Magdalena Jacobson
email: magdalena.jacobson@kv.slu.se.

A Aspan

A Nordengrahn

M Lindberg

P Wallgren

Monitoring of Lawsonia intracellularis in breeding herd gilts

Keywords: Lawsonia intracellularis, biosecurity, epidemiology, pig, PCR, serology

In modern pig production, proliferative enteropathy is a common cause of diarrhoea and poor growth in young animals. This study aimed to determine the possible spread of L. intracellularis through the sale of replacement gilts and the possibility to protect the herds by adequate biosecurity measures. This was achieved by repeated sampling of 50 gilts in an infected multiplying herd, from the last day in the farrowing pen and until sale. Further, 60 gilts sold from this herd were tested during their stay in quarantine in a recipient herd. To confirm freedom from infection, 100 growing pigs in the recipient herd were also tested. Individual faecal (n=748) and blood (n=728) samples were analysed by PCR and ELISA, respectively.

Transmission of L. intracellularis from the sows to their offspring was not demonstrated.

However, the possible transmission between herds by replacement gilts was demonstrated.

Peak shedding occurred at 12 and 15 weeks of age, and single animals were also PCR-positive at 24 to 36 weeks of age in the multiplying herd and in the quarantine in the recipient herd.

Further, the possible occurrence of chronically infected carrier animals was suggested.

Although L. intracellularis is widely spread, it appears possible to avoid the transmission between herds by employing adequate bio-security measures. Thus, it would be advisable to establish herd profiles in breeding herds to avoid the selling of infected animals as well as to establish the health status of the recipient herd. Further, the health status of the recipient herds should be known.

A c c e p t e d M a n u s c r i p t 1. Introduction Proliferative enteropathy, manifested as diarrhoea and poor performance in growing pigs, or as acute haemorrhagic enteropathy with high mortalities in gilts and finisher pigs, is widespread in pig production (Guedes et al., 2002b;Jacobson et al., 2003b). The causative organism, L. intracellularis, has been demonstrated globally with herd prevalences ranging from 48 to 100% [START_REF] Lee | Prevalence of porcine proliferative enteropathy and its control with Tylosin in Korea[END_REF][START_REF] Jacobson | The prevalence of Brachyspira spp. and Lawsonia intracellularis in Swedish swine herds and in the wild boar population[END_REF]. The microbe was first described in 1993 [START_REF] Gebhart | Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species[END_REF] and still, features of the disease such as the transmission within and between herds, remain to be fully elucidated [START_REF] Lawson | Proliferative enteropathy[END_REF][START_REF] Mcorist | Global patterns of porcine proliferative enteropathy[END_REF]. The microbe may be viable for 14 days outside the host [START_REF] Collins | Studies on the ex vivo survival of Lawsonia intracellularis[END_REF] and rodents are thought to only play a minor role in the spread [START_REF] Mcorist | Global patterns of porcine proliferative enteropathy[END_REF]. Thus, pig-to-pig transmission is considered as the main route of infection [START_REF] Jordan | A Lawsonia intracellularis transmission study using a pure culture inoculated seeder-pig sentinel model[END_REF] and the possibility that single gilts transmit the microbe to their offspring has occasionally been suggested [START_REF] Jensen | Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces[END_REF][START_REF] Kroll | Proliferative enteropathy: a global enteric disease of pigs caused by Lawsonia intracellularis[END_REF]. A few infected piglets will carry the bacterium to the grower-finisher facilities were the main shedding and spread will occur [START_REF] Bronsvoort | Management factors associated with seropositivity to Lawsonia intracellularis in US swine herds[END_REF][START_REF] Jensen | Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces[END_REF]. Within these units, the infection may persist due to poor sanitation measures and short empty-time space between batches [START_REF] Smith | Questionnaire survey of proliferative enteropathy on british pig farms[END_REF][START_REF] Mcorist | Monitored control programme for proliferative enteropathy on British pig farms[END_REF]. Experimentally, shedding has been detected 1-12 weeks post inoculation, whereas antibodies have been detected in sera from 2-13 weeks [START_REF] Smith | Development of persistent intestinal infection and excretion of Lawsonia intracellularis by piglets[END_REF][START_REF] Guedes | Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis[END_REF]. However, only a few studies have addressed the sequential shedding (Guedes et al., 2002b;[START_REF] Stege | Infection dynamics of Lawsonia intracellularis in pig herds[END_REF][START_REF] Jensen | Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces[END_REF] or seroconversion in conventional herds [START_REF] Just | Monitoring of Lawsonia intracellularis by indirect serum immunofluorescence assay in a commercial swine production system[END_REF][START_REF] Marsteller | Monitoring the prevalence of Lawsonia intracellularis IgG antibodies using serial sampling in growing and breeding swine herds[END_REF]. In these, peak shedding occurred between 10-16 weeks of age and persisted for 2-6 weeks. Seroconversion was seen from 10-24 weeks of age, although serum antibodies only persisted for 1-6 weeks in single animals. In the spread between herds, the replacement animals are considered to be of major importance [START_REF] Smith | Questionnaire survey of proliferative enteropathy on british pig farms[END_REF] , 2003). However, the actual infection status of breeding animals close to the introduction into new herds has rarely been investigated [START_REF] Friendship | The effect of porcine proliferative enteropathy on the introduction of gilts into recipient herds[END_REF].

Hence, the aim of this study was to determine the possible spread of L. intracellularis through the purchase of infected breeding herd gilts. Shedding and the development of circulatory antibodies were determined during rearing and at the time of transfer from a breeding herd selling gilts at various ages. Further, the possibility to protect the recipient herd from the infection by adequate biosecurity measures was investigated by repeated sampling of gilts sold from the breeding herd during their stay in quarantine in a recipient herd.

Materials and methods

The study was approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden.

Herds

Pigs from the breeding herd had tested positive for L. intracellularis by necropsy and by PCR on faecal samples 3 months prior to the start of the study. Batches of 18 sows farrowed at 4 weeks interval. Piglets were weaned at five weeks of age and remained in the farrowing pen for one additional week before they were transferred to the grower facilities in a separate building.

