collected for all animals present on the farms. Figure 2 shows the number of animals in each pen on the farm over a six year period. For every single day during this time period, animal location on the farm was documented. All diagnostic infection data, strain typing data, herd management, demographic and production data was maintained in a relational database (Microsoft Access).

ELISA

Upon receipt at the laboratory, blood tubes were centrifuged for 10 min at 900 × g and the plasma was separated. Harvested plasma was placed in three 1.5-mL screw capped vials (with a rubber O-ring seal) labeled with farm identification, cow identification, and collection date and stored in a −20°C freezer or stored in a Figure 1 Sampling scheme in the three RDQMA farms. All cows in the three farms were sampled at least twice a year from first calving. At each sampling event, both serum and fecal samples were taken. At the time of culling, animals were tagged with special RDQMA ear tags. When these tags were recognized at slaughter, further samples were taken after slaughter. The harvested tissues included two lymph nodes located at the ileocecal junction and two pieces of ileum, one taken from 20 cm proximal to the ileocecal valve and the other taken from very near the ileo-cecal valve. A final fecal sample was also taken at the time of slaughter. refrigerator for up to 3 days when they could not be processed immediately. Stored plasma samples were taken from the freezer and allowed to come to room temperature before they were processed for ELISA testing. The sample vial was inverted several times to ensure complete mixing. Plasma samples were evaluated with the ParaCheck (Prionics USA Inc., La Vista, NE; formerly CSL/ Biocor) ELISA to monitor serologic status of the cows.

Microbiology

For fecal samples, 2 g was placed in a 50-mL plastic tube containing 35 mL of water (fecal-water tube). The contents were shaken vigorously and placed on a mechanical shaker for a minimum of 30 min. Following mixing, the sample was allowed to stand at room temperature for 30 min. A 5-mL sample from the top portion of the fecal-water tube was transferred to a second 50-mL plastic centrifuge tube containing 25 mL of 0.9% hexadecylpyridinium chloride in half-strength brain heart infusion broth solution (final concentration of hexadecylpyridinium chloride = 0.75%). Next, tubes were incubated at 35 to 37°C for 18 to 24 h (decontamination or germination step). Following the germination, tubes were centrifuged for 30 min at 900 × g, the supernatant was discarded, and the pellet was resuspended by adding 1 mL of antibiotic brew (1 L of half-strength brain heart infusion broth, 18.5 g/L; amphotericin B, 50 mg/L; nalidixic acid, 100 mg/L; vancomycin, 100 mg/L) followed by vortexing. In the next step (incubation step), the resuspended pellet was incubated overnight or up to a maximum of 3 days at 35 to 37°C. Following incubation, 4 tubes of Herrold's egg yolk media (2 in-house and 2 commercial (BD Diagnostics)) were inoculated with 0.2 mL per tube and then incubated in a slanted position at 37°C. The tubes were read every 2 weeks with the final reading at 16 weeks. Slightly raised white-yellow colonies were evaluated for typical acid fastness and morphological appearance of MAP. Each culture with colony growth was subcultured for mycobactin dependency before reporting the culture positive for MAP.

MAP Shedding pattern analysis

Animals with at least four data points on bacterial shedding were included in an analysis on MAP shedding patterns. Two types of shedding were recognized, progressors and non-progressors. One group of MAP shedders were indicated as progressors, these animals showed an increase in cfu of MAP over time. In these progressors, no samples were obtained from these animals that were negative for MAP (0 cfu) after previous samples where MAP bacteria were identified. Nonprogressors were defined as cows with no increase in cfu of MAP shedding and measurement of absence of shedding in between fecal samples that showed MAP shedding were present in these animals.

Molecular epidemiology, strain typing methods

The most frequently used method for MAP straintyping is based on sequencing of multilocus short-sequence-repeats (MLSSR). This sequence based method is a highly discriminatory method that has been used for typing M. avium subsp. paratuberculosis isolates and many other bacteria [22]. While only a limited number of crosssectional studies have used this method, and with a restricted set of isolates, it has been recognized that the use of well-designed longitudinal studies using several herds in multiple states is essential for applying the MLSSR sequencing technique to understand the epidemiology of M. avium subsp. paratuberculosis [5]. Six loci were selected due to their highest genetic diversity indices and were identified as the most discriminatory, stable, and informative SSR loci [5]. PCR amplification was carried out with extracted DNA for all isolates using the previously published primers for the six loci [5].

Estimation of adult exposure as it relates to infection status at slaughter

Using the demographical data for each cow, it was possible to evaluate for every cow in Farm A the daily pen location (see Figure 2 for farm pen data) and therefore the daily configuration of cows in each pen. Combining the pen location data with fecal culture results and molecular typing allowed for strain specific estimation of exposure of each individual cow on a daily basis. For each cow in the herd, we calculated a strain-specific estimate of exposure-days and days without exposure and then regressed this against the cow's strain-specific infection status at slaughter as the outcome variable (see below).

Statistical methods

All data were stored in databases and evaluated for missing or unlikely values. Data quality was checked on a continuous basis. Statistical analysis were done in SAS v. 9.3. All data were analyzed using descriptive methods. Data on the risk of infection at slaughter as it relates to exposure to MAP as adult cows was analyzed using logistic regression analysis. The risk of strain-specific MAP infection at slaughter was modeled as a binary variable, and the number of 100 day periods that a cow was in the same pen as a shedder of the same strain was used as the predictor variable. The logistic regression model was then:
Logit MAP-infected Strain i ð Þ ¼ β 0 þ β 1 Ã Days exposed to strain i þ error
These analyses on the risk of infection as an adult cow were only done for the dominant strains in farm A.

Results

Duration of measurements on farms was different between the three farms. Farm A was studied for approximately 10 years and is still being followed. Herd B was in the study for a total of 8 years and herd C was in the study for a total of 7 years. During the years of sampling, a total of approximately 7000 fecal samples, 13 000 serum samples and 1500 tissue samples were collected. These data are summarized in Table 1.

