Dr Michael Jeffrey Toscano
email: mj.toscano@bris.ac.uk

Leanne Sait

Frieda Jørgensen

Christine J Nicol

Claire Powers

Michael Bailey

Thomas J Humphrey

A L Smith

Sub-clinical infection with Salmonella in chickens differentially affects behaviour and welfare in three inbred strains

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

There is increasing government and public pressure on agriculture to provide environments that ensure good animal health and welfare. Given the banning, or discouragement, of prophylactic antibiotic use, identifying how an animal adapts to infection is an area where production must meet government and public demands for enhanced animal health. In terms of animal welfare, a dramatic decrease in health, or a method of coping that is either ineffective or detrimental to long-term fitness in response to a perceived or actual challenge, is likely to have a detrimental impact [START_REF] Dawkins | From an animal's point of view: Motivation, fitness, and animal welfare[END_REF][START_REF] Broom | Animal welfare: concepts and measurement[END_REF]. Thus it is important to assess not only the final impact of an illness (survival, weight loss, etc.) but also the means by which an animal adapts to infection. Recent work has suggested that particular coping styles of animals are related to immunity and disease susceptibility [START_REF] Koolhaas | Coping style and immunity in animals: Making sense of individual variation[END_REF] indicating a differentiation of responses with associated outcomes. By identifying the genetic contribution to coping style and the innate means that are most important for animals to overcome or avoid infection, producers may be able to select genotypes that are more resistant to or deal with infection in a manner that is less costly to health and welfare.

Our experiment focused on Salmonella, which causes an infection often described as 'subclinical', because poultry frequently appear to show little to no behavioural or pathological symptoms [START_REF] Efsa | Trends and sources of zoonoses, zoonotic, agents and antimicrobial resistance in the European Union in 2004[END_REF]. However, we suggest that, by measuring a broad set of indicator variables, it may be possible to detect and characterise flock responses to infection and to determine how subtle physiological and behavioural changes within individual birds interact to produce a particular outcome. A greater understanding of the overall impact of apparently 'sub-clinical' infections may also facilitate the design of environments that exploit an animal's natural ability to reduce risks of infection, resulting in improved animal health and welfare. The current study used three distinct genetic strains of poultry in an effort to characterise between-and within-strain variation in indicators of competitive ability, glucocorticoids, inflammatory protein, antigen-specific immunoglobulins and bacterial load in response to a Salmonella challenge.

Of the three genetic lines (referred to as P, 0 and N), line N is the most studied and has been used in investigations of Salmonella resistance [START_REF] Bumstead | Genetics of resistance to Salmonella typhimurium in newly hatched chicks[END_REF]Wigley et al., 2002;Tilquin et al., 2005). Line N has been shown to be relatively resistant to Salmonella infections [START_REF] Bumstead | Genetics of resistance to Salmonella typhimurium in newly hatched chicks[END_REF], though resistance varied with age and subspecies (Tilquin et al., 2005). Published information on salmonella resistance in lines P and 0 are not available. Each of the SUB CLINICAL INFECTION WITH SALMONELLA lines is known to be susceptible to a wide range of diseases, although two were originally selected for resistance/susceptibility to Marek's disease virus (lines N and P) and lack of endogenous avian leukosis virus (ALV) genes (line 0). Breeding coefficients range from 0.5 (N) to 0.6 (P and 0). Our choice of line was based on an unpublished experiment by our group comparing the three strains that found different suseptibilities to enteric Salmonella colonisation, as well as evidence that each strain exhibited different behavioural profiles, for example, line P appeared more flighty.

Our expectation was that genetic lines would differ in the collected measures and indicate a variation in immune function, behaviour and physiology that might relate to pathogen presence. Data were interpreted in a manner that sought to develop profiles of genetic strains to identify potential causal relationships between responses and salmonella susceptibility for future experimental work. We also aimed to determine the extent of correlations between the measured responses and the extent of bacterial invasion into infected tissues in individually challenged birds.

We predicted that decreased susceptibility to infection would be associated with a response profile that limited the invasion and spread of the pathogen while conserving energy. Specifically, birds with reduced infection would have a response characterised by: increased serum glucocorticoid, inflammatory protein, glucose and creatinine concentrations while weight gain, general behavioural activity and behaviour during competition tests would be reduced. Our predictions are based on the assumption that birds with reduced indications of infection will respond by directing resources towards supporting the immune response, for example, glucocorticoids help to mobilise stored energy [START_REF] Genuth | The Endocrine System[END_REF]. Following the acute response to infection and reflecting the chicken's effort to reduce salmonella colonisation [START_REF] Revolledo | Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks[END_REF], increased immunoglobulin concentrations would indicate a more robust immune response.

MATERIALS AND METHODS

Animals and infection

The three inbred chicken lines (P, 0, and N), all single-comb White Leghorns, were maintained at the Institute for Animal Health (IAH, Compton Laboratories, Berkshire UK). Line 0 was initiated in 1979, was SPF and was selected for the absence of endogenous ALV-E proviral genes and for resistance to endogenous but susceptibility to exogenous ALV [START_REF] Bacon | A review of the development of chicken lines to resolve genes determining resistance to diseases[END_REF]. Line N, housed at IAH, originated at Cornell University, was obtained from the Avian Disease and Oncology Laboratory, USDA, in 1982 and was highly resistant to Marek's disease (MD). Line P, housed at IAH and obtained from Lelystad in 1996, originated at Cornell and was highly susceptible to MD [START_REF] Cole | Studies on genetic resistance to Marek's disease[END_REF][START_REF] Bacon | Genetic resistance to Marek's disease[END_REF].

Female and male birds were hatched at IAH and transported to the University of Bristol School of Veterinary Science within 3 d of hatching. Birds of the three lines were randomly divided into two groups placed in separate large rooms where they were maintained in mixed-strain groups of approximately 100 birds (1 group/room) with ad-libitum access to water and food (160 g protein/kg). At 35 d of age, the birds were sexed and female birds only were placed into 4 groups in rooms that each contained 4 pens. Each pen measured 1.5 x 1.22 m and had open top feeders and drinkers that provided ad libitum food and water. Pens were also provided with a perch and wood shavings approximately 60 mm in depth. Three of the 4 pens were allocated 6 pullets from a single line, that is, 6 birds per pen. The fourth pen contained two pullets of each strain (2 line P, 2 line 0 and 2 line N), though a shortage of line P birds resulted in only a single bird being placed in two of the 4 pens. The SUB CLINICAL INFECTION WITH SALMONELLA fourth pen was included in the experimental design P as part of a separate study and results pertaining to these mixed genotype pens were not included in the present analyses. Feeders were topped up and pens cleaned daily by animal care staff. Light was provided between 07.00 and 19.00 and the temperature was maintained between 16 and 22°C.

At 56 d of age (hereafter referred to as 0 d post infection (dpi)), all birds in two of the 4 rooms received 1.8 x10 8 of Salmonella enterica Typhimurium 4/74 via oral gavage (SAL treatment; n = 36 birds not including mixed genotype pens). Our choice of dosage was based on previous studies by the authors and others comparing dose ranges [START_REF] Barrow | Faecal shedding and intestinal colonization of Salmonella enterica in in-bred chickens: the effect of host-genetic background[END_REF][START_REF] Beal | Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to rechallenge[END_REF] and was directed at achieving a subclinical infection. Birds in the remaining two rooms served as a control (CON treatment; n = 36 birds not including mixed genotype pens) and received an equivalent volume of salmonella-free broth. Personnel entering the facility were required to wear boiler suits and rubber boots. Boots were dipped into an antimicrobial bath before entering rooms. In addition, to minimise accidental infection of controls, rooms of each treatment were located in identical suites separated by a central corridor.

Behavioural observations

During the period between allocation to the various groups at six weeks of age and post-mortem examination seven weeks later, behavioral differences between strains, homogeneity of strains in the pen and response to salmonella infection were examined.

Competition for limited resources requires the expenditure of energy and involves social risk. It is therefore a good discriminator between individual animals and provides a response measure that should be sensitive to any detrimental effects of infection. We devised a resource competition test and used this to (i) examine strain differences (ii) determine whether inter-bird differences in resource competition during the pre-inoculation period were associated with infection outcome in SAL birds and (iii) monitor the effects of infection by comparing resource competition behaviour pre-and post-salmonella challenge.