Between each batch, all units were washed, disinfected and left empty for one week. Litters were not mixed. The pigs remained in the growing unit for 4-6 weeks and thereafter, gilts were moved to an un-insulated barn and mixed in two large pens with deep straw litter bedding. Within this unit, gilts were moved and mixed 3-4 times. On average, 10-15% of the gilts were sold as pre-pubertal gilts (i.e. ~12-14 weeks old), 10-15% were sold as young gilts (~22 weeks of age), 10-15% as gilts for mating (~30 weeks of age) and 55-70% as pregnant gilts (~42 weeks of age). The recipient herd was a 160-sow, piglet-producing herd where L. intracellularis had never been demonstrated (Jacobson et al., 2003b). Usually, six young gilts and six gilts for mating were bought every second month from the multiplying herd and introduced into the quarantine, located at a one-km distance from the main herd. Separate equipment, clothing and footwear were used. The gilts remained here until they were ready for mating (three to nine weeks). In the main herd, the sows farrowed in batches of 30 sows into nursery units that had previously been washed with hot water under high pressure followed by disinfection with Virkon S (Pharmacia & Upjohn Animal Health AB, Helsingborg, Sweden). Piglets were weaned at 5 weeks of age and remained in the nursery for 1-2 additional weeks, before they were moved to the grower facilities, mixed and sorted according to weight. The piglets were sold at 75 days of age (~30 kg b. w.). The post-weaning mortality was 0.4 to 1.5% and the average piglet growth rate was 500 g/day.

Animals and experimental design

In the breeding herd, 50 individually ear-tagged, crossbred Yorkshire × Swedish Landrace gilts (1-3 per litter), born within one week and in one farrowing unit, were included in the study.

Faecal and blood samples were collected every 3rd week from the last day in the farrowing pen (i.e. one week post weaning) and until sale. The pigs were sold to eight different herds.

The recipient herd only bought gilts from the breeding herd. All of the 60 gilts (22-35 weeks old) bought during one year were included in the study. Sampling was performed at arrival, after 3 weeks stay in quarantine, on one occasion also after 6 weeks stay in quarantine, and before the gilts were transferred to the main herd. To further confirm the infection status of the herd, samples were obtained from 20 growing pigs in the age of 10-12 weeks on five occasions A c c e p t e d M a n u s c r i p t 6 during a 1 ½-year period (in total 100 pigs). With one exception, individual rectal faecal samples and blood samples were collected at each occasion.

Analysis

Faecal samples were stored at -80°C until analysis by nested PCR as previously described [START_REF] Jacobson | Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR[END_REF]. All reactions were performed in 25-L reaction volume and 1 L of an internal control (mimic) was added to each tube (Jacobson et al., 2003a). Positive and negative controls were included in each run. Inhibited samples were re-analysed by PCR once or twice, and if results were still not obtained, faecal DNA was extracted by a commercial kit (QIAamp DNA Stool Mini Kit, Qiagen Inc., Valencia, California, USA) and analysed by conventional PCR [START_REF] Jacobson | Routine diagnostics of Lawsonia intracellularis performed by PCR, serological and post mortem examination, with special emphasis on sample preparation methods for PCR[END_REF].

Blood samples without additives were centrifuged and sera were stored at -20 C until analysis by blocking ELISA according to the instructions given by the manufacturer (Svanova Biotech, Uppsala, Sweden, and Bioscreen GmbH, Münster, Germany). Validation of this method has not been published in a scientifically scrutinised paper, but the stated sensitivity was 96.5% and the specificity, 98.7% (Svanova biotech, unpublished results). The resulting OD value was read at 450 nm and converted to the calculated percent inhibition (PI). Values above PI 35 were regarded as positive.

Results

Sampling and analysis

In total, 492 faecal samples and 484 sera were obtained from the breeding herd (Table 1). Six animals were sold as prepubertal gilts (88-94 days old), three pigs were sold as young gilts (24 weeks of age), and 23 animals as pregnant gilts. Three pigs were culled at 40 weeks of age A c c e p t e d M a n u s c r i p t 7 because of poor growth. In addition, seven animals died or were culled for other reasons. The study was terminated 8 months after the first sampling (in total 14 sampling occasions), when only eight animals remained. From the recipient herd, 256 faecal samples and 244 sera were obtained from in total 60 gilts and 100 growing pigs.

Following the first PCR analysis, a total of 214 samples (29%) were inhibited. Of these, 32 (4%) remained inhibited following the second analysis and 4 samples were prepared by the commercial kit before being re-analysed.

Faecal shedding of L. intracellularis in the breeding herd

In total, 49 of 50 pigs shed L. intracellularis at least once and no shedding was detected on the first sampling occasion (in the farrowing unit). The results are shown in Table 1. In 25 pigs, shedding was detected on one occasion only. In 11 pigs, shedding was detected on two consecutive samplings, in 2 pigs, shedding was detected on three consecutive samplings and in one pig, shedding was detected on four consecutive samplings. In 10 pigs, shedding occurred non-consecutively (Table 2). One of these pigs shed the microbe on two consecutive samplings, followed by seven negative samplings, and then shed the microbe again at 36 weeks of age (Table 2).

Clinical signs in the breeding herd

During the study, 21 pigs (42%) exhibited clinical signs that might have been related to proliferative enteropathy, with diagnoses such as runt or "small" pigs, unthriftiness, and/or diarrhoea. Ten of fourteen pigs suffering from unthriftiness or diarrhoea were simultaneously PCR-positive for L. intracellularis. Fourteen pigs, seven of which also had suffered from diarrhoea, were noted as being "runt" or "small" at various time points during the study.