Fecal results

Prevalence data for fecal results in both fecal samples and in tissues harvested at slaughter are shown in Figure 3 and Table 1. Prevalence in fecal samples ranged between 1.2% and 5.0% of samples with an average of 2.2%. These prevalences are in line with observed prevalence in many surveys [10,18]. However prevalence in tissues was much higher, and ranged between 8% and 30% of all animals with tissue culture results, averaging a prevalence of MAP culture positive of 17% of all cows with tissue culture results.

Shedding patterns of 58 individual animals with at least four positive MAP culture results are shown in Figure 4. In Figure 4, animals are split into two groups of shedding patterns. One group of MAP shedders are indicated as progressors (n = 16), these animals show an increase in cfu of MAP over time. In these progressors, no samples were obtained from these animals that were negative for MAP (0 cfu) after previous samples where MAP bacteria were identified. Non-progressors (n = 42) were defined as cows with no increase in cfu of MAP shedding and measurement of absence of shedding in between fecal samples that showed MAP shedding.

These individual shedding patters were used for evaluation of shedding patterns in the companion paper by Mitchell et al. [23]. These longitudinal patterns are valuable to show infection progression, or lack thereof, over time. It appears from these data that animals that eventually become high shedders may be identified early based on their MAP shedding pattern [23].

Molecular strain typing data

In Figure 5, results of MLSSR sequencing technique for discrimination of M. avium subsp. paratuberculosis isolates are shown to describe strain diversity on three farms. It is clear from this figure that multiple infection transmission patterns are present in these herds. Herds A and C show the presence of a dominant strain that is responsible for a large proportion of the observed MAP infections. In contrast, herd B shows the presence of multiple strains at approximately the same frequency. Without having identified the exact transmission pathways, these data appear to indicate that transmission in herds A and C was predominantly due to contagious infection patterns. In herd B this is less obvious, and it may be hypothesized that animals may become MAP infected from multiple sources.

Serological results

Serological results are shown in Figure 6 Figure 7. There was a linear increase in ELISA OD value with increasing CFU in animals that showed an infection progression. Animals that belong to the the non-progressors, had ELISA of values that were low and not significantly different from cows that never shed MAP. In Figure 8, the relationship between cfu of MAP in culture results and ELISA OD value of samples taken at the same time is shown. There was a strong correlation between cfu of MAP and OD ELISA values. Particularly in samples with a cfu close to or more than 100 cfu showed high OD ELISA values. As shown in Figure 7, these higher OD values are virtually only present in animals that show infection progression.

Adult risk of infection with MAP

Data analysis on adult cow physical proximity to shedders and the risk of infection at time of slaughter are shown in Figure 9. Cows that were infected at the time of slaughter with a given strain of MAP were significantly more exposed as adults to cows shedding the same strain of MAP compared to cows that were culture negative for MAP at slaughter. The statistical analysis of these data presented in Table 2 indicated that per 100 days exposed to a heavy shedder, the odds of being infected at slaughter increased significantly with an odds ratio of 1.12 (1.06-1.18). The model fitted the data well, based on the observed AIC value. These results were analyzed for each of the dominant strains on each of the farms (Figures 5 and 9).

Discussion

Particularly with infectious disease with a slow progression such as infections of ruminants with MAP, longitudinal data are essential to understand infection dynamics. In this manuscript, approximately 10 years of longitudinal data with multiple measurement per year was available to understand MAP infection dynamics. Previous reports on MAP infection dynamics assume infection transmission routes through in-utero infection [24], and infection in early life [25]. Typically an exponential decay in the risk of MAP infection with age is  In this graph MAP straintyping is based on sequencing of multilocus short-sequence-repeats (MLSSR). Six loci were selected due to their high genetic diversity. PCR amplification was carried out with extracted DNA for all isolates using the previously published primers for the six loci [5]. Strain types were coded using an uninformative coding system, coding sequence types from 1 to 15. Strain diversity per farm is shown in this figure.

assumed, with a zero risk of infection after the first year of life [2,14,19]. Based on the longitudinal data presented in this manuscript, it has been possible to rethink some of the infection transmission routes of MAP in endemically infected herds. First, the concept of very low prevalence in endemically infected herds was again dismissed based on the observed data. As with many other studies [10,26], the actual number of cows with positive diagnostic tests at any given point in time is low, in our data anywhere from 1% to approximately 5%. If these prevalence data would reflect the true prevalence of MAP infection, it might be expected that many herds just by chance would be able to eliminate MAP infections from the herd [12]. However, the reality is that such herds that eliminated MAP do not exist in large numbers. Unique in the RDQMA data is the longitudinal follow up where a number of animals were followed all the way into the slaughterhouse. It turned out that these data on tissue MAP infection status showed a much higher prevalence, where an overall prevalence of approximately 20% was observed. These results that show high MAP prevalence are in line with slaughterhouse studies of Wells et al. [27] and Vazquez et al. [26]. To match the fecal and serum prevalence data with the tissue culture results, it is necessary that many animals are actually infected, but that only a relatively small portion of these animals are showing fecal shedding or a sero-response. Or, many MAP infected animals show a latent or intermittent shedding stage where no MAP isolated can be found using the current diagnostic methods. Still, this high prevalence is likely necessary to maintain infection in the herd. Further understanding on the importance of latent MAP infections is one of the key research areas to work on in the coming years.