For identification purposes, all birds had a numbered wing band. To ease identification of birds within a pen, each bird was also marked on its back using stock marker in a unique pattern. In the week before resource competition tests began, birds were cclimatized to the dish on which food would later be presented and to the observer by performing the procedure twice, though no measurements were taken. The resource competition test was conducted on three occasions over two days prior to infection (on days -4 and -3 dpi) and on three occasions over two days afterwards (between days + 3 and +10 dpi). For the test, a single observer approached the pen and waited immediately outside it for 60 s to allow the birds to acclimatise to her presence. At the end of this period, the observer placed a small ceramic dish (diameter: 60 mm) that contained several waxworms (Achroia grisella) in a corner of the pen. Following worm placement, birds within a pen were continuously observed for 5 min during which frequency of behaviours were recorded. Observed behaviours were mutually exclusive and included: aggressive pecks, running, approaching worms, feeding on and pecking at worms, feeding on pellets, sitting on feeder, litter pecking, perching, preening, sitting and/or standing alert, threats, drinking, ground scratching and flying. At the conclusion of the test, all data were summed across the 5-min period and entered into a spreadsheet for later analysis. Data were further consolidated into pre-and post-infection periods in an effort to produce data with a normal distribution. Behaviours not included in analysis due to infrequent SUB CLINICAL INFECTION WITH SALMONELLA occurrences were aggressive pecks, sitting on the feeder, mild pecks, sitting alert, threats and flying. Reported units are the total number of times the behaviour was performed during the observation period.

To measure the impact of infection on behaviour, we compared the general behavioural profile of each treatment (SAL and CON) one week after infection, using scan sampling techniques. Each pen was filmed for 3 h x 3 times in the week following infection using colour closed-circuit TV cameras. Each three-hour segment of footage was observed using 10-min interval scan samples for each animal where the animal's activity was classified using the mutual exclusive categories of active, inactive or feeding/drinking. The total number of times each category was performed by an individual during each of the three-week sections of footage was expressed as a fraction of the total number of observations (that is, 10-min intervals) made throughout that section.

Collection of faeces, blood and bird weight

Faeces were collected at -13, -6, +1 and +8 dpi. To minimise stress associated with collection, faeces were collected passively by placing the animal in a custom built faecal collection unit. The unit consisted of 4 plywood rectangular panels (120 x 160 mm) connected by 4 wooden dowels at each corner and separated by a gap of 160 mm resulting in a three dimensional rectangle. The base and the sides of the unit were wrapped in chicken wire. Units were placed alongside one another during collections providing visual contact with birds in adjacent units. Birds of the same pen underwent the collection protocol at the same time. Birds were placed individually in the collection units and after 20 min the birds were returned to their home pen by which time approximately 90% of birds had defaecated in the area below allowing for samples to be collected and placed on ice At the conclusion of collections, samples were placed in a -80˚C freezer until analysis. Collections were begun at approximately 09.00 h and concluded within 3 h to minimise effects of the natural diurnal rhythm of the glucocorticoid on sample variation. Room-to-room collection order was changed for each of the time points.

Blood was collected at -6, +1 dpi and +15 dpi (post-mortem). Room-to-room collection order was changed for each of the time points. For serum collection at -6 and +1 dpi, approximately 1-3 ml of whole blood was removed from a wing vein using a syringe and 25 G needle, injected into an empty vacutainer, and placed on ice. Samples were refrigerated for 24 h, centrifuged at 700 g for 10 min, and the serum removed and portioned appropriately. Serum collected postmortem was separated as described above.

All birds were weighed at -21 and -9 dpi. The change in weight for each bird was obtained by calculating the difference between these two weights.

Hormone and metabolite analysis

Glucocorticoid metabolites were determined in faeces using a commercially available assay kit (07-120-103, MP Biomedicals, Illkirch, France). Samples were assayed in duplicate and any resulting in a coefficient of variation (CV) greater than 10% were re-determined. For extraction of metabolites, a protocol adapted from Denhard et al. (2003) was used. Approximately 0.3 g of sample was combined with 5 ml of 90% methanol and manually separated by vortexing and tapping for 10 s. All samples were individually weighed immediately before placement into methanol. Samples were shaken for 30 min (130 rpm, 25ºC) with an orbital shaker and then centrifuged (10 min, 1200 g). Supernatant (4 ml) was transferred to a new tube containing 5 ml of water, thus diluting samples to 40 % methanol. After completion of the assay, the Commercially available assays were used to determine the concentration of various serum constituents including: α 1 -acid glycoprotein (AGP) (P0801-1, Cardiotech Services, Louisville, KY, USA), glucose (1070-125, BDS International Diagnostics, Schwetzingen, Germany), and creatinine (500701, Cayman Chemical, Ann Arbor, MI, USA).

Enzyme-linked immunosorbent assay (ELISA) of immunoglobulins

For production of soluble salmonella lysate antigen, preparations of soluble S. enterica Typhimurium 4/74 nalr lysate (STAgP) were prepared as described by [START_REF] Beal | Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to rechallenge[END_REF]. Overnight cultures of STm 4/74 nalr were used to inoculate a 250 ml Erlenmeyer flask containing 100 ml of LB medium and incubated overnight at 37 °C in an orbital incubator (150 rpm). Bacterial cells were pelleted (4 000 g) and washed twice with an equal volume of phosphate-buffered saline (PBS; pH=9.6) and resuspended in 20 ml PBS. The bacterial suspension was subjected to three freeze-thaw cycles in liquid nitrogen followed by sonication (9 × 20 s bursts with 1 min cooling on ice between bursts) in a 10 ml volume at an amplitude of 15 µm using a Soniprep 150 (MSE Scientific Instruments, Crawley, UK). The suspension was centrifuged at 4080 g for 20 min at 4°C followed by centrifugation of the supernatant at 30 000 g for 20 min at 4°C to remove insoluble fractions. The supernatant was retained. Protein concentrations of the soluble antigen preparation were measured using the Bradford protein determination kit (Merck, Poole, UK) and aliquots were frozen (-20 °C) until used.

Concentrations of serum antibody (IgM and IgY) reactive against STAgP were measured by ELISA as described by [START_REF] Beal | Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to rechallenge[END_REF]. Flat-bottomed 96-well ELISA plates (BD Biosciences, Oxford, UK) were coated overnight at 4°C with 50 µl per well of STAgP preparation (diluted to 14.73 µg/ml in carbonate/bicarbonate buffer (pH 9.6)). Plates were washed with PBS Tween-20 (0.05% by volume) (PBS-T) before blocking with PBS-T supplemented with 3% skimmed milk powder for 1 h at 37°C. Serum samples were diluted to 1:400 in blocking buffer (for both IgY and IgM detection) and 50 µl per well was added to the plates before incubation at 37 °C for 1 h. Plates were washed and bound immunoglobulins were detected by incubation at 37 °C for 1 h with horseradish peroxidase conjugated to either goat anti-chicken IgM (1:1000) (Serotec, Oxford, UK) or rabbit anti-chicken IgY (1:2000: Sigma) diluted in blocking buffer (50 µl per well). Plates were washed and a solution containing 2,2-azino-di(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and hydrogen peroxide (50 µl/well) was added. Plates were incubated at room temperature in the dark for 30-60 min and the reaction was stopped by addition of 1% SDS (50 µl per well). Absorbances were read at 405 nm on a Benchmark microplate reader (Biorad, Hemel Hempstead, UK).

Post mortem examination and microbiological analysis

At +15 dpi, all birds were killed. The presence of salmonella recovered from homogenised spleen and caecum were determined by plating on to modified Brilliant Green Agar (mBGA) using two samples per site. The limit of accurate detection was 2 log 10 cfu g -1 tissue and any sample containing fewer bacteria was pre-enriched in buffered peptone water (CM509, Oxoid Ltd, Basingstoke, UK) followed by selective enrichment in Rappaport Vassiliadis broth (CM669, Oxoid Ltd, Basingstoke, UK).

Statistical analysis

All non-microbiological data (body weight, change in weight, IgM, IgY, faecal corticosterone metabolites, glucose, AGP, and behaviour) were checked to assume a normal distribution and log transformed if necessary. Any data requiring log transformation were back-transformed for presentation of data and variation presented as the width of the 95% confidence interval. PROC GLM of SAS (SAS Inst. Inc., Cary NC) was used for analysis and the following independent variables were included in the model: strain nested in treatment by room combination, strain, treatment, and strain by treatment interactions. Strain nested in treatment by room combinations was identified as a random variable by the RANDOM statement of GLM and the TEST option was used to generate the appropriate test statistic for model components [START_REF] Littell | Statistical analysis of repeated measures data using SAS procedures[END_REF]. Separate analysis was conducted for measures collected on different days to avoid multiple levels of nesting. P-values equal to or less than 0.05 were considered statistically significant.