Development of detectable antibodies to L. intracellularis in the breeding herd

The results are shown in Table 1 and Figs. 12. Most pigs (83-85%) were seropositive at 12 and at 15 weeks of age (average PI 55 and 50, respectively). Thereafter, the number of seropositive animals, as well as the average PI values (Fig. 2), gradually decreased. On the 7 th sampling occasion, at 24 weeks of age, the average PI was 27 and only 18% of the animals were seropositive. A second, lower "peak" occurred on the 10 th to 12 th sampling occasion (59 and 52% of the animals, average PI 40 and 34, respectively).

In total, 36% of the animals were seropositive on the first demonstration of shedding. Further, 50% of the animals seroconverted three weeks after the first positive faecal sampling. Five pigs (13%) were seronegative despite that the microbe had been demonstrated in faeces three weeks earlier and one pig never seroconverted despite that L. intracellularis was demonstrated in faeces on three consecutive samplings. In 15 pigs, positive titres were only demonstrated for three weeks following shedding. However, in two pigs, detectable levels of antibodies were demonstrated for 30-33 weeks following the demonstration of L. intracellularis. Five pigs were seropositive on the first positive faecal sampling occasion; thereafter detectable levels of antibodies were below the cut-off value for 3-9 weeks, followed by the demonstration of the microbe in faeces. One pig only was PCR-negative on all sampling occasions (n=13), and with the exception of the sampling at 33 weeks of age when a weak positive titre (PI 46) was demonstrated, this pig was also seronegative throughout.

Faecal shedding of L. intracellularis in the recipient herd

Altogether, samples from 60 gilts and 100 growers were obtained on 21 sampling occasions.

All growing pigs and three groups of gilts were negative with respect to faecal PCR. In the last two groups of gilts, one gilt per group (27 and 32 weeks old, respectively) were PCR-positive A c c e p t e d M a n u s c r i p t 9 at arrival. Both these animals were negative on the consecutive samplings, but on the next sampling occasion three other gilts within these two groups were positive. Two of these latter gilts remained in quarantine for six weeks and samples were collected a third time. At that time, both gilts were negative by PCR. The third gilt stayed in quarantine for three weeks and was PCR-positive the day before being moved to the main herd. Piglets from her first litter were mixed with other pigs and sampled at 12 weeks of age (in total 20 animals). All samples were negative.

Clinical signs in the recipient herd

On three sampling occasions, altogether eight growing pigs had diarrhoea. Furthermore, four gilts in quarantine had "loose stools". None of these animals were positive for L. intracellularis by PCR.

Development of detectable antibodies to L. intracellularis in the recipient herd

In the recipient herd, 58 of 60 gilts were seropositive (PI>35) on all sampling occasions. Two gilts had PI values of respective 32 and 34 at arrival, and on the consecutive samplings the PI had increased to 85 and 88, respectively. The pigs that were PCR positive had PI values ranging from 40-82 on all occasions. All growing pigs were seronegative on all sampling occasions.

Discussion

We were not able to demonstrate any transmission from the sows to their offspring in the breeding herd. All piglets included in the study were negative in the farrowing unit, both with respect to the presence of L. intracellularis in faeces and detectable antibodies in sera. Instead, the major spread of L. intracellularis occurred in the grower facilities and during the first three A c c e p t e d M a n u s c r i p t 10 weeks in the deep straw litter bedding pens. Probably, once the microbe had gained entrance into the herd, the short time that the pens were left empty between each batch facilitated the persistence of the infection within the grower units and the deep straw litter bedding pens.

However, a possible transmission between herds by breeding herd gilts was clearly demonstrated: The six pigs sold at 13 weeks of age were all PCR positive on the previous sampling occasion and could thus have carried the microbe into the new herd. Furthermore, despite that most animals shed the microbe at 9-12 weeks of age, shedding was also detected at 24 and 36 weeks of age (Table 2). This was also illustrated by the demonstration of L. intracellularis in faeces from two 27-and 32-week-old gilts at arrival to the quarantine in the recipient herd. Although L. intracellularis is widely spread, there are still a number of herds that may not suffer from the infection. Considering the large economical consequences of the disease, it would be desirable to minimize the risk of introducing the infection into these herds.

The better understanding of the infection status of the breeding animals and the recipient herds for the establishment of adequate quarantine periods may prohibit outbreaks of the acute form of the disease [START_REF] Friendship | The effect of porcine proliferative enteropathy on the introduction of gilts into recipient herds[END_REF]. Therefore, it might be advisable to establish herd profiles to avoid selling infected animals. Such profiles should identify the age category where the main shedding occurs and the pattern of shedding in particular herds. Alternatively, animals "at risk" may be kept in quarantine for a prolonged period in the recipient herd. A further possibility would be to eradicate the microbe from single herds [START_REF] Harm Nielsen | Attempt to eradicate Lawsonia intracellularis by medication in 3 sow herds[END_REF][START_REF] Conradsen | Elimination of Lawsonia intracellularis; the way to high performance and low usage of antibiotic[END_REF], although the risk of re-introducing this endemically spread microbe must be considered [START_REF] Johansen | Attempt to eradicate Lawsonia intracellularis by medication in 9 sow herds preliminary results[END_REF].

It seemed plausible, that the microbe would be most easily detected in the grower facilities in the recipient herd. However, despite repeated sampling (in total 100 piglets), L. intracellularis Furthermore, serum antibodies to L. intracellularis were not demonstrated in the growers. In addition, some as yet unknown factors related to protection may be present in the herd.

The interval between the shedding in faeces and demonstration of antibodies in sera generally varied from 0-6 weeks. This is largely consistent with other reports, where serum antibodies were detected 1-3 weeks after infection [START_REF] Guedes | Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis[END_REF][START_REF] Stege | Infection dynamics of Lawsonia intracellularis in pig herds[END_REF][START_REF] Collins | Re-challenge of pigs following recovery from proliferative enteropathy[END_REF]. However, in two pigs, positive antibody titres were not demonstrated until 21 and 24 weeks post onset of shedding, and one pig never seroconverted. Similar results have been noted by others and it is possible that an infection limited to the enterocytes may not elicit a systemic immune response (Guedes et al., 2002b). Thus, the detection of antibodies may correlate with the presence of profound intestinal lesions rather than with exposure (Guedes et al., 2002a). Hence, single subclinically infected animals may not be detected.