Vazquez et al. [17,26] presented very similar results on MAP prevalence, in a cross section study on 333 randomly selected Holstein-Friesian cows at slaughter, an infection prevalence based on pathology, histology, serology and rtPCR was estimated. Approximately 53% of cows showed histological lesions associated with paratuberculosis, 29% of cows were positive in rtPCR, 14% of cows were positive in tissue culture, while only 6% of cows was positive in serum ELISA. There was a clear increase in immune responsiveness as lesion severity increased [26], very similar to the observed relationship between ELISA results in progressors and non-progressors as observed in our data, although both Vazquez et al. [26] and Wells et al. [27] were both cross-sectional studies. Vazquez et al. [26] introduced the terms latent and patent infections. Here, latent infected animals show focal granulomatous lesions with our without the presence of MAP and little or no ELISA positivity, while patent infected animals show advanced lesions with MAP presence and very high ELISA positivity. Although the Vazquez study was a cross-sectional study, the observed infection dichotomy appears to coincide with progressors and non-progressors in our data.

The distribution where a large proportion of hosts are infected but only very few shed very high numbers of pathogens and suffer clinical signs is also observed with macro-parasite infections [28,29]. This aggregation of shedding patterns is represented by the negative binomial distribution, where a measure of aggregation,  together with the mean, is used to describe the distribution of infectious organisms between hosts [28]. This widely observed aggregation in organism burden arises from heterogeneities in host populations or in infection pressure. These heterogeneities may be generated by changes in the climate over time or space; genetic differences between hosts [30]; heterogeneity in infection levels, because of host or physiological (age, sex) diffe-rences. It is argued [28,29] that the presence of both infectious organisms and the immune response in hosts produces more stable dynamics and lower host population sizes than that observed in the absence of infectious organisms. In evolutionary analyses [30] it can be shown that parasite fecundity is an evolutionarily stable strategy. Phenotypic polymorphisms with respect to immunity in the host species are common and expected in  Lifetime exposure for MAP infected and MAP free cows. Days exposed and not exposed to MAP shedders for both MAP infected and MAP infection free cows. Where ○ indicates cows that are tissue and fecal culture negative at slaughter, ■ indicates cows that were culture positive in either tissue or fecal at slaughter. Regression analysis showed that per 100 days exposed, cows had an odds ratio of 1.12 (1.06-1.18) to be MAP infected at slaughter compared to MAP unexposed cows. evolutionary stable host strategies. These similarities in infection and host response profiles in macroparasites and microparasites would suggest that endemic infectious diseases such as MAP have an evolutionary background and operate as population tools that result in more stable host populations.

A second observation that may change our thinking on MAP epidemiology is the apparent occurrence of new infections in adults. Based on molecular data, we concluded earlier that such adult infection are indeed likely [5], and now a more quantitative argument where cows exposed to high shedders as an adult, were significantly more likely to be MAP infected at slaughter with the same strain (see also Figure 9). These precise MAP exposure data, combined with previously reported molecular epidemiological data [5] support the occurrence of new MAP infections in adult animals in endemically infected herds. The data available for analysis of exposure information was limited to cows with complete information. These cows differed from all cows particularly in more of these cows being present earlier in the study. This may have resulted in a bias towards cows born earlier in the study. It is not expected though that the biology of MAP infection has changed over the years of the study.

Although adult exposure may lead to a detectable MAP infection, the role of these adult infected animals in infection transmission is still unclear. It may be expected that these adult infections are less likely to show high shedding or severe clinical disease [19]. Still, transmission in utero and transmission from dam to daughter may still be considered realistic possibilities. Certainly the observed relationship between adult exposure and MAP infection creates a much larger window of infection opportunity. Currently known infection routes are then in-utero [24], from dam to daughter [25], from calf-to-calf [20], and young adults and adults from other adults [this manuscript , 5].

These data also emphasize the value of precise infection data, where not only repeated observations per year were used, but also where all MAP isolates where typed using molecular typing methods. These molecular typing methods have recently become available [22] and are valuable to better understand infection dynamics. In reality, on the farms that we observed, multiple infection dynamics occur simultaneously. As a consequence strain specific transmission studies would be necessary. Hence in future observational studies, molecular typing of obtained isolates is essential to understand infection dynamics on farms.

Genetic selection of animals, where animals are preferably selected that are not infected with MAP may need to be re-evaluated [31,32]. When in reality a very large proportion of animals are MAP infected, and show very little immune response or clinical signs, then selection against such an infection state may not be valuable or even feasible. With such high MAP infection prevalence as reported in this study and by the studies of Vazquez et al. [17,26] and Wells et al. [27], elimination of MAP may not be a reasonable objective for genetic selection programs or control programs. Instead, genetic selection against progressors (our data), high shedders or patent infected animals [17] would be more successful in terms of population progression. In a follow-up study to their initial survey, Vazquez et al. [26] evaluated the genetic associations between phenotype and genotype. It may be hypothesized that non-progressors (latent infections) are a preferred response to MAP infection. These non-progressors show limited lesions and a limited humoral response often combined with low or no MAP presence and represent an immune response that prevents animals from developing more severe forms or even bacteriological cure. In contrast, progressors (patent infections) correspond to true failures with a high antibody production and a high MAP load, and detectable and consistent MAP shedding. Genetic selection should then logically be focused on identifying genetic association with the progressor phenotype. As shown by Vazquez et al. [17], a difference in genetic control between MAP infection phenotypes may be present in the population.

Mycobacterial diseases such as Johne's are extremely difficult to control due to long latent periods, poor diagnostic sensitivity, wildlife and environmental reservoirs of infection, and heterogeneous strain infectiousness.