To identify changing patterns in birds due to infection, a paired t-test (SAS Inst. Inc., Cary NC) was used to compare the average of each individual's behaviour in the pre-and post-infection period. Comparisons were made separately for the SAL and CON treatments to determine whether the changes could be attributed to age or time effects rather than treatment.

To identify patterns of responses associated with the site(s) of isolation of S. Typhimurium post-mortem, animals in the SAL treatment were identified as either positive or negative based upon the presence of Salmonella for each of the collected tissues: spleen, caecum or both tissues combined. For instance, an animal that was positive in the spleen by direct plating only would be considered positive in the spleen, negative in the caecum and positive in the combined class. Resulting data were analysed using Proc GLM to identify differences between the positivity groupings in the various physiological, immunological and behavioural responses. Analysis was only conducted for the SAL treatment; the model was similar to the above with the exception that genetic strain was nested in room only and treatment was not included in the model.

Microbiological data from the SAL treatment were analysed using Fisher's Exact test of Proc FREQ within SAS (SAS Inst. Inc., Cary NC) to determine whether the number of animals positive for Salmonella was independent of genetic strain, as would be expected under the null hypothesis. Fisher's Exact test is intended for data sets where the expected frequency of 1 or more cells is less than 5 [START_REF] Severino | Proc Freq: It's more than counts[END_REF]. Positivity analysis was conducted separately for the spleen, caecum or both tissues combined. When an association between strain and positivity was found, the binomial option of Proc Freq within SAS (SAS Inst. Inc., Cary NC) was used to construct post-hoc tests that tested whether there was a statistical difference between the number of positive and negative animals of each strain within the SAL treatment. No salmonella were found in CON birds.

RESULTS

Resource competition test

During the pre-infection period, pecking of waxworms differed between genetic strains with P line birds exhibiting the least of these activities (P = 0.03). Birds of the P line also drank more than those from the other lines (P = 0.008) (Table 1). Feeding on waxworms occurred more frequently in the SAL treatment (P = 0.02; data not shown; SAL = 0.53 ± 0.24, CON = 0.37 ± 0.24). Feeding on pellets showed a strain SUB CLINICAL INFECTION WITH SALMONELLA by treatment effect where line P birds within the CON treatment fed more frequently than all other lines by treatments (data not shown; line P of SAL = 2.1 ± 0.31, remaining treatment combinations < 0.7± 0.31). During the post-infection period, line P birds from the SAL treatment stood alert more than their CON counterparts while the opposite was seen in line 0 birds (P<0.05) (Figure 1). In comparing pre-and postinfection data of the resource competition test, SAL birds revealed a significant decrease in several behaviours (drinking, feeding on pellets and litter pecks) (P<0.05) (Table 2) between the pre-and post-infection periods. In contrast, pecking at waxworms increased in the SAL birds (P = 0.004) (Table 2). Comparing the pre-and post-infection periods of the CON treatment revealed feeding on waxworms increased (P < 0.04). Grouping SAL birds by salmonella positivity at post-mortem examination revealed a tendency for birds that were positive in the spleen and/or caecum to perform less feeding on waxworms during the post-infection period (P = 0.07; data not shown; Salmonella-negative = 0.9 ± 0.6, Salmonella-positive = 0.3 ± 0.3).

Scan samples of bird activity

Statistical analysis from scan sample data of general bird behaviour in the week following infection showed no significant effect of treatment on activity level or feeding and drinking behaviour. Birds of line N were significantly less active than line 0 birds during the same period (P<0.01, data not shown).

Glucocorticoid metabolites

There were no line differences in faecal glucocorticoid metabolites during the preinfection period (P>0.3). Birds which were to be infected with salmonella had a reduced (P = 0.04) concentration of glucocorticoid metabolites at -13 dpi but there was no difference (P = 0.4) between the CON and SAL birds at -6 dpi (Table 3). During the post-infection period, no differences (P>0.3) between strains were observed. Birds of the CON treatment showed a dramatic increase (P<0.0001) in glucocorticoid metabolite concentration at +1 dpi, which was not observed in the SAL. By +8 dpi the glucocorticoid metabolite concentrations of the CON birds had returned to normal, so there was no longer a difference (P = 0.3) between treatments (Table 3). No effect of positivity was found for glucocorticoid metabolites on any of the collection dates (P > 0.1).

α 1 -Acid Glycoprotein

During the pre-infection period, line 0 birds had serum AGP concentrations that were reduced in comparison to the lines P and N (P<0.0001; line P: 424 ± 58, line 0: 245 ± 52, line N: 445 ± 54). All other pre-infection comparisons revealed no statistically significant differences (P>0.1). In the post-infection period, salmonella-infected birds had a tendency for reduced AGP concentrations compared to controls (P = 0.06, SAL = 280 ± 64 ug/mL, CON = 375 ± 64 ug/mL) at +1 dpi. At +15 dpi, each genetic line had different AGP concentrations that were independent of treatment with the lines P and 0 having the greatest and least, respectively (P<0.05; data not shown; line P = 365 ± 55 ug/mL, line 0 = 162 ± 50 ug/mL; line N = 253 ± 56 ug/mL). Positivity comparisons found birds which were positive for salmonella in the spleen and/or caecum at post-mortem had a reduced concentration of AGP at -6 dpi (P = 0.04) with a tendency for a similar pattern at +15 dpi (P = 0.07) (Fig. 2). Birds which were positive for salmonella in the spleen only at post-mortem examination had a reduced concentration of AGP at +15 dpi (P = 0.006) and a tendency to be reduced at -6 dpi (P 3). All other comparisons were similar between positive and negative animals (P > 0.19).

Immunoglobulins Y and M

Absorbance measures of the antigen-specific IgY and IgM during the pre-infection period did not differ between lines or treatment (P>0.4). During the post-infection period, all absorbance readings for IgM (Table 4) and IgY (Table 5) indicated greater concentrations in infected birds compared to controls (P 0.05) with the exception of birds from line N which were similar between treatments for IgY (P = 0.09). Comparison within the SAL treatment revealed that lines P and 0 birds had greater absorbance readings than line N birds for both IgM and IgY (P<0.002). All other comparisons between strains revealed no differences (P>0.1). No effect of salmonella positivity on IgY or IgM concentrations was found within the SAL treatment during either the pre-or post-inoculation periods (P>0.5).

Weight and weight change

Weight and weight change during the pre-infection period differed between all three strains at each time point. The greatest weight and weight gains (P<0.04) were in line N followed by lines 0 and P (Table 6). Birds that were to receive salmonella were heavier at -6 dpi (P = 0.01; data not shown; SAL: 515 ± 11 g, CON: 494 ± 11 g). Bird which were positive for salmonella in the spleen and/or caecum post-mortem tended to weigh more at -6 dpi than birds that were negative (P = 0.07; positive = 334 ± 12 g, negative = 293 ± 21 g). All other comparisons of positivity for the collected weights and weight gain were similar (P>0.1). No weight data were collected during the post-infection period.

Salmonella infections

More birds from lines 0 and N were positive for salmonella when positivity for the caecum and spleen were combined (P<0.04) (Table 7). When findings from the spleen were considered independently, line 0 had more animals positive for salmonella (P = 0.003). In each of these cases, the number of negative birds was greater than the number of positive. All other comparisons between strains for the number of birds found to be positive for Salmonella were similar (P>0.07).

Non-significant results

Serum glucose and creatinine concentrations were unaffected by strain, treatment, or positivity of the bird at post-mortem (P>0.09).

DISCUSSION

The current work served two objectives: firstly, comparing the response of three genetically different poultry strains to a sub-clinical salmonella challenge and secondly, identifying an underlying behavioural, physiological and immunological profile within or across genetic strains that resulted in reduced susceptibility to that challenge. Genetic differences are known to influence a variety of factors that impact on disease susceptibility including gut microflora [START_REF] Hart | The role of the gut flora in health and disease, and its modification as therapy[END_REF] and immune function (Xavier and Rioux, 2008). Despite this knowledge, many questions remain regarding how non-genetic animal factors (acquired behavioural tendencies) affect disease susceptibility or how these genetic and acquired factors may interact.

As part of our first objective, we expected that the chosen strains would differ independently of a salmonella challenge and thus pre-infection data were collected to SUB CLINICAL INFECTION WITH SALMONELLA determine baseline differences between strains. Consistent with our hypothesis, comparison of pre-infection measures suggested that birds from line P were more timid in terms of their behavioural style and exhibited a pattern of energy use that was more conservative. Our characterisation is based on line P birds pecking less at the waxworms, drinking more and reduced weight and weight gain compared to the other strains. With regards to drinking, line P drank at a frequency 5 times greater than the other lines which may reflect a lack of interest or shift in activity due to a fear of approaching the waxworms during this test rather than an actual physiological need to drink. Line P birds also had a higher AGP concentration than line 0 birds though similar to line N. Feeding on pellets was greatest in line P birds, which were to receive salmonella, although this was likely to be a suite effect as it was not consistent across pens containing line P birds. Future work will be needed to identify whether a causal relationship exists between behaviour, weight gain and concentrations of inflammatory proteins during periods of positive health. If such a relationship were found, identifying the mechanisms involved may help producers to develop poultry lines with improved ability to overcome infections, resulting in improved animal welfare.