The results in Fig. 1 might indicate the development of protective immunity to L. intracellularis. However, 15 pigs shed the microbe in faeces consecutively for 6-9 weeks or intermittently for 15-24 weeks, despite being seropositive. Furthermore, 15 pigs remained seropositive (PI>35) for 3 weeks only. Hence, the systemic antibody response does not seem to be entirely correlated to protection, as also discussed by Guedes et al. (Guedes et al., 2002b).

Further, the results may also be caused by constant re-exposure to the microbe. The second shedding at 24 weeks of age might indicate re-infection. Alternatively, some pigs were latently infected, or shed the microbe in amounts too low to be detected by PCR, and thus were asymptomatic, chronic carriers [START_REF] Guedes | Onset and duration of fecal shedding, cell-mediated and humoral immune responses in pigs after challenge with a pathogenic isolate or attenuated vaccine strain of Lawsonia intracellularis[END_REF]. This may explain the detection of positive antibody titres in some pigs during 30-33 weeks. Hence, the three pigs that were culled because of poor growth at 42 weeks of age might have been chronically infected. These pigs shed the microbe at 9-15 weeks of age, and in one case also at 36 weeks of age, and antibodies to L. intracellularis were demonstrated on almost all sampling occasions.

In the multiplying herd, diarrhoea and unthriftiness was correlated to shedding of L. intracellularis in 20% of the pigs. Furthermore, after 40-weeks rearing period, three pigs (6%) were culled because of poor growth. Thus, it seems reasonable to believe that losses in this herd were substantial, although figures on average growth rate were not available. On the contrary, the recipient herd that was negative with respect to L. intracellularis had a good average performance.

In the breeding herd, one pig only remained PCR-negative throughout the study. The low levels of serum antibodies demonstrated on one single occasion might have been a false positive reaction and this single pig appears to have escaped the infection. Alternatively, this pig might have contracted the infection subclinically for a short period only.

Conclusion

Although L. intracellularis is widely spread, it appears possible to avoid the transmission between herds by employing adequate bio-security measures. Therefore, it would be advisable to establish herd profiles in breeding herds to avoid the selling of infected animals as well as to

  . It is possible, that the low incidence of disease in the age category of gilts bought, combined with the prolonged quarantine period for particularly the youngest of these animals, have protected the herd from infection. Other possible explanations include poor sensitivity of the diagnostic methods used. However, the PCR has previously been shown to demonstrate as few as 10 2 -10 3 organisms/g faeces and the use of an internal control enabled the demonstration of suboptimal reactions caused by the presence of inhibitory factors.

Figure 1 .

 1 Figure 1. The development of positive (PI >35) serum antibody titres demonstrated by an

Figure 2 .

 2 Figure 2. The average serum antibody levels (PI) to Lawsonia intracellularis demonstrated by

  Figure 1.

Acknowledgements

This study was financed by grants from the Swedish Farmer's Foundation for Agricultural Research. We also want to thank Maria Persson for skilful technical assistance.

Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. A c c e p t e d M a n u s c r i p t


Alois Čížek

Monika Dolejská

Radana Sochorová

Kateřina Strachotová

Veronika Piačková

Tomáš Veselý

Antimicrobial resistance and its genetic determinants in aeromonads isolated in ornamental (koi) carp (Cyprinus carpio koi) and common carp (Cyprinus carpio)

Keywords: Aquaculture, Aeromonas spp., Antimicrobial resistance, tet resistance genes, Integrons

The aim of this study was to evaluate antimicrobial susceptibility of Aeromonas spp. isolates from common carp and koi carp coming from randomly chosen farms. The isolates were tested for susceptibility to 8 antimicrobial agents using the standard agar dilution susceptibility test. In all isolates, PCR was used to detect the presence of tet(A-E) genes, integrase genes, and gene cassettes. From the total 72 isolates of motile aeromonads sampled from koi carp, 36 isolates (50%) were resistant to oxytetracycline, 18 (25%) to ciprofloxacin, 5 (7%) to chloramphenicol, 5 (7%) to florfenicol, and 11 (15%) to trimethoprim. Among 49 isolates of motile aeromonads collected from common carp, 20 (41%) were resistant to oxytetracycline, 3 (6%) to Manuscript chloramphenicol, and 3 (6%) to florfenicol. The resistance of aeromonads isolated from koi carp was significantly higher to ciprofloxacin (P = 0.00024). The presence of class 1 integrons was detected in these isolates only (P = 0.00024). Tet genes were detected in 40% (48/121) of isolates, with tet(E) being the most dominant. Our results demonstrated a significant difference in the incidence of resistant isolates collected from koi carp and common carp (P = 0.00042).

This difference can be ascribed to a distinct antibiotic policy established on consumer fish farms versus ornamental fish farms. The potential risk for resistant bacteria to spread and transmit infection to humans should be considered in cases of technological crossover between the two types of fish farms.