The key to controlling these diseases is an integrated approach to understand the pathways through which pathogen transmission occurs at all levels in an ecosystem: within animals, between individual animals, between livestock and wildlife, and between livestock and the environment [21]. As we are studying agricultural systems, which must be commercially viable, economic decisions play an important role in contact structures, cattle life histories, and control measures. Thus, we must consider the effects of economic determinants on the transmission dynamics of these systems, as well. Traditional a single discipline approach fails to consider the system as a whole. We believe that an ecological approach, simultaneously considering the impact of all aspects of the disease ecosystem, combined with economic analysis will the offer many advantages over past approaches. Based on observations in our RDQMA data and other studies [10,17,27], MAP control programs may need to be refocused towards control rather than elimination of MAP infections. The more realistic objective would be to reduce or eliminate clinical disease and production losses due to MAP [33,34]. Such programs aimed at control rather than elimination would likely have many similar characteristics compared to current programs. Reduction of exposure in young animals would be a key characteristic, just as identification and culling of known high shedders [12,13], particularly identified progressors. However, additional components would include reduction of exposure in young animals and in adults, again through identification and elimination of progressors. Genetic selection against progressors would be an additional program component as well as identification of strain types in identified MAP isolates. Based on the identified strain types in a given population, more or less aggressive diagnostic and culling programs may be implemented. Clearly, communication of realistic goals to dairy producers in these control programs would be essential. Further research to develop and evaluate such refocused MAP controls will certainly be necessary.

To carry out such research requires not only detailed longitudinal data, such as the RDQMA data described in this paper, but also to develop the methodology to accurately investigate the transmission of pathogens. Recent improvements of typing techniques, which allow differentiation of MAP strains within cattle herds, have allowed researchers to evaluate within-farm distribution of MAP strains [5,22, this manuscript]. Estimation of transmission based on large scale sequencing data has not been applied to MAP modeling efforts, although the results of a recent study using MAP isolates suggested that estimation of transmission pathways using sequencing data is currently feasible and should be used for estimation of transmission pathways of MAP [15]. As bioinformatics tools become faster, easier, and more affordable, their application to disease research has the potential to expand beyond outbreak investigation to elucidating the fundamentals of disease ecology and transmission. However, new methodologies will be needed to tie these emerging tools to existing methods of analysis, including classical epidemiologic models.

The ideal outcome of such research would be to develop a methodology for incorporating whole genome sequencing results in MAP bacterial transmission models [35,36], involving agricultural systems, cattle lifehistories, environmental and wildlife reservoirs, and economic decisions. This will allow us to predict the role of each potential source of infection and to recommend control options targeting these sources, expanding the toolbox available to decision makers.

Longitudinal data are essential to understand infection dynamics of slowly progression infections. Infection dynamics of MAP in three US dairy herds was studied for approximately 10 years and these observations resulted in a number of important changes in our understanding of MAP infection dynamics. First, a much higher prevalence of MAP infection was observed in these herds due to lifetime follow up of cows, including slaughter. Approximately 20% of cows turned out to be MAP infected. Second, two distinctly different infection patterns were observed, so called non-progressors and progressors. Non-progressors were characterized by intermittent and low shedding of MAP bacteria and a virtual absence of a humoral immune response. Progressors were characterized by continuous and progressive shedding and a clearly detectable and progressive humoral immune response. Strain typing of MAP isolates on the three farms identified on two of three farms a dominant strain type, indicating that some strains are more successful in terms of transmission and infection progression. Based on these observations, control programs including specific genetic selection may need to be refocused. Continuous high quality longitudinal data collection turned out to be an essential tool in our understanding of pathobiology and epidemiology of MAP infections in dairy herds.

Figure 2
2Cow location per pen from 1/1/2004 and 12/31/2010. Precise data on cow location within farm was available here for all cows and all pens on one of the farms in the RDQMA study. Shown in this figure are the number of cows present per day in three lactating pens, dry cow pen and fresh pen.

. As expected, the vast majority of animals have low OD values, only a small proportion of animals have OD-values above .2. Although the cut-off for positive results is batch specific, the approximate value for a positive test is an OD value of .2 or greater. Across all farms, only 1.4% of samples were considered serologically positive. Serological results for cows showing infection progression and not showing progression are shown in

Figure 3 Figure 4
34Serology and culture based MAP prevalence on three RDQMA farms. Prevalence of MAP in all samples collected throughout a 10 year follow-up study in three dairy farms in New York, Pennsylvania and Vermont. Prevalence in three farms and overall prevalence are shown. Prevalence of MAP in serum samples is measured through ELISA, prevalence in fecal samples is measured using MAP culture methods, prevalence in tissues, collected at slaughter, is measured through culture of four tissues samples. Longitudinal shedding pattern of cows. Cows in the top graph are progressing to be a high shedder, while cows in the lower graph are shedding intermittently and low numbers of cfu. Progressors (n = 16) were defined as cows with an increasing cfu of MAP shedding over time. Non-progressors (n = 42) were defined as cows with no increase in cfu of MAP shedding and measurement of no shedding in between measurements with MAP shedding.

Figure 5
5MAP strain types based on short sequence repeat typing.

Figure 6
6ELISA MAP results of all samples collected in the study. Plasma samples were evaluated with the ParaCheck (Prionics USA Inc., La Vista, NE; formerly CSL/Biocor) ELISA. In this figure the optical density value as measured at the end of the ELISA process is shown. Although a cut-off is defined for each batch of samples, the approximate cut-off value for samples to be considered MAP positive is a value greater than 0.20 optical density units. Overall prevalence of positive samples is approximately 1.4%.

Figure 7
7Distribution of ELISA OD values for progressors and non-progressors. ELISA optical density values are shown for MAP progressors and Non-progressors. Progressors (n = 16) were defined as cows with an increasing cfu of MAP shedding over time. Non-progressors (n = 42) were defined as cows with no increase in cfu of MAP shedding and measurement of no shedding in between measurements with MAP shedding.

Figure 8
8Relationship between cfu MAP and OD ELISA value. For this analysis, only samples where Fecal and serum sample were taken on the same day were included in the analysis. CFU values were calculated as the average number of colonies from four culture plates. Values below 1 are possible due to single colonies in only one or more culture plates, but not in all four plates. A total of 10.510 samples were included in the analysis.