Because we were focussing on sub-clinical infections we did not expect a severe response following infection, although the broad measures we employed did reveal differences between treatments. Potential responses that we anticipated include a well-established collection of behaviours that animals typically adopt during infection including: reduced feeding, changes in activity and social interactions in combination with huddling, shivering and other behaviours that conserve energy and improve bodily processes to combat disease [START_REF] Millman | Sickness behaviour and its relevance to animal welfare assessment at the group level[END_REF]. In addition to behavioural responses during periods of illness, we also expected animals to have an immunological and physiological response including release of inflammatory proteins (such as AGP) [START_REF] Holt | Comparison of the effects of infection with Salmonella enteritidis, in combination with an induced molt, on serum levels of the acute phase protein, alpha1 acid glycoprotein, in hens[END_REF], immunoglobulins [START_REF] Beal | Antibody response to Salmonella: its induction and role in protection against avian enteric salmonellosis[END_REF] and glucocorticoids (Webster and Sternberg, 2004). Although our expectations were only partially met in terms of these responses, birds that received salmonella adopted a profile that was less active, with reductions in drinking, feeding on pellets and litter pecks. In the SAL treatment, pecking at waxworms increased although consumption of worms remained unchanged, suggesting a decrease in hunger motivation. In terms of an immune response, IgM and Y increased confirming that the infected birds were experiencing an immunological challenge. In contrast, glucocorticoids and AGP were altered in a manner that was opposite to our predictions and potential explanations are discussed below. Feeding on waxworms increased in CON birds possibly as a result of acclimatisation to the test procedure.

Following infection with salmonella, we hypothesized that genetic strains would differ in the resulting bacterial load. In support of this hypothesis, line P birds appear to have cleared salmonella more effectively as shown by the line 0 and N having a greater proportion of birds positive for salmonella in the combined tissue grouping (spleen and caecum). In the spleen only, a trend existed for line P to have a greater number of animals negative for salmonella. Although our interpretation is conjecture, the possibility exists that line P benefited in the form of a heightened immune response from a pre-infection profile characterized by timidity, slower growth, and a relatively high serum AGP concentration. Post-infection measures regarding line P appeared to further confirm the possibility of enhanced immune function for combating salmonella as suggested by the line P having serum concentrations of AGP and immunoglobulins that were greater than line N. Additionally, birds of the line P in the SAL treatment stood alert more than the CON treatment whereas line 0 Our findings are based entirely on correlations that are subject to interpretation but they form the basis for a subsequent hypothesis-based approach to ensure the presence of an actual relationship. For instance, line 0 birds were all positive for salmonella in terms of the spleen only and combined caecum and spleen positivity measure yet had a statistically similar immunoglobulin concentration to line P at +15 dpi. If our hypothesis that line P was adopting a profile that was effective in providing energy for immune function, we would expect that line 0 birds would have comparatively reduced immunoglobulins. It is also possible that birds in neighbouring pens or the adjacent rooms may have influenced one another, though footbaths outside the rooms and physical barriers between pens were present to minimize this possibility. Whether birds of each genetic strain are adopting different strategies with different grades of success in terms of salmonella clearance will need to be determined with future research.

In pursuit of our second objective to identify correlations between measured responses and the extent of bacterial invasion, we hypothesized that birds with a reduced bacterial load post-mortem would manifest specific changes in the measured responses. Our hypothesis was based on an expectation that these responses reflect a more aggressive immune response and, as a result, reduced invasion following infection. In support, birds negative for salmonella at in the spleen post-mortem examination had comparatively more AGP at +15 dpi suggesting that those that were better able to limit invasion had greater serum AGP concentrations. Additionally, serum AGP concentrations during the pre-infection period mirror those at +15 dpi, supporting the concept of a profile, i.e., certain birds may have mechanisms better able to reduce invasion of bacteria before exposure. The finding that birds positive in the spleen and/or caecum at post-mortem examination tended to weigh more at -6 dpi also suggests that birds with reduced weight gain may benefit from improved immune function. In contrast to our findings, [START_REF] Holt | Comparison of the effects of infection with Salmonella enteritidis, in combination with an induced molt, on serum levels of the acute phase protein, alpha1 acid glycoprotein, in hens[END_REF] reported a positive correlation between AGP and presence of Salmonella Enteritidis in the faeces of poultry undergoing feed withdrawal at 3dpi. The difference in results may be related to our focus on invasiveness in tissues at a later date while the work by [START_REF] Holt | Comparison of the effects of infection with Salmonella enteritidis, in combination with an induced molt, on serum levels of the acute phase protein, alpha1 acid glycoprotein, in hens[END_REF] quantified the presence of bacteria in faeces during the acute phase of infection. A greater concentration of AGP during the acute phase of infection may indicate the severity of disease while concentrations at later stages reflect an ability to clear Salmonella and limit its spread into central tissues.

In addition to those discussed above, several findings were contrary to our expectations, notably faecal glucocorticoid metabolite and AGP concentrations. Glucocorticoids are normally reported to increase in concentration following bacterial challenges (Webster and Sternberg, 2004). In contrast to this expectation, the average faecal glucocorticoids at +1 dpi in the CON animals was nearly three times greater than concentrations for either of the treatments at any other time point suggesting the CON animals had the most aggressive response to the imposition of treatment. Concentrations of faecal glucocorticoid metabolites are a reliable indicator of plasma glucocorticoids [START_REF] Dehnhard | Measurement of plasma corticosterone and fecal glucocorticoid metabolites in the chicken (Gallus domesticus), the great cormorant (Phalacrocorax carbo), and the goshawk (Accipiter gentilis)[END_REF], thus the collection medium is unlikely to be a factor in our findings. Nonetheless, several factors should be considered. First, although glucocorticoid metabolite concentrations were greater in CON compared to SAL animals, both treatment groups were handled and received a broth, albeit a sham broth in the former. Thus, the increased glucocorticoid metabolite concentrations SUB CLINICAL INFECTION WITH SALMONELLA observed in the CON treatment at +1 dpi in comparison to the previous time points may be attributed to the handling when receiving the broth. The lack of a similar change in SAL animals may be related to our extraction method which did not employ a process to remove water content as others have reported the step to result in similar concentrations regardless of its inclusion (Wasser et al., 2000). One explanation to consider is that animals in the SAL treatment had a higher incidence of diarrhoea resulting in greater water content in their faeces. Although this would not have affected the absolute concentration of glucocorticoid metabolites in the faeces, it may have increased the weight used in calculating the concentration of metabolite/g of faeces. Secondly, there was a detectable difference in faecal glucocorticoid metabolites before treatments were applied at -13 dpi, thus there may have been a preexisting condition that was exacerbated by receiving the broth, although this is unlikely as pre-infection differences were eliminated by -6 dpi.

A second unexpected finding was a tendency for increased AGP concentrations in CON birds at +1 dpi, given that AGP is normally greater in animals undergoing a bacterial challenge [START_REF] Fournier | Alpha-1-acid glycoprotein[END_REF]. The observed pattern may reflect greater clearance of AGP in the SAL treatment, although this is an untested hypothesis. In addition to the unexpected pattern seen between treatments, the magnitude of the change in concentrations suggests a minimal AGP response. Related work by [START_REF] Holt | Comparison of the effects of infection with Salmonella enteritidis, in combination with an induced molt, on serum levels of the acute phase protein, alpha1 acid glycoprotein, in hens[END_REF] reported that leghorn hens infected with Salmonella Enteritidis, a different and more invasive serovar, had AGP concentrations four times greater than their control counterparts at 3 dpi. In our study, the concentration of AGP in CON animals was only 1.5 that of the SAL treatment suggesting a small difference between treatments. We had chosen a relatively low dose of salmonella to induce a subclinical response and the possibility exists that the dose used was not adequate to provoke a major response in AGP concentrations. More critically, the concentrations had actually decreased from the pre-infection concentrations observed at -6 dpi as suggested by the reported means.