A c c e p t e d M a n u s c r i p t

Introduction

The Czech Republic is a traditional producer of common carp (Cyprinus carpio). Its annual production is approximately 18,000 tons, half of which is exported. Recently, the development of ornamental (koi) carp (Cyprinus carpio koi) farming has appeared in the country, both on small-scale farms and large production farms. Intensive carp farming is associated with risk for the incidence and spread of infectious diseases commonly associated with therapeutic and prophylactic use of antibiotics. In general, two or three agents have been granted marketing authorisations in EU countries [START_REF] Smith | Antimicrobial resistance in aquaculture[END_REF]. In the Czech Republic, for example, oxytetracycline and flumequine are permitted, with the former used extensively on common carp farms. On the other hand, the regulation of antibiotic use is not enforced on such ornamental fish farms as koi carp operations. Therefore, the ornamental fish producers tend to administer antibiotics in a non-systematic and uncontrolled manner, thus making the selection and spread of antibiotic-resistant bacteria possible. The results of existing studies show that administering antibiotics in "aquacultures" leads to higher antibiotic resistance across the entire A c c e p t e d M a n u s c r i p t 3 microbial water ecosystem [START_REF] Schmidt | Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms[END_REF][START_REF] Sørum | Effects of oxytetracycline containing feed on pond ecosystem and health of carp (Cyprinus carpio L.)[END_REF] and thus also to higher risk for transmitting genetic resistance determinants to bacteria that are pathogenic for fish. Indicator bacteria suitable for studying the incidence and development of antibiotic resistance on fish farms include motile aeromonads. These bacteria are interconnected with the water ecosystem, colonize fish, and can cause various infectious processes in them [START_REF] Austin | Bacterial fish pathogens. Diseases of farmed and wild fish[END_REF]. Sporadically, aeromonads have been proven to cause human infections [START_REF] Janda | Aeromonas species in septicemia: laboratory characteristics and clinical observations[END_REF][START_REF] Ko | Clinical features and therapeutic implications of 104 episodes of monomicrobial Aeromonas bacteraemia[END_REF].

To date, very little has been published on the susceptibility of bacteria isolated from common and koi carp worldwide (Guz and Kozińska, 2004;[START_REF] Taylor | Multiple antimicrobial resistance in a chronic bacterial infection of koi carp[END_REF], while no data are available at all about bacterial susceptibility in the Czech Republic. The aims of this study were to identify the levels of antimicrobial resistance and to define resistance determinants in motile aeromonads isolated from koi and common carp coming from randomly chosen farms in the Czech Republic.

Material and methods

Bacterial cultures

A total of 138 koi and common carp were tested in 2005 (7 localities) and 2006 (7 localities).

Fish were subjected to bacteriological examination for differential diagnosis of KHV (koi herpes virus). No increase of morbidity or mortality was observed. Gills and skin swabs from euthanized fish were cultured on Columbia agar (CM331, Oxoid Ltd., Basinstoke, UK), supplemented with 5% sheep blood, in order to isolate Aeromonas spp. If the fish had ulcerous changes on skin or gills, samples were taken from these ulcers preferentially. Inoculated agars were incubated at 28 ± 2 °C for 24-48 hours. Primary bacterial cultures thus obtained were pre-identified according to morphology of colonies, Gram reaction, motility, catalase and oxidase production, and resistance to vibriostatic agent O/129 [START_REF] Rahman | Identification and characterization of pathogenic Aeromonas veronii biovar sobria associated with epizootic ulcerative syndrome in fish in Bangladesh[END_REF].

Presumptive cultures of Aeromonas spp. were examined using API 20E test (bioMérieux, France). Esculin hydrolysis and growth on triple sugar iron agar slant were used as A c c e p t e d M a n u s c r i p t 4 supplementary tests. Phenotypic identification was done using Aerokey II [START_REF] Carnahan | Aerokey II: A flexible key for identifying clinical Aeromonas species[END_REF]. Identified cultures were stored in cryoprotective medium at -80°C and tested for susceptibility to antimicrobials and the presence of resistance determinants. For these tests, no more than 1 Aeromonas spp. isolate from each fish was used. Quality control was done with reference strains Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. samonicida ATCC 33658.

Determination of susceptibility to antimicrobials

Susceptibility tests were performed by agar dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) guidelines [START_REF] Clsi | Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals[END_REF] and using the following antibacterial substances: chloramphenicol, ciprofloxacin, florfenicol, oxolinic acid, oxytetracycline, trimethoprim, spectinomycin, and streptomycin (Sigma Aldrich, Prague, Czech Republic). Antimicrobial agents were incorporated into Mueller-Hinton agar (Oxoid Ltd., Basingstoke, UK), with each plate containing the agent in log2 doubling dilutions of the following ranges: chloramphenicol, florfenicol (2-64 µg/ml); ciprofloxacin, oxolinic acid and trimethoprim (0.25-16 µg/ml); oxytetracycline (1-64 µg/ml); streptomycin (0.5-64 µg/ml); and spectinomycin (2-1024 µg/ml). Frozen Aeromonas spp. isolates and control strains were inoculated on nutrient agar (CM3, Oxoid Ltd., Basingstoke, UK) and incubated at 28 ± 2°C for 24 hours. Growth was re-suspended in 2 ml sterile PBS using a sterile cotton-tipped swab at a concentration of 1.5 x 10 8 CFU/ml, in accordance with 0.5 McFarland standard. The turbidity was adjusted with a photometer (Densi-La-Meter, LIAP, Latvia). Plates were inoculated using a multipoint inoculator (Trios, Czech Republic), which delivered 10 5 organisms per spot. Inoculated plates were incubated at 28 ± 2 °C for 24 to 28 hours.

The cultures were compared with a growth control plate containing no antibiotic, and minimum inhibitory concentration (MIC) for each agent was determined as the lowest concentration of an antimicrobial that inhibited the growth of a given culture. Each isolate was examined three times and each batch of media was checked using the aforementioned control strains. The

Detection of resistance genes, integrons and integron-associated genes

All isolates were tested by PCR for tet(A-E), the class 1 and 2 integrase genes intI1 and intI2, respectively, the variable region of class 1 and class 2 integrons (designed by primers 5CS-3CS and Hep74-Hep51), gene cassettes inside the integron structure, and the sul1 gene which is an essential part of the class 1 integron. This test technique has been previously described by Dolejska et al. (2007) and [START_REF] Wilkerson | Antibiotic resistance and distribution of tetracycline resistance genes in Escherichia coli O157:H7 isolates from human and bovines[END_REF].

Data analysis

The results obtained were statistically evaluated by chi-square test using MS Excel® software with the Analyse-it module. Differences with p ≤ 0.05 were considered as significant.