Figure 9
9Figure 9 Lifetime exposure for MAP infected and MAP free cows. Days exposed and not exposed to MAP shedders for both MAP infected and MAP infection free cows. Where ○ indicates cows that are tissue and fecal culture negative at slaughter, ■ indicates cows that were culture positive in either tissue or fecal at slaughter. Regression analysis showed that per 100 days exposed, cows had an odds ratio of 1.12 (1.06-1.18) to be MAP infected at slaughter compared to MAP unexposed cows.

Table 1
1Number of samples collected during the course of RDQMA studyFarms

A
B
C
Total

Herd size
330
105
145
580

Fecal results

Negative
3905
1366
1076
6347

Positive
71
16
56
143

% positive
1.79%
1.16%
4.95%
2.20%

Tissue results

Negative
1274
911
358
1592

Positive
318
76
155
318

% positive
20.0%
7.7%
30.2%
16.7%

Serological results

Negative
7531
2509
2807
12847

Positive
105
17
55
177

% positive
1.38%
0.67%
1.92%
1.36%

Table 2
2Logistic regression results of risk of infection at slaughterCow-specific exposure during their adult life was regressed against risk of infection with the same MAP strain identified at slaughter was.Parameter
Estimate (SE)
Chi-square
P-value
Odds ratio (95% CI)

Intercept
−5.25 (.48)
118.69
<.0001

MAP exposure strain i
0.11 (.027)
16.56
<.0001
1.12 (1.06 -1.18)

AcknowledgementsThe authors acknowledge the collaboration of the three dairy farmers and their staff within the Regional Dairy Quality Management Alliance. Without their input and support, the data collection in this study would not have been possible. The authors also acknowledge the support of the Within-host modeling of MAP infections Working Group at the National Institute for Mathematical and Biological Synthesis, sponsored by the National Science Foundation, the U.S. Department of Homeland Security, and the U.S. Department of Agriculture through NSF Award DBI-1300426, with additional support from The University of Tennessee, Knoxville.Competing interestsThe authors declare that they have no competing interests.Authors' contributions YHS, AB, YTG and RMM wrote the manuscript, RHW, JvK, DW collected data, performed diagnostic testing and reviewed the manuscript. All authors read and approved the manuscript.
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Effect evaluation of a heated ambulance mattress-prototype on body temperatures and thermal comfort -an experimental study

Jonas Aléx
Stig Karlsson
Britt-Inger Saveman
Effect evaluation of a heated ambulance mattress-prototype on body temperatures and thermal comfort -an experimental study
O R I G I N A L R E S E A R C H Open Access
Background: Exposure to cold temperatures is, often, a neglected problem in prehospital care. One of the leading influences of the overall sensation of cold discomfort is the cooling of the back. The aim of this study was to evaluate the effect of a heated ambulance mattress-prototype on body temperatures and thermal comfort in an experimental study. Method: Data were collected during four days in November, 2011 inside and outside of a cold chamber. All participants (n = 23) participated in two trials each. In one trial, they were lying on a stretcher with a supplied heated mattress and in the other trial without a heated mattress. Outcomes were back temperature, finger temperature, core body temperature, Cold Discomfort Scale (CDS), four statements from the state-trait anxietyinventory (STAI), and short notes of their experiences of the two mattresses. Data were analysed both quantitatively and qualitatively. A repeated measure design was used to evaluate the effect of the two mattresses. Results: A statistical difference between the regular mattress and the heated mattress was found in the back temperature. In the heated mattress trial, the statement "I am tense" was fewer whereas the statements "I feel comfortable", "I am relaxed" and "I feel content" were higher in the heated mattress trial. The qualitative analyses of the short notes showed that the heated mattress, when compared to the unheated mattress, was experienced as warm, comfortable, providing security and was easier to relax on. Conclusions: Heat supply from underneath the body results in increased comfort and may prevent hypothermia which is important for injured and sick patients in ambulance care.

Introduction

Prehospital patients are vulnerable to cold exposure especially in situations of grave danger such as serious injury situations or severe illnesses [1]. Hypothermia is an independent predictor of increased morbidity and mortality regardless of injury severity [2]. Several studies state the physiological effects of hypothermia [3][4][5]. Factors that may impair the body's natural thermoregulatory mechanisms are fatigue, central or peripheral nervous system injuries, medication, and the influence of alcohol or drugs [5,6]. Additionally, age, female gender, chronic illness, trauma, starvation, and endocrine diseases [5,7] are regarded as aggravating circumstances. Traditionally, hypothermia has been defined as body core temperature <35°C. Because of poor prognosis in the combination of trauma and hypothermia the level has been set to <36°C [8].

A review study described hypothermia as initiated by cold exposure and peripheral vasoconstriction leading to peripheral blood being shunted to the central body regions in order to support the vital organs and to retain heat [9]. Initially, the cold is experienced and observed in the hands and feet, but the back also cools quickly [1]. In general, after being cold, it is difficult to get warm and it takes longer to recover than it does to become cold [6]. Although there are major shifts in temperature in the environment, the human body strives for a constant temperature between 36-38°C, which is the temperature at which physiological functions operate optimally [9,10].

Patients can be expected to have a negative experience even before the body reaches a temperature low enough to qualify as hypothermia [11]. Aléx et al. [12], showed in their study that patients injured outdoors in cold environments experienced anxiety of freezing to death. Despite injury coldness gradually became the primary problem. They expressed that it felt good to receive active heat supply and warmth spreading throughout their whole body.

A review study by Kober, Scheck, Fulesdi, Lieba, Vlach, Friedman and Sessler [4], showed increased anxiety and perceptions of pain in participants when exposed to cold. After the patients had been wrapped with a warm blanket, they stressed that they experienced decreased anxiety and had a more positive experience because it increased the thermal comfort. Similar results were seen in elderly patients who experienced an external heat supply to be positive. The heat supply increased satisfaction and decreased experience of fear and restlessness [13,14].