In conclusion, our results suggest the existence of a coping profile involving a suite of attributes that were potentially associated with an improved clearance of salmonella. These attributes include: drinking, pecking at the waxworms during the resource competition test, weight and weight gain, serum AGP and immunoglobulin concentrations. In addition, our findings suggest that these genetic lines offer a useful resource to identify genetic and environmental influences on susceptibility to subclinical salmonella infections. Future work using hypothesis-based testing will be required to determine any causal associations of the identified correlations with improved clearance. *Signifies a difference between number of negative and positive birds within a genetic line (P < 0.05).

F

**Signifies a trend between the number of negative and positive birds within a genetic line (P <0.07). † Totals are different due to laboratory contamination and counts not being able to be completed.

  WITH SALMONELLA resulting metabolite concentration was multiplied by 113.5 to obtain the concentration of the initial dilution and then divided by the weight of the faecal sample. Resulting units are expressed as ng glucocorticoid metabolites/g of faeces collected.

Formatted

Formatted

Formatted

  WITH SALMONELLA displayed the opposite pattern. A profile characterized by greater production of immunoglobulins and AGP and a reduced activity level may have benefited the line P during the challenge.

Formatted

  the total number of times per 15 min the behaviour was performed during the three post-infection competition tests subtracted from the total during the three preinfection tests. Positive values indicate behaviours were performed more in the preinfection compared to the post-infection period.

Figure 1 .

 1 Figure 1. Standing alert during the competition tests in post-infection period.

Figure 2 .

 2 Figure 2. Concentrations of α 1 -acid glycoprotein for positivity grouping in the spleen before and after salmonella challenge at day 0.

Figure 3 .

 3 Figure 3. Concentrations of α 1 -acid glycoprotein for positivity grouping in the spleen and/or caecum before and after salmonella challenge at day 0.

Table 1 .

 1 SUB CLINICAL INFECTION WITH SALMONELLA Tilquin, P.,Barrow, P.A., Marly, J., Pitel, F., Plisson-Petit, F., Velge, P., Vignal, A., Baret, P.V., Bumstead, N. & Beaumont, C. (2005) A genome scan for quantitative trait loci affecting the Salmonella carrier-state in the chicken. Genet Sel Evol, 37: 539-561. Wasser, S.K., Hunt, K.E., Brown, J.L., Cooper, K., Crockett, C.M., Bechert, U., Millspaugh, J.J., Larson, S. & Monfort, S.L. (2000) A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. General and Comparative Endocrinology, 120: 260-275. Strain differences in resource competition tests during the pre-

	o r
	P
	e
	e r
	R
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	w
	O n l
	y

Table 2 .

 2 Behaviours revealing a difference between pre-and post-infection periods during the resource competition test Treatment

	¥	Behaviour	t-value	Probability	Difference between
					pre-and post-
					infection †
	SAL	Drinking	2.68	0.01	0.343
	SAL	Feeding on pellets	4.45	<0.0001	0.451
	SAL	Litter pecks	3.00	0.01	1.235
	SAL	Pecking at waxworms	-2.79	0.01	-0.980
	CON	Feeding on waxworms	-2.22	0.03

Table 3 .

 3 Faecal glucocorticoid metabolite concentrations (ng of metabolite/g of faeces) in salmonella and control treatments before andafter Salmonella challenge at day 0 Treatment

	Day relative
	to infection	SAL*	CON	P-value
	-13	0.40 ± 0.09	0.55 ± 0.14	0.04
	-6	0.44 ± 0.09	0.50 ± 0.07	0.4
	+ 1	0.33 ± 0.08	2.09 ± 1.20	>0.0001
	+ 8	0.78 ± 0.18	0.59 ± 0.15	0.3
	SAL, salmonella infected; CON, control.

Table 4 .

 4 Absorbance (490 nm) indicating antigen-specific immunoglobulin M concentrations for each treatment and strain at postmortem (+15 dpi) following a Salmonella challenge Treatment

				P-
	Strain	SAL	CON	value †
	P	1.85 ± 0.40 a	0.27 ± 0.05 a	<0.0001
	O	1.75 ± 0.34 a	0.23 ± 0.04 a	<0.0001
	N	0.98 ± 0.20 b	0.24 ± 0.05 a	<0.0001
	† P-value denotes comparison for treatments
	ab Values within a column not sharing a common superscript are
	significantly different (P < 0.0001)

Table 5 .

 5 Absorbance (490 nm) indicating antigen-specific immunoglobulin Y concentrations for each treatment and genetic strain at post-mortem examination (+15 dpi) following a Salmonella challenge Treatment

	Strain	SAL	CON	P-value †
	P	1.14 ± 0.36 a	0.35 ± 0.10 a	<0.0001
	O	0.93 ± 0.26 a	0.41 ± 0.11 a	<0.0001
	N	0.56 ± 0.17 b	0.43 ± 0.13 a	0.09
	† P -value denotes comparison for treatments
	ab Values within a column not sharing a common superscript are significantly
	different (P < 0.002)

Table 6 .

 6 Differences between genetic strains in weight and weight change (g) during the pre-infection periodStrain

	Days relative to infection	P	O	N	Pooled SE
	-21 dpi	290 a	334 b	352 c	9.0
	-6 dpi	443 a	508 b	562 c	14.5
	change	153 a	175 b	210 c	7.8

Table 7 .

 7 Number of animals that were negative/positive for Salmonella at post-mortem (+15 dpi) within different tissue groupings for genetic line †

		Spleen or	Spleen		Caecum
	genetic		Cecum
	strain	-	+	-	+	-	+
	P	5	4	6 ** 2	4	4
	O	0 * 12	0 * 12	6	6
	N	2 * 9	4	6	6	4

E-mail: br.poultsci@bbsrc.ac.uk URL: http://mc.manuscriptcentral.com/cbps British Poultry Science


David Frank Bibby

Iain Mcelarney

Judith Breuer

Duncan Clark

Keywords: Diagnosis, Evaluation, Novel

published or not. The documents may come

L'archive ouverte

Table II Primer and probe sequences for each of the ten targets detected by the real-time respiratory RT-PCR system. The ADV primer-probe set is derived from those described in [START_REF] Heim | Rapid and quantitative 247 detection of human adenovirus DNA by real-time PCR[END_REF], and the MPNV set from [START_REF] Maertzdorf | Real-time reverse transcriptase 266 PCR assay for detection of human metapneumoviruses from all known genetic 267 lineages[END_REF].

The remaining primers and probes were developed in-house. To evaluate the prospective utility of Seeplex® DPO technology in respiratory virus 6 diagnosis, a panel of 99 respiratory samples positive by real-time RT-PCR for one or 7 more viruses was assayed by the Seegene Seeplex® RV12 system. As well as being 8 able to detect all ten viruses in the real-time RT-PCR system with the exception of 9 enteroviruses, RV12 can also distinguish between the two subgroups of RSV and 10 detect two subgroups of coronaviruses. 11

F

Seven of the 9 viruses in common with the RT-PCR were detected reliably by RV12. 12 Eleven samples RT-PCR-positive for Metapneumovirus and 5 samples positive for 13 Influenza B were not detected by RV12. Seegene developed a second-generation 14 system, RV15, which not only allowed detection of 3 additional viruses, but also 15 addressed the potential problems with RV12 specificity. To address these concerns, 16 84 respiratory samples positive for a range of viruses by real-time PCR were assayed 17 with RV15. The results of this evaluation improved significantly upon those seen with 18

RV12. 19

The high throughput capabilities and potential lower technical requirements afforded 20 by the Seeplex® system may offer an alternative to real-time RT-PCR systems. 21 in settings as diverse as haematopoietic stem cell transplant recipients [Jalal et al., 25 2007;[START_REF] Piralla | 285 Multicluster nosocomial outbreak of parainfluenza virus type 3 infection in a 286 pediatric oncohematology unit: a phylogenetic study[END_REF], childhood bronchitis and pneumonia [START_REF] Shay | Bronchiolitis-293 associated mortality and estimates of respiratory syncytial virus-associated 294 deaths among US children, 1979-1997[END_REF]26 Michelow et al., 2004] and the seasonal influenza epidemics implicated in substantial 27 annual mortality [START_REF] Mook | 279 Surveillance of influenza and other respiratory viruses in the United Kingdom: 280 October 2007 to[END_REF]. As such infections often present with similar 28 symptoms, laboratory analysis is essential. 29 Many laboratories rely still on non-molecular approaches such as direct 30 immunofluorescence and viral culture. However, in the UK, various real-time PCR-31 based systems have been employed for a number of years, their utility having been 32 demonstrated largely by comparison with these more traditional methods [Templeton 33 et al., 2004;[START_REF] Bustin | Real-time reverse transcription PCR (qRT-PCR) and its 228 potential use in clinical diagnosis[END_REF][START_REF] Freymuth | Comparison of multiplex PCR assays 240 and conventional techniques for the diagnostic of respiratory virus infections 241 in children admitted to hospital with an acute respiratory illness[END_REF]van de Pol et al., 2007;34 Mahony et al., 2007;[START_REF] Kuypers | Comparison of 262 conventional and molecular detection of respiratory viruses in hematopoietic 263 cell transplant recipients[END_REF] (reviewed in [START_REF] Mahony | Detection of respiratory viruses by molecular methods[END_REF]. Emerging 35 technologies thus have to demonstrate significant benefit against these existing tools 36 to achieve widespread adoption [START_REF] Pabbaraju | Comparison of the Luminex 282 xTAG respiratory viral panel with in-house nucleic acid amplification tests for 283 diagnosis of respiratory virus infections[END_REF][START_REF] Arens | Comparison of the Eragen Multi-Code (R) Respiratory Virus Panel 223 with conventional viral testing and real-time multiplex PCR assays for 224 respiratory viruses[END_REF]. 37