Results

Susceptibility to antimicrobials

In total, 121 presumptive isolates were identified as A. sobria (60 isolates), A. hydrophila (55 isolates) and A. veronii subsp. sobria (6 isolates). MICs obtained for 8 antimicrobial agents are demonstrated in Table 1. Seventy-three of these isolates (60%) were resistant to one or more antibiotics. Significant differences were detected between the prevalence of resistant isolates in koi carp and common carp (P = 0.0042). Of 72 isolates of motile aeromonads sampled from koi carp, 36 (50%) were resistant to oxytetracycline, 18 (25%) to ciprofloxacin, 5 (7%) to chloramphenicol, 5 (7%) to florfenicol, and 11 (15%) to trimethoprim. Among 49 isolates of motile aeromonads collected from common carp, 20 (41%) were resistant to oxytetracycline, 3 (6%) to chloramphenicol, and 3 (6%) to florfenicol. Comparison of MIC results indicated significant difference in susceptibility to ciprofloxacin (P = 0.00024). Significant difference A c c e p t e d M a n u s c r i p t 6 was detected also between the isolates of the two origins in the prevalence of simultaneous resistance to more than one antibiotic (P = 0.02559).

Tet genes, integrons and integron-associated genes

All isolates of aeromonads were screened for the presence of tet(A-E) genes. Tet(E) was identified in 33% (40/121) of these isolates. Moreover, tet(E) was detected in combination with tet(A) (3 isolates), tet(D) (3 isolates), and both tet(A) and tet(D) (2 isolates). One isolate contained the tet(D) gene. The isolates with detected tet genes had MICs for oxytetracycline in a range from 1.0 to >64 mg/L. No tet(A-E) genes were proven in 12 (21%) of 56 oxytetracycline-resistant isolates.

The intI1 gene was found in 17 (14%) of 121 isolates. The presence of class 1 integrons was detected only in the isolates from koi carp (P = 0.00024). These intI1 positive isolates were significantly related to elevated MICs for spectinomycin (in a range from 32 to >1028 mg/L).

MICs for streptomycin of these isolates ranged from 1 to >64 mg/L. All of these intI1-positive isolates contained the sul1 gene. Four types of class 1 integrons with the following size of the variable region gene cassettes were determined: 1 kb with an aadA2 gene cassette, 1.6 kb with a dhfr12-aadA2 gene cassette, 1.7 kb and 2 kb with an aadA1 gene cassette. One isolate with the intI1 and sul1 genes and a dhfr12-aadA2 cassette was negative for the variable region detected by 5CS-3CS primers. All isolates were negative for the intI2 gene.

Discussion

The aim of this study was to evaluate susceptibility of Aeromonas spp. isolates from common and koi carp coming from randomly chosen farms. Standard methods for testing susceptibility of bacteria isolated from fish have been published only recently [START_REF] Clsi | Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals[END_REF]. However, no criteria for evaluating susceptibility in bacteria isolated from aquatic animals have yet been accepted and published. Moreover, the needs of veterinarians and epidemiologists vary [START_REF] Bywater | Antimicrobial breakpoints -Definitions and conflicting requirements[END_REF][START_REF] Miller | Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data[END_REF]. In this study, criteria published in medicinal products for therapy and prohibits using any other products. In the Czech Republic, antibiotic therapy of infections in common carp is based solely on oxytetracycline that is administered orally in granulated feed at doses dependent on the size of the fish stock in a pond. However, therapeutic use of oxytetracycline leads to contamination of pond sediments with residues of this antibiotic (Svobodová et al., 2006), which can contribute to the selection of bacteria resistant to oxytetracycline. Our results have confirmed that resistance to oxytetracycline and the incidence of tet genes among isolates of aeromonads from common carp was rather high. Similar findings were previously described in various species of fish kept on intensive farms [START_REF] Alcaide | Occurrence of drug-resistant bacteria in two European eel farms[END_REF][START_REF] Nawaz | Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish[END_REF]Jacobs and Chenia, 2007). This study proved that in the isolates of motile aeromonads from common carp and koi carp, the resistance to oxytetracycline was most often coded by the tet(E) gene or by a combination of the tet(E) gene with tet(A) and tet(D) genes. The 12 oxytetracyclineresistant isolates that were negative for the tested tet(A-E) genes could possess one of the less frequent tet or otr genes that also have been described in Aeromonas spp. isolates [START_REF] Chopra | Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance[END_REF][START_REF] Gordon | Complete sequence of the floR-carrying multiresistance plasmid pAB5S9 from freshwater Aeromonas bestiarum[END_REF]. Potential mutation inside the tet gene or another antimicrobial resistance mechanism are other explanations.

Recently, tet(E) has been detected in Aeromonas spp. strains on Danish fish farms. The tet(E) gene occurs on large plasmids that can be horizontally transmitted to E. coli (Agersø , 2007). This finding confirms the possibility of spreading tetracycline resistance to other bacterial pathogens in a fish farm ecosystem. In this sense, the possible overlap of farming technologies for koi and common carp (e.g. hatcheries, circulation systems, handling of fish, etc.) should be viewed as a very serious epidemiological problem. This can lead not only to transmission of pathogens (e.g. koi herpes virus) but also of resistant bacterial clones. In this study, resistant aeromonad isolates with class 1 integrons were detected in koi carp produced by 2 out of 4 large farmers who, at the same time, produced also common carp (the results are not presented here).

Evaluating the susceptibility of bacteria isolated from fish to fluoroquinolones can be problematic, as no binding breakpoints have yet been approved. In general, one substance should be selected that would function as a "reporter" of susceptibility to other fluoroquinolones. For this purpose, nalidixic acid has been preliminarily accepted (Rodriguez-Avial et al., 2005). MIC results for oxolinic acid did not differ significantly in dependence upon the origin of isolates. Detailed comparison of our results, however, showed a shift of MIC values between the isolates of different origin. These results will need to be evaluated as per epidemiological cut-off values (ECOV) estimated by [START_REF] Miller | Epidemiologic cutoff values for antimicrobial agents against Aeromonas salmonicida isolates determined by frequency distributions of minimal inhibitory concentration and diameter of zone of inhibition data[END_REF].