In nursing, preventing thermal discomfort can be seen as an important way to satisfy fundamental needs [15]. Thermal comfort seems to improve patients' psychological and physiological status and is also important for prehospital patients. From a nursing perspective warmth seems to contribute to experiences of comfort and safety. In prehospital settings there are studies concerning the supply of heat sources such as chemical heating pads, hot water bottles, charcoal heater [16], hot air blankets, and reflective blankets on top of the body [4,5]. Prehospital rewarming with heated intravenous fluids is not efficient and intravenous fluids cool rapidly and aggravate hypothermia [5,17]. In prehospital care, in Sweden, warm intravenous fluids are available, but if the ambient temperature is lower than 37°C the intravenous fluids will adapt to the ambient temperature and instead contribute to a decreasing body temperature [17]. Polyester blankets used in ambulance care have a bad insulation value in windy conditions compared to indoor use [18].

Despite these findings, limitations exist concerning equipment for reducing risks causing decreased thermal comfort for patients in modern ambulances. There is a lack of research about the effect of heat supply from underneath the body. We assume that it would be beneficial to prevent conductive heat loss by using a heated mattress underneath the patient because of the back's large surface.

Exposure to cold temperatures is often a neglected problem in prehospital care [1]. Cooling of the back is one of the leading influences of the overall sensation of discomfort from cold [19]. Exposure to a cold environment is by far, the largest contributor to hypothermia [9]. Thus, in prehospital care, it is important for nursing staff to identify and prevent heat loss to avoid patients' thermal discomfort and hypothermia [1,12,20].

In the wintertime, when the stretcher or equipment is taken out of the ambulance, the patient compartment and associated equipment is cooled down quickly, and the patients are also exposed to cold by conduction [11]. Conduction is the most effective method of heat and cold transfer [17].

It seems important to investigate the effect on thermal comfort using active heat supply from underneath. Can a heated mattress have a positive impact on body temperatures and increase thermal comfort for persons requiring ambulance transport? The aim of this study, therefore, was to evaluate effects of a heated ambulance mattress-prototype on body temperatures and thermal comfort in an experimental study.

Method

Design

Both quantitative and qualitative data were used to obtain a more comprehensive picture than what either method would reveal used alone [21].

Setting

Data were collected during four days in November, 2011 inside and outside of a cold chamber. The cold chamber had a constant temperature (+2°C). Outside the cold chamber, the temperature was approximately +21°C. Data were collected by three ambulance nurses. Each nurse cared for one participant at a time. In the cold chamber, each nurse had a separate area from the other nurses when collecting data from the participants. The stretchers were in different rooms and the participants had no possibility to communicate with each other.

Participants

Twenty-three of approximately 50 students from the Umea University Nursing Department agreed to participate in the experiment, 20 women and three men. The average participant age was 24, varying between 19 and 46. Exclusion criteria were being pregnant, smoking or snuff user, having cold-induced asthma, and being medicated with circulation affecting drugs. None of the 23 students were excluded.

Experimental procedure

All participants participated in two trials each. In one trial, they were lying on a stretcher supplied with a heated mattress and in the other trial without a heated mattress. Three stretchers were used, of which one had a heated mattress. The participants performed the experiment three at a time with an ambulance nurse each of their side during the experiment. The first trial began with measurements ( Figure 1) at room temperature, i.e., baseline. Participants were then exposed to cold temperature in the cold chamber for 10 minutes. The participants were told to wear only one layer of clothing, i.e., trousers and a thin shirt, and to stand up without moving their hanging arms. The individual measurements were repeated after 10 minutes in the cold chamber. They then left the cold chamber and lay down in a room with 21°C on an ambulance stretcher with a sheet and wrapped with a blanket. They were randomly selected to lie down on an ambulance stretcher supplied with a heated mattress (n = 8) or without a heated mattress (n = 15). The heated mattress is 150 cm long and is connected to 12 volts with a temperature of + 30°C. After ten minutes on the stretcher, a third measurement was performed. Thereafter, the participants had 15 minutes to recover their body temperature with warm clothes until they felt that they returned to their original temperature at baseline ( Figure 1). The second trial was identical except that the participants now used the opposite stretcher, either supplied with a heated mattress or no heated mattress.

At each measurement time, temperatures were measured, back temperature between the shoulders and finger temperature, on outer fingertips, left hand were measured with an IR thermometer with dual laser points indicating the measurement area, CIR 8819. Measurements were taken from approximately 7 cm from the measurement surface/area (3.5 cm Ø). Core body temperature was measured via internal ear, infrared light (IR), with a Braun Thermo Scan, Exac Temp IRT 4520, Germany.

To measure thermal discomfort, Cold Discomfort Scale (CDS) was used; it has been used in previous studies [7,22,23] and validated [24]. The scale ranges from 0-10; 0 is equal to no discomfort from the cold, and 10 is the worst possible discomfort from the cold. Further, four statements from the STAI [25] were used. The statements "I am tense", "I feel comfortable", "I am relaxed", and "I feel content" were measured. The answers to the four statements from the STAI instrument were estimated from 1-4 where 1 = Not at all, 2 = Somewhat, 3 = Moderately So, and 4 = Very Much So [25]. At the end of the day, the participants (n = 21) wrote down short notes about their experiences of laying on the different mattresses. No structured questions were asked in the notes section.

Quantitative data analysis

Sample size calculation showed that a minimum of 17 participants was required in each trial. The endpoint finger temperature was assumed to differ 0.5°C between the trials with a power of 80% and a significant level of 5%. To examine differences between trials, the data were separated according to the heated mattress trial (n = 23) and the unheated mattress trial (n = 23). Paired-Samples t-Test and Wilcoxon signed rank test were used. The difference between having spent 10 minutes in the cold chamber and then lying on the mattresses 10 minutes was calculated on an individual level for each parameter/statement. Difference in mean, standard deviation, significance level, and effect size were calculated. The statistical analyses were performed with SPSS software (version 18.0, SPSS Inc., Chicago IL, USA).