The novel Seeplex® system exploits Dual Priming Oligonucleotides (DPOs) as PCR 38 primers, each comprising a standard primer-length 5' portion which initiates priming, 39 separated by multiple inosine residues from a shorter, 6-12 nucleotide 3' component 40 which determines duplex extension. As well as facilitating melting temperature 41 regulation during primer design, mispriming and PCR artefacts such as primer 42 dimerisation are almost eliminated, allowing simultaneous and efficient amplification 43 of multiple complex targets in single reactions [START_REF] Chun | Dual priming 230 oligonucleotide system for the multiplex detection of respiratory viruses and 231 SNP genotyping of CYP2C19 gene[END_REF]. 44

The system summary in Figure 1 The RV12 system enables simultaneous detection of 12 respiratory viruses in 2 50 reactions per sample. Its performance when compared to traditional diagnostic 51 methods has been demonstrated frequently [Yoo et al., 2007;[START_REF] Drews | Use 233 of the Seeplex RV Detection kit for surveillance of respiratory viral outbreaks 234 in Toronto, Ontario, Canada[END_REF]Roh 52 et al., 2008;[START_REF] Kim | Rapid detection and identification of 12 respiratory 259 viruses using a dual priming oligonucleotide system-based multiplex PCR 260 assay[END_REF], with potential time and resource savings when 53 compared to more modern methods, particularly in high throughput settings where 54 prompt diagnosis is important. More recently, the RV15 kit has been developed that 55 targets 15 viruses in 3 reactions. 56

No studies evaluating either system against real-time PCR-based respiratory virus 57 detection methods have yet been published. The results of a comparative evaluation of 58 serial iterations of the Seeplex® system with the multiplex PCR-based method that 59 has been used for 5 years as the primary diagnostic assay for respiratory virus 60 detection in a major London hospital are described. 61 Additionally, all samples that underwent easyMAG extraction were pre-incubated at 73 56°C for 10 minutes with Buffer AL and 30mAU/ml proteinase K (both Qiagen). 74 Nucleic acid extracts were stored at -80°C between extraction and analysis. 75

Real-Time RT-PCR. 76

Each sample extract was subjected to seven simultaneous real-time RT-PCRs, with 77 each reaction detecting between one and three targets (Table I). The seventh reaction 78 constituted a duplex control reaction, with primers and probes targeting both a human 79 gene from the ras family (K-Ras) [START_REF] Hilbe | Automated real-time PCR to 251 determine K-ras codon 12 mutations in non-small cell lung cancer: 252 comparison with immunohistochemistry and clinico-pathological features[END_REF] and Protein H (haemagglutinin) 80 of the PDV internal control RNA. 81 Mastermixes and samples were dispensed robotically into the Rotorgene Gene-Disc 82 reaction vessels by a CAS1200 liquid handler (both Qiagen). 83 Quantitect Multiplex RT-PCR Mix NoRox (Qiagen), specific primers and probe 84 combinations (Table II, all obtained from Sigma-Genosys, Gillingham, UK), and 10µl 85 of nucleic acid extract were mixed in 25µl reactions. Real time cycling, detection and 86 III). 98

Random hexamer-primed cDNA synthesis products were generated using the 99 Revertaid system (Fermentas, York, UK), according to the manufacturer's 100 instructions, and stored at -20°C until use. 101

Each cDNA preparation was subjected to the RV12 PCR procedure according to the 102 manufacturer's instructions (Seegene, Seoul, South Korea). Briefly, parallel 20µl 103 reactions were set up, each containing RV12 mastermix, 8-MOPS contamination 104 control reagent, and 3µl of cDNA. One of each pair was supplemented with 4µl 105 primer mix A, the other with 4µl primer mix B. Thermal cycling conditions were as 106 follows: 15 minutes at 94°C, followed by 40 cycles of 94°C for 30s, 60°C for 90s, and 107 72°C for 90s, followed by a single incubation of 10 minutes at 72°C. Completed 108 reactions were analysed using the MultiNA platform (Shimadzu Corporation, Kyoto, 109 Japan), coupled to a dedicated Seegene software template. Analysis output comprised 110 viruses detected by RT-PCR were also detected by RV15, including all instances of 159 MPNV (7/7). Inverse correlations between C t and RV15 band intensity were less 160 pronounced than that seen with RV12. 161 Three of 9 ADV-positive samples were not detected by RV15. All four FLB-positive 162 samples that had also been tested with RV12 remained FLB-positive by RV15. 163 However, a further 5 previously untested FLB-positive samples failed to signal with 164 RV15. Again, viruses were detected by RV15 that were not detected by the RT-PCR: 165 HRV, enterovirus, coronavirus, and bocavirus (1, 1, 2, and 1 sample respectively). 166

The RT-PCR system did not target either coronavirus or bocavirus. Table V details the RT-PCR system, detected by poorly-amplifying PDV target, occurs with a not 205 insignificant frequency; it is essential to report that a lack of virus detection is not due 206 to any technical deficiency within the system. 207

In the laboratory setting, the Seeplex® system looks to compare favourably with 208 existing technologies where high throughput, ease of use, low technical requirements, 209 and a fully kit-based format are factors. Equally useful is the ability of the RV15 210 system to detect bocavirus and coronaviruses, whose clinical impact is becoming 211 increasingly clear [START_REF] Esposito | Impact of human bocavirus on children and 237 their families[END_REF][START_REF] Brodzinski | Review of new and newly discovered respiratory 226 tract viruses in children[END_REF]Garcia-Garcia 212 et al., 2010]. 213

Since the completion of this work, the Seeplex® RV15 kit now includes the 214 redesigned influenza B primer set, human RNase P whole process control and now 215 comprises a one-step RT-PCR system rather than the two-step systems employed in 216 these evaluations. 217

In summary, the Seeplex® RV15 produces results comparable to the in house system. 218

The high throughput capabilities and low technical requirements afforded by 219 Seeplex® recommend it as a practical alternative to real-time RT-PCR. 220

Figure 2 :

 2 Figure 1: Workflow diagram of the Seeplex® diagnostic system. 30x56mm (300 x 300 DPI)

  diagnosis of respiratory virus infection is essential to patient care 24

  shows a standard nucleic acid extraction, a cDNA 45 step, and then a DPO PCR, in which the products of each target virus have different Post-PCR product analysis employs an automated bench-top 47 system with which target detection interpretations are inferred from PCR product 48 sizes. 49

  aspirates, bronchio-alveolar lavages, or respiratory 63 swabs (nose and/or throat) submitted to the laboratory for routine testing. Aspirates 64 and lavages were diluted in saline and centrifuged to separate cellular material from 65 mucoid and liquid components. Swabs were transported in viral transport medium and 66 extracted directly. Two automated nucleic acid extractors were employed at different 67 times during the course of this evaluation -the NucliSENS easyMAG platform 68 (Biomérieux, Marcy l'Etoile, France) and the QIAamp One-For-All Nucleic Acid Kits 69 on the Qiagen BioRobot MDx DSP (Qiagen, Crawley, UK). Immediately prior to 70 extraction, samples were supplemented with a fixed quantity of phocine distemper 71 virus (PDV), obtained from cell culture supernatant and stored in lots at -80°C. 72

  analysis were performed on the 72-well Rotorgene platform (Qiagen) with 87 fixed fluorescence thresholds, allowing direct comparison both between samples and 88 between runs. Positive controls (a panel of plasmids containing the specific PCR 89 target as inserts) for each respiratory virus target were included in each run, and 90 compared to expected threshold cycle (C t ) values. For each sample, C t values for the 91 PDV and K-Ras control reactions were compared to expected values before parallel 92 PCR results were considered valid. 93 Seeplex® RV12. 94 Ninety-nine respiratory extracts stored at -80°C were selected that previously had 95 tested positive by RT-PCR for one or more respiratory virus targets. With the 96 exception of enterovirus and the internal control virus (PDV), which were not 97 detected by RV12, all viral targets in the RT-PCR were represented (Table