Unfortunately, the range of oxolinic acid concentrations tested in this study was beyond ECOV for wild-type isolates. This problem may be solved by the proving of quinolone-resistance determinants.

The incidence of antibiotic-resistant aeromonads in freshwater fish is generally viewed as a potential risk of infection for human consumers of fish [START_REF] Alderman | Antibiotic use in aquaculture: development of antibiotic resistance-potential for consumer health risks[END_REF]. In this study, a high incidence of antibiotic-resistant aeromonads was confirmed in koi carp. Fish of this kind can be a source of contamination for recreational water reservoirs, which can pose a potential risk for human health.

  values obtained were used to calculate MIC 50 , MIC 90 and the MIC range. Criteria published in A c c e p t e d M a n u s c r i p t 5 Standards for susceptibility tests for bacteria isolated from animals (CLSI, 2008) were used to evaluate susceptibility of isolates to chloramphenicol, ciprofloxacin, florfenicol, oxytetracycline, and trimethoprim. Cut-off values for interpreting MICs for oxolinic acid, streptomycin, and spectinomycin were not determined.

7

  Standards for susceptibility tests for bacteria isolated from animals[START_REF] Clsi | Standards for antimicrobial disk and susceptibility tests for bacteria isolated from animals[END_REF] were therefore used to evaluate susceptibility of isolates to selected antibiotics.Depending upon their origin, aeromonads examined in this study differed in their susceptibility to antibiotics and the presence of class 1 integrons. Statistically significant differences between two groups of isolates investigated in this study can be explained by wholly different antibiotic policies. While producers of koi carp are not at all restricted in using antimicrobials, common carp farmers must observe Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products. The Directive permits the use of only authorized

  Distribution of MICs in Aeromonas spp. isolates from Koi carp (n = 72) and Common carp (n = 49) CHLchloramphenicol; CIPciprofloxacin; FFCflorfenicol; OAoxolinic acid; OTCoxytetracycline; SPTspectinomycin; Sstreptomycin; Ttrimethoprim; NTnot tested; shaded areas denote MIC breakpoints (CLSI, 2008); a MIC breakpoints for oxolinic acid, streptomycin and spectinomycin were not determined

Acknowledgements

This study was supported by Project QH71057 under the National Agency for Agricultural Research (NAZV) and Grant No. MSM 6007665809 of the Ministry of Education, Youth and Sports of the Czech Republic.


DVM, MPH April J Johnson
email: johnsonaj@purdue.edu

Lori Wendland

Terry M Norton

Bill Belzer

Elliott R Jacobson

Development and Use of an Indirect Enzyme-linked Immunosorbent Assay for Detection of Iridovirus Exposure in Gopher Tortoises (Gopherus polyphemus) and Eastern Box Turtles (Terrapene carolina carolina)

Keywords: Reptile, Turtles, Iridovirus, Ranavirus, Enzyme-linked immunosorbent assay

Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (Gopherus polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of Eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only one turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response.

Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence.

A c c e p t e d M a n u s c r i p t

Introduction

Iridoviruses of the genus Ranavirus are well known for causing mass mortality events of fish and amphibians with increasing reports of infection in reptiles. Prior to 2003, only two occurrences of chelonian infection with a Ranavirus were known in the United States; a freeranging gopher tortoise (Gopherus polyphemus) in Florida (Westhouse et al., 1996) and a collection of captive box turtles (Terrapene carolina carolina) in North Carolina [START_REF] Devoe | Ranavirusassociated morbidity and mortality in a group of captive eastern box turtles (Terrapene carolina carolina)[END_REF]. Since then, several additional cases and outbreaks have been identified and described in both free-ranging native and captive exotic chelonian species including Eastern and Florida box turtles (Terrapene carolina carolina and T. carolina bauri), gopher tortoises, and Burmese star tortoises (Geochelone platynota) [START_REF] Allender | Intracytoplasmic inclusions in circulating leukocytes from an easter box turtle (Terrapene carolina carolina) with iridoviral infection[END_REF][START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. Experimental transmission studies have confirmed that ranaviruses are in fact primary pathogens of chelonians [START_REF] Johnson | Experimental transmission and induction of ranaviral disease in Western Ornate box turtles (Terrapene ornata ornata) and red-eared sliders (Trachemys scripta elegans)[END_REF]. The method of transmission has yet to be elucidated, but conspecific amphibians have been shown to be infected with identical or very closely related viruses, suggesting that amphibians may serve as a source of infection for chelonians [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. The prevalence and incidence of infections in free-ranging chelonians remains unknown.

Indirect enzyme linked immunosorbent assays (ELISA) have been used to detect exposure of various species of reptiles to specific pathogens [START_REF] Schumacher | Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease[END_REF][START_REF] Origgi | Enzyme-linked immunosorbent assay for detecting herpesvirus exposure in Mediterranean tortoises (spur-thighed tortoise [Testudo graeca] and Hermann's tortoise [Testudo hermanni])[END_REF][START_REF] Brown | Detection of antibodies to a pathogenic mycoplasma in American alligators (Alligator mississippiensis), broadnosed Caimans (Caiman latirostris), and Siamese crocodiles (Crocodylus siamensis)[END_REF][START_REF] Jacobson | Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to west Nile virus in American alligators (Alligator mississippiensis) in Florida[END_REF] and have been used to detect exposure of amphibians to iridovirus infections (Whittington et al., 1997;[START_REF] Gantress | Development and characterization of a model system to study amphibian immune responses to iridoviruses[END_REF][START_REF] Maniero | Generation of a long-lasting, protective, and neutralizing antibody response to the ranavirus FV3 by the frog Xenopus[END_REF]. To determine iridovirus exposure in free-ranging gopher tortoises in the United States, an indirect ELISA was developed using a previously described mouse anti-desert tortoise IgY monoclonal antibody as the secondary antibody [START_REF] Schumacher | Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease[END_REF]. We describe the assay and the results of a larger serological survey of free-ranging gopher tortoises from various sites in Alabama, Florida, Georgia, Louisiana and Mississippi. Additionally, 55 surviving Eastern box A c c e p t e d M a n u s c r i p t 4 turtles (Terrapene carolina carolina) from a population in Pennsylvania that experienced an outbreak of illness that killed 16 turtles were sampled to determine whether any of the box turtles were exposed that might have survived infection.