Qualitative data analysis

The participants were asked to make their own short notes at the end of the experiment. The short notes, including a few sentences per participant, were analysed according to manifest qualitative content analysis [26], where the short notes were categorized in four categories, corresponding to the aim of the study.

In the present study, the qualitative and quantitative data were analysed separately. Using qualitative and quantitative datasets may provide greater insight than would have been possible only considering each dataset separately [21].

Ethical considerations

The study was approved by the Regional Ethical Review Board in Umeå, (reference number 2011-343-31 M). Participation was voluntary, and participants were informed both orally and written about the experimental approach, and that they could withdraw from the study at any time. No one outside the research team had access to the material. All material from the observations was treated confidentially.

Results

Quantitative results

A statistically significant increase and a large effect size were seen in back temperature between the heated mattress trial compared to the unheated mattress trial. The difference concerning finger temperature, ear temperature, and CDS showed no statistical significance (See Table 1.)

There was a statistically significant decrease in the participants rating of the statement "I am tense" (p = 0.011, ES = 0.37) from the heated mattress trial compared to the unheated mattress trial; a medium effect size. There was a statistically significant increase and medium effect size of the participants' statements "I feel comfortable" (p = 0.012, ES = 0.37), "I am relaxed" (p = 0.029, ES = 0.32), and "I feel content" (p = 0.008, ES = 0.39) from the heated mattress trial compared to the unheated trial.

Qualitative results

The heated mattress was positively experienced. The analysis resulted in four categories. The analysis from text to category, exemplified with quotations, is described in Table 2. 1. Being warm and comfortable. The experiences of the heated mattress were expressed as warm and pleasant, especially on thighs and back, and laying on the heated mattress was a good experience and the whole body was warmed up. 2. Provides security. It was a good experience to be on the heated mattress. It was expressed that the heated mattress transferred heat and safety. 3. Being unrelaxed. It was expressed that it was difficult to relax on the unheated mattress because of the cold, and that it was cold to lie on. 4. Not being warm. It was expressed that it took a long time to get warm on the unheated mattress, and some participants did not feel any warmth for a long time afterwards.

Discussion

The aim of this study was to evaluate effects of a heated ambulance mattress-prototype on body temperatures and thermal comfort in an experimental study. It has been a contentious issue whether hypothermic patients should be warmed or not in prehospital care. The justification against heating has been a risk of complications, but there is also a lack of equipment and monitoring methods to control the effect. Increasingly, studies [3,12,16,[27][28][29] show positive results using different active warming methods already in the ambulance. It has become clear that this is a safe and profitable complement to other medical interventions. However none of the previous studies have investigated active heat delivered from underneath the body.

Several studies from hospital settings have investigated different methods to prevent and treat cold exposure, e.g., warm blankets [13,14]. Warming polyester blankets has been shown to be ineffective and the initial benefit dissipated in about 10 minutes [30].

In the present study, the participants show that the heated ambulance mattress-prototype had an effect on thermal comfort. The result show that the participants had a higher back temperature, higher rating on the CDS, and felt less tense, more relaxed, more comfortable and more content in the heated mattress trial compared to the unheated mattress trial.

The back temperatures were higher in the heated mattress trial compared to the unheated trial. There is a lack of research of active heat delivered from underneath the body in prehospital care; however, there are intraoperative studies stating that using an underbody warming system is effective in preventing hypothermia [31,32]. We argue that a constant active heat source is preferable to no active heat source in prehospital care. A chemical heating pad supplied on the chest to prehospital patients showed a more positive experience, compared to only supplying blankets [12,22]. As in our study, similar results using warm blankets in operation units showed increased comfort and reduced anxiety [4]. Sessler and Schroeder [30] described how 90% of persons in cold environments who were given an electric blanket on top of the body experienced it to be comfortable. Electric blankets were also shown to more effectively reduce acute low back pain for patients being transported to hospital when compared to woolen blankets [33]. Results from our previous study show that the unheated ambulance mattress appears to be an important factor in cooling the patients. The patients seem to warm up the stretcher, and not the other way around [11]. We argue that the supply of active heat is important because the synthetic blankets used in prehospital care that patients are wrapped in, are almost worthless in windy conditions as 8 m/sec [18] and in environments with cold temperatures [11]. We also argue that when using heat from underneath, the staff has better access to examine the patient compared to when using resistive blankets.

Participants in the present study showed no significant decrease on the estimate of the CDS scale in the heated mattress trial compared to the unheated mattress trial. However, the qualitative result showed that the heated mattress felt much warmer compared to the unheated mattress.

The statements showed a decrease of the statement "I'm tense" and an increase on the statements "I feel comfortable", "I am relaxed", and "I feel content". The statements can be seen as increased thermal comfort associated with the heated mattress. Candas and Dufour [34] clearly state that there is a relationship between skin temperature and discomfort. Cooling of the back is one of the leading influences of the overall sensation of discomfort from cold [19]. Similar to our study, other studies states that active heating during ambulance transport increases thermal comfort, core temperature, reduces pain and anxiety, and improves overall patient satisfaction in prehospital care [4,14,22]. Following the results from our study and other research results [3,16,[27][28][29], we argue that it is important to provide active heat in prehospital care, such as in the ambulance and that thermal comfort might contribute to the possibility for the patient to rest in a stressed situation. Our study on a heated mattress has shown positive results on thermal comfort.

The short notes from the participants showed that they experienced the heated mattress to be warm, safe, and easier to relax on. They found it pleasant to be Table 2 Examples of findings from the qualitative data

Area Categories Citations

The heated mattress Being warm and comfortable "The stretcher felt immediately warm and the heat spread out through the body. After a short while the body was warm again", "The heated mattress felt much warmer even in the neck."