  respiratory extracts stored at -80°C testing positive for one or more 114 RT-PCR virus targets (TableIV) were subjected to cDNA synthesis using the Mu-LV 115 system (Invitrogen, Paisley, UK), according to the manufacturer's instructions. 116Reaction products were stored at -20°C until use. Eight samples of the 84 had been 117part of the previous RV12 analysis.118 Each cDNA preparation was subjected to the RV15 PCR procedure according to 119 Seegene's instructions. Briefly, parallel 20µl reactions were set up for each sample, as 120 per the RV12 kit, but with three reactions per sample (primer mixes A, B & C) rather 121 than the two in the earlier kit. Thermal cycling conditions were as per the RV12 kit. 122Completed reactions were analysed using the Tape Station platform (Lab901, 123 Edinburgh, UK) linked to a specific Seeplex® RV15 interpretation software module. 124 Again, results were presented both qualitatively and semi-quantitatively, according to 125 the migration rate and band intensity of each species present within completed 126 reactions. 127 Redesigned Influenza B Primers. 128 During the evaluation, a new panel of influenza B primers was developed by Seegene 129 to replace those in the RV15 kit. The cDNA preparations of the 19 FLB-positive (by 130RT-PCR) samples analysed by RV15 were subjected to amplification using these 131 primers under the same conditions as for the primer mixes of the RV12 and RV15 132 kits, but in monoplex reactions. Analysis was performed as per RV15, above. 133 by the RT-PCR were also detected by Seeplex® RV12 in 88 of 136 the 99 nucleic acid extracts tested with the Seeplex® RV12 system. In 7 of the 137 remaining 11 samples, single infections were missed (4xFLB, one each of HRV, PF1, 138 and MPNV), and in four samples where two viruses had been detected by RT-PCR, 139 the virus target with the higher C t was missed by RV12 (2xMPNV, 1xPF3 and 140 1xADV). Comparison of MultiNA peak heights and RT-PCR C t values for each virus 141 target revealed a broad negative correlation between the two parameters, with the 142 exception of HRV, where no correlation was observed (Figure2). 143In all instances where PF2 (7 samples), FLA (15), and/or RSV (15) were detected by 144 RT-PCR, these virus targets were detected with RV12 also. For each instance where 145 PF1, PF3, HRV, and/or ADV were detected by RT-PCR, all except one of each virus 146 target were detected by Seeplex® RV12. Each instance of undetected virus 147 represented a sample whose C t was found in the higher part of the range for that virus. 148 FLB and MPNV targets were detected less frequently. Four of the 5 FLB-positive 149 samples with the highest C t values, and the 3 MPNV-positive samples with the 150 highest C t values were not detected by RV12. 151 Conversely, ADV, RSV, HRV and MPNV were detected by RV12 in extracts where 152 the RT-PCR system had failed to do so (in 3, 4, 4, and 1 sample respectively), and 153 with the exception of the RSV detections, had high peak heights, consistent with high 154 target concentrations in the initial samples. 155 Seeplex® RV15. 156 Eighty four extracts from respiratory samples RT-PCR-positive for at least one 157 respiratory virus were tested by RV15. With the exception of ADV and FLB, all 158

  167 the discrepant results between the RT-PCR and Seeplex® systems. 168 Redesigned Influenza B Primer Set. 169 Using the new primer set designed to overcome the FLB-detection limitations seen 170 with RV12 and RV15, all FLB-positive samples in which FLB had been detected by 171 RV15 were FLB-positive by the new primer set, as were four of the five samples 172 FLB-negative by RV15. The single sample where FLB was not detected by Seeplex® 173 had given a C t of 30 by RT-PCR. The lack of amplification controls in this assay 174 series precluded detection of PCR inhibition. 175 the Seeplex® system is its internal control facility. The artificial 201 targets included in each PCR mastermix allows validation only of the PCR step, 202 whereas the addition of PDV to each extraction allows the RT-PCR system also to 203 control for both the RNA extraction and the reverse transcription step. Inhibition of 204

  AC, van Loon AM, Wolfs TF, Jansen NJ, Nijhuis M, Breteler EK, 300 Schuurman R, Rossen JW. 2007. Increased detection of respiratory syncytial 301 virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time 302 PCR in samples from patients with respiratory symptoms. J Clin Microbiol 303 45:2260-2262 304 Yoo SJ, Kuak EY, Shin BM. 2007. Detection of 12 respiratory viruses with two-set 305 multiplex reverse transcriptase-PCR assay using a dual priming 306 oligonucleotide system. Korean J Lab Med 27:420-427 307

John Wiley & SonsJournal of Medical Virology


Laura Elizabeth Cox

Onder Arslan

Jean-Pierre Allain

Önder Arslan

Characterisation of hepatitis B virus in Turkish blood donors, and the prevalence of the 1 SP1 splice variant. 2 3

Keywords: Turkey, HBV, sequencing, splicing Turkey, HBV, sequencing, splicing 21 22

published or not. The documents may come

Characterisation of hepatitis B virus in Turkish blood donors, and the prevalence of the SP1 splice variant.

Introduction

36

Hepatitis B virus (HBV) carrier prevalence in Turkey is reported to be around 4% 37 [START_REF] Degertekin | Horizontal transmission of hepatitis B virus in Turkey[END_REF], which is in line with other Mediterranean and Middle Eastern 38 countries and meets the criteria for intermediate endemicity level. HBV belongs to the family 39 Hepadnaviridae, and has a 3.2kb genome that, when encapsidated within the virion, is a relaxed 40 circular DNA molecule that is only partially double-stranded. It was first reported in 1989 that

41

HBV could undergo splicing by the host's machinery in human hepatoma cells transfected with 42 full-length HBV [START_REF] Su | Hepatitis 294 B virus transcript produced by RNA splicing[END_REF]. This 2kb variant is commonly termed SP1, and contains a 1223 43 nucleotide deletion beginning at the last codon of the core gene and spanning through to the 44 middle of the S gene. To date, this splice variant has been identified in chronic hepatitis B 45 patients infected with HBV genotypes A and D, and an additional 10 splice variants have been

63

HBsAg-positive plasma samples were collected during 3 separate periods in 2004,2005 64 and 2006, with a total collection period of 13 months. Samples were heat-inactivated at 60 O C for 65 30 min prior to shipping on dry ice, and upon arrival in the UK were stored at -80 O C until ready 66 to use. Viral DNA was extracted and quantified as described previously [START_REF] Allain | The risk of hepatitis B virus 228 infection by transfusion in Kumasi, Ghana[END_REF] 67 [START_REF] Garmiri | Deletions and recombinations 246 in the core region of hepatitis B virus genotype E strains from asymptomatic 247 blood donors in Guinea, west Africa[END_REF]. To generate sequence for the entire HBV genome from 50 samples, the 68 Expand High Fidelity PCR system (Roche) was used for two nested PCR assays. Firstly, a nearly 69 full-length fragment of approximately 3000bp, minus a 50bp precore section was amplified as 70 previously described [START_REF] Zahn | 306 Molecular characterization of occult hepatitis B virus in genotype E-infected 307 subjects[END_REF]. A second 300bp fragment including the basic core 71 promoter/precore (BCP/PC) 50bp gap was produced to complete the full genome [Candotti et 72 al., 2006]. PCR products were purified using the EZNA cycle pure kit (Omega; Crawley, UK).

73

A semi-nested PCR was used to screen for the SP1 variant, using newly designed primers 74 located in the core and S genes. SP1_core (5'-GTCGCAGAAGATCTCAATCTCGGG-3'; position 75 2421-2444) was used as the sense primer in both first and second round PCR; SP1_R1 (5'-

96

Sequencing and phylogenetics analysis was performed using Seqman, SeqBuilder for HBsAg (187 male: 13 female), giving a prevalence of 1% for males and 0.6% for females.

108

Plasma was obtained for 199 HBsAg positive samples, and quantification of HBV DNA load by Q-

109

PCR was successful in 176 (88.4%) of these samples. The median viral load was 1.19 x10 3 IU ml -1 110 (range 1.62 x10 1 -8.95 x10 8 IU ml -1 ). In the remaining 23 samples (11.6%), HBV DNA was 114

Complete genomic sequences were obtained for 50 samples (GenBank Accession 115 numbers: JF754586-JF754635), from 57 randomly selected samples in which this was attempted.