Provides security "I felt secure when embedded on the stretcher."

The unheated mattress Being unrelaxed "It was more difficult to relax on the unheated mattress."

Not being warm "It felt good to have a blanket but the heat took time to return."

warm on thighs and back, and they expressed laying on a heated mattress as a good experience and that the whole body was warmed. This result provides a more comprehensive picture to the quantitative findings. There were no significant decreases on ear temperature. The temperature effect (30°C) of the heated mattress is probably too low to increase body core temperature in 10 minutes. The mattress is, however, intended to be used on all patients, aiming to prevent thermal discomfort and to avoid contact with cold materials for sick and injured patients. When caring for hypothermic patients we recommend using the heated mattress in combination with other active heat sources.

Methodological considerations

In the present study, we found it was useful to use both quantitative and qualitative methods to provide a more comprehensive picture, which also is highlighted by others [35].

The cold chamber had a constant temperature (+2°C), which meant that all participants were exposed to the same temperature. Therefore, variations due to situational factors such as wind, snow, and rain were eliminated. The time in the cold chamber and on the stretchers was constant for all participants. The same type of equipment and devices were used as in an ordinary ambulance care, and all participants wore the same type of clothing during the experiment. We consider the study and the measurement accuracy and reliability to be high in terms of choice of method and relevance and that validity of the study is good.

We are aware that most students in the nursing department are women. Women have, in general a lower hand temperature and are more sensitive to cold exposure by air temperature [36]. However, in the present study we have focused on the differences between the two mattress types and not on gender differences. Data were collected by three ambulance nurses, and even with a careful approach before the study, there is still a risk that the measures were collected differently. However, we believe it could be more of a strength because this data collection is more controlled and more reliable with its clear instructions and few researchers when compared with measurements performed in patient care, e.g., by various ambulance nurses. The significance between trials may have been larger if using a larger sample size or if the time in the cold chamber had been extended. Still, we believe that the results and significant effect size have clinical importance.

The participants considered themselves to be healthy without the influence of trauma, disease, alcohol, or medication. With the exclusion criteria, we considered that some factors that affect reactions to cold were eliminated. This means that our conclusions can only be generalized to that specific group. Despite this, we believe the results are transferable to prehospital care because the experiment was set in controlled conditions without influences of fear and pain that can influence the vasoconstriction and the feeling of chill/cold; and the results show an effect of active heat. Thermal comfort for injured and unhealthy patients can be different from healthy people because of the physical disability that will affect thermophysiology, thermal sensation, blood flow metabolism regulatory response as vasomotor control of body skin temperature, or the ability to sweat [37]. Injured or sick patients may have a worse experience of thermal discomfort in a decreased temperature compared to young and healthy students.

Clinical implication and future research

Future research is needed on patients in ambulance care. Other studies together with present results are evidences enough for expediting the process of clinical implementation with active heat supply in ambulance care.

Conclusion

As shown in this study, using a heated mattress after being exposed to cold increases thermal comfort. There are several positive aspects of using active heat from underneath in the form of mattresses for use in today's ambulances. Heat supply from underneath the body results in increased comfort and may prevent hypothermia which is important for injured and sick patients in ambulance care.

Figure 1
1Observation schedule. Two trials, in and outside the cold chamber.

Table 1
1*Difference between having spent 10 minutes in the cold room and then lying on the stretcher 10 minutes. A negative value denotes a decrease.˜F rom Paired-Sample t-Test. Effect Size (Eta Squared) 0.01 = small or no effect, 0.06 = moderate effect, 0.14 large effect. † †From Wilcoxon signed-rank test. †Effect Size (Cohen) 0.1 = small effect, 0.3 = medium effect, 0.5 large effect.Difference in mean* of back temperature, cold discomfort scale (CDS), ear and finger temperatures between
the heated mattress trial (n = 23) and the unheated mattress trial (n = 23)

Unheated mattress trial
Heated mattress trial

Mean (SD)
Mean (SD)
Difference in mean (%)
p-value
ES

Back temperature (°C)
12.0 (2.98)
14.4 (3.70)
2.4 (20.0)
0.009˜˜0.27Ẽ

ar temperature (°C)
−0.05 (0.42)
−0.01 (0.34)
0.04 (80)
0.69˜˜0.00F

inger temperature (°C)
8.9 (5.15)
8.2 (5.31)
0.7 (8)
0.867 † †
0.35  †

Cold Discomfort Scale (CDS)
−3.5 (1.73)
−4.2 (1.78)
0.7 (20.0)
0.063 † †
0.38 †

http://www.sjtrem.com/content/22/1/43
AcknowledgementsWe would like to thank Västerbottens County Council for funding parts of the study, Umeå Ambulance Service and Roger Westling, for making it possible to do the experimental study. Erika Olofsson and Kristina Forsberg for contributing to data collection. The grocery store, ICA Glunten, Umeå, Sweden for using their cold chamber and Dr. Ulf Isaksson for statistical discussions. http://www.sjtrem.com/contentCompeting interestsThe authors declare that they have no competing interest.Authors' contributions JA: Planning the study, data collection, analysis and writing of the manuscript. SK: Analysis and supervising of the manuscript. B-IS: Supervising and planning the study, analysis and writing of the manuscript. All authors have participated in the manuscript according to the criteria for authors. All authors read and approved the final manuscript.
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Effect evaluation of a heated ambulance mattress-prototype on body temperatures and thermal comfort -an experimental study. Aléx , Resuscitation and Emergency Medicine. 2243Scandinavian Journal of TraumaCite this article as: Aléx et al.: Effect evaluation of a heated ambulance mattress-prototype on body temperatures and thermal comfort -an experimental study. Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2014 22:43.