116

Phylogenetic analysis identified all strains as belonging to HBV genotype D; 47 clustered as 117 subgenotype D1 (94%), 2 as subgenotype D2 (4%), and 1 as subgenotype D3 (2%). Sequence 118 analysis of the S gene revealed the predominant serotype to be ayw2 (n=41), 5 strains specified 119 ayw3 and 1 strain was ayw4. An additional strain belonged to serotype adw, but could not be 120 classified further, and serotype determination was not possible in one strain due to a deletion in 121 this region (Table I). 148

The mean overall amino acid divergence within the Pre-S/S region was 2.7% (range 0-149 6.9%). The major hydrophilic region (positions 100-169) was more variable, with a mean amino 150 acid substitution rate of 5.3% (range 0-20%

176

is subgenotype D1 and serotype ayw2 [START_REF] Bozdayi | Complete genome sequence and phylogenetic analysis of hepatitis B virus 234 isolated from Turkish patients with chronic HBV infection[END_REF][START_REF] Sertoz | Restriction fragment length 280 polymorphism analysis and direct sequencing for determination of HBV 281 genotypes in a Turkish population[END_REF]. In previously published complete HBV genomes from chronic Hepatitis B patients from 178

Turkey, the G1896A variant was identified in 18% of samples, and these patients were HBeAg 179 negative [START_REF] Bozdayi | Complete genome sequence and phylogenetic analysis of hepatitis B virus 234 isolated from Turkish patients with chronic HBV infection[END_REF]. In this cohort from healthy volunteer donors, the G1896A 180 precore stop variant was identified in 66% of samples. This is in keeping with data that shows a 181 high prevalence of the G1896A mutation in samples from Mediterranean populations [Funk et 182 al., 2002]. G1896A has previously been documented to be present in 15% of HBeAg-positive 183

Turkish patients, compared to 85% of HBeAg-negative patients [START_REF] Bozdayi | Polymorphism of precore region of hepatitis B virus DNA among patients 231 with chronic HBV infection in Turkey[END_REF]Bozdayi et 184 al., 2005]. This mutation is of interest as it results in the prevention of HBeAg synthesis, and 185 seroconversion of HBeAg to anti-HBe is correlated with a decline in viral load [Tedder et al., 186 2002]. The core promoter dual mutations (A1762T, G1764A) were identified previously in 3/11 187 chronic-HBV infected patients, one of whom was HBeAg positive [START_REF] Bozdayi | Complete genome sequence and phylogenetic analysis of hepatitis B virus 234 isolated from Turkish patients with chronic HBV infection[END_REF], and was 188 identified in 20% of the strains sequenced in this study. The double mutation has been

189

implicated with a reduction in the generation of precore mRNA and HBeAg production, and is 190 more prevalent in patients with advanced liver disease [START_REF] Poustchi | Clinical significance of precore and 267 Page 11 of 15[END_REF]. Given the patients, and have correlated its presence with chronic infection and hepatocellular carcinoma 202 [START_REF] Lin | Structural and functional analysis of 2.2 kb 254 spliced variant of hepatitis B virus genomes isolated from liver tissues from 255 hepatocellular carcinoma patients[END_REF]. The association between prevalence of the HBV splice variant and severity of 203 liver disease is not yet elucidated; one study reported an association between a high proportion

204

of spliced HBV: wild-type HBV in patients with more severe liver necroinflammation [Soussan et 205 al., 2008], whereas a different study found no association between spliced HBV prevalence and

206

disease status [START_REF] Preiss | Defective 272 hepatitis B virus DNA is not associated with disease status but is reduced by 273 polymerase mutations associated with drug resistance[END_REF]. Decreased levels of SP1 have been observed in patients with 207 a lamivudine-resistant HBV strain, suggesting that antiviral therapy disrupts the balance 208 between circulating HBV splice variants and full-length HBV genomes [START_REF] Preiss | Defective 272 hepatitis B virus DNA is not associated with disease status but is reduced by 273 polymerase mutations associated with drug resistance[END_REF].

209

In addition, a novel 10.4kDa protein (HBV splice-generated protein; HBSP) arose from 210 the fusion of a section of the polymerase and a new ORF created downstream of the splicing 211 event [START_REF] Soussan | In vivo 283 expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF]. Detection by western blot analysis has revealed the presence of 212 this protein in 46% of serum samples from chronic HBV carriers, as opposed to <1% of healthy

213

HBV-negative controls [START_REF] Soussan | The expression of hepatitis B spliced protein (HBSP) 291 encoded by a spliced hepatitis B virus RNA is associated with viral replication 292 and liver fibrosis[END_REF]. Functionally, this protein has been demonstrated 214 in vitro to increase hepatocyte apoptosis [START_REF] Soussan | In vivo 283 expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF], and in humans and transgenic

  , first-time, replacement blood donors collected in Ankara, Turkey,

93

  TOPO® XL vector (Invitrogen; Paisley, UK) as per the manufacturer's instructions. Following 94 transformation of chemically competent E. coli cells, plasmid DNA was isolated using a QIAprep 95 spin miniprep kit (Qiagen), and a minimum of 10 clones were sequenced.

97(

  DNASTAR) and MacVector software. PAUP* version 4.0 beta 10 was used to calculate genomic 98 distances. Microsoft Excel and Prism (version 4) were used for analysis of HBV DNA and HBsAg

111

  undetected by Q-PCR. However amplification of the BCP/PC region was achieved by nested-PCR 112 in one of the 23 samples, meaning detection of HBV DNA was possible for a total of 177/199 113 samples (89%).

  a deletion of 37 nucleotides, resulting in a frameshift mutation 125 and premature protein truncation. In another strain, insertion of two adenine nucleotides in the 126 precore region caused a frameshift mutation, which is predicted to result in the inclusion of 31 127 amino acids in the precore protein, whilst leaving the core protein unaffected. Cloning of the 128 BCP/PC for this strain identified the double adenine insertion in all 10 clones sequenced. Serum 129 testing of these two samples identified the presence of anti-HBc. Another sample contained a 130 single adenine insertion within the core region, introducing a premature stop codon and protein 131 was not available for anti-HBc testing of this sample. Seven samples (14%) 132 contained mutations that abolished the precore initiation codon. The G1896A mutation, which 133 introduces a premature stop codon and abolishes precore protein and HBeAg synthesis, initiation codon of Pre-S2 were identified in 3 samples (6%).138 Five samples contained deletions in the Pre-S/S transcript, ranging in size from 6 to 51 139 nucleotides. These were present throughout the three ORFs. Three samples contained nonsense 140 mutations that are predicted to result in premature truncation of the protein. One further 141 sample contained a 66 nucleotide deletion spanning the Pre-S1/Pre-S2 boundary, in addition to 142 a nonsense mutation leading to premature truncation of the S protein at amino acid residue 69. 143 Testing for the presence of anti-HBs in the serum from which this strain was obtained was 144 negative. Cloning of this sample identified the presence of wild-type species, in addition to a 145 much smaller product with a 1223 nucleotide deletion (positions 2449-489), predicted to result 146 from splicing of viral mRNA. This deletion started at the final codon of the core gene and 147 extended into the middle of the S gene.

191

  relatively high frequency of these mutations in this study population, and their clinical 192 implications, it may be of use to sequence the viral genome in subjects testing positive for HBV-193 infection in Turkey. 194 This is the first time that screening of the SP1 variant has been conducted in 195 asymptomatic blood donors. The data demonstrated that the SP1 variant was present in more 196 than half of the samples screened (21/40), and was ubiquitous in samples with viral loads ≥ 10 6 . 197 However, that SP1 is detected at a much lower frequency in samples with low viral loads (3/10 198 VL < 10 4 IU/mL) may not be directly linked to viral load, but may be a matter of the ratio at 199 which the transcripts are present and the ability of the PCR to detect very low levels of the 200 spliced variant. Previous studies have examined the prevalence in symptomatic HBV-infected 201

313Figure 1 :

 1 Figure 1: Representative gel of SP1 screening. Amplification of full-length sequence results in a

Table I :

 I Molecular and serological characteristics of HBV strains from Turkish blood donors.

	311 312	F o F o o core promoter mutations in genotype D hepatitis B-related chronic liver disease. J F Legends:
		r r r
		P P P
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	215 216 217	mice to induce a T-cell response [Mancini-Bourgine et al., 2007]. R e v i e w R e v i e w R e v i e w

John Wiley & SonsJournal of Medical Virology

Acknowledgements

This study was supported by grant BS05/2 from the National Health Service Blood 222 and Transplant.