Materials and Methods

Positive and Negative Reference Plasma and Virus Isolate

In July of 2003, three of five captive Burmese star tortoises (BST) became ill with clinical signs consisting of nasal discharge, conjunctivitis, frothing from the mouth, cervical subcutaneous edema, and oral plaques. One of the three tortoises died and histologic lesions were observed that were consistent with those seen in iridovirus infections in fish, amphibians and other reptiles, including the presence of intracytoplasmic inclusion bodies [START_REF] Reddacliff | Pathology of hematopoietic necrosis virus (EHNV) infection in rainbow trout (Onchorhynchus mykiss Walbaum) and redfin perch (Perca fluvius)[END_REF]Westhouse et al, 1996;[START_REF] Bollinger | Pathology, isolation and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan[END_REF][START_REF] Docherty | Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events[END_REF]). Molecular investigations demonstrated the presence of Ranavirus in various tissues [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF].

This Ranavirus here termed BSTRV, was used as the antigen in the development of the ELISA.

Surviving tortoises were treated with acyclovir (Glaxo Wellcome, Brentford, Middlesex, United Kingdom) and supportive care and all four tortoises survived. Plasma was collected at the time of the onset of clinical signs of disease during July and then again two months later in September 2003. Plasma from one of these tortoises collected in September was used as the positive control in development of the ELISA. Plasma from a Burmese star tortoise from a zoological collection with no known history of disease was collected to serve as negative reference plasma for the ELISA.

Antigen Preparation

Terrapene heart-1 (TH-1) cells were grown to confluency in 225cm 2 tissue flasks (Costar, Corning, NY). Cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (Gibco), gentamicin (60mg/liter; A c c e p t e d M a n u s c r i p t 5 Sigma, St. Louis, MO), penicillin G (120,000 U/liter), streptomycin (120,000 U/liter) and amphotericin B (300µg/liter; Sigma). Cells were inoculated with a fourth passage of BSTRV and incubated at 28ºC in the presence of 5% CO 2 . When cytopathic effects (CPE) consisting of cell rounding and detachment from the flask were observed in over 80% of cells, the cells and media were transferred to 15ml tubes and centrifuged at 4,500xg for 30 minutes. Supernatant was then discarded and the cell pellets were resuspended in residual media, and frozen and thawed three times to release virus from the cells. Tubes were vortexed before and after each freeze cycle and following the final thaw. Subsequently, they were centrifuged again at 4,500xg for 30 minutes.

Supernatant containing concentrated amounts of virus was then transferred to a 4ml sterile cryotube. A Bradford protein assay was performed to determine the final protein concentration of the antigen [START_REF] Bradford | A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding[END_REF]BioRad, Hercules, CA). A previously described polymerase chain reaction (PCR) test was used to confirm the presence of the Ranavirus major capsid protein (MCP) gene [START_REF] Marschang | Isolation and characterization of an iridovirus from Hermann's tortoises (Testudo hermanni)[END_REF]. Uninfected flasks were concurrently processed in the same manner to serve as control antigen to detect any background cross reactivity of plasma to cellular proteins.

ELISA Procedure

A checkerboard optimization strategy was used to determine the optimum concentrations of both antigen and plasma to be used in the ELISA. Antigen concentrations were evaluated at dilutions of 1:100, 1:250, 1:500, and 1:1000. Plasma concentrations evaluated were two-fold serial dilutions from 1:50 to 1:1600. The following procedure was found to be optimal utilizing the crude cell lysate antigen. Each well of a high protein binding 96 well microplate (Maxisorp F96; Nunc, Kamstrup, Denmark) was coated with 50l of infected or uninfected cell lysate diluted to 1:100 in 0.01M sodium phosphate buffer (pH 7.2) containing 0.15 M NaCl and 0.02% A c c e p t e d M a n u s c r i p t 6 sodium azide (PBS/Az). Plates were incubated overnight at 4C. Antigen was then aspirated off and wells were washed four times with ELISA wash buffer (PBS/Az with 0.05% Tween 20).

This washing process was performed between each of the following steps. Wells were then blocked against non-specific binding with 300l of Superblock blocking buffer by Pierce (Rockford, IL) for one hour at room temperature (RT). Each remaining step was incubated for 1hr at RT. Plasma samples diluted 1:100 in blocking buffer were added at 50l volumes to wells in triplicate. One well was coated with uninfected cell lysate, while the other two wells were coated with infected cell lysate. Next, a biotin-conjugated monoclonal antibody produced against the desert tortoise IgY light chain [START_REF] Schumacher | Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease[END_REF] was diluted to a final concentration of 0.5g/ml in PBS/Az and added to each well in 50l volumes. Alkaline phosphataseconjugated streptavidin (Zymed Laboratories, Inc., San Francisco, California) was applied to each well at 50l of a 1:5000 dilution in PBS/Az. Next, the ELISA was developed with 100l per well of a 1.0mg/ml P-nitrophenyl phosphate prepared in 0.01M sodium bicarbonate buffer containing 2mM MgCl 2 and plates were stored in the dark. The absorbance of each well was read at A 405 using a StatFax 3200 microplate reader (Awareness Technology, Palm City, Florida) at 30 minutes.

Each plasma sample was read in triplicate. Plasma was placed on one well originally coated with uninfected cell lysate and on two wells coated with infected cell lysate. The average absorbance reading of the two wells coated with infected cell lysate was calculated and the positive/negative (P/N) ratio value of each sample was determined by dividing the mean absorbance of the duplicate average by the absorbance reading of the well from the uninfected cell lysate. This subtracts out any background noise, or non-specific binding that may have occurred as a result of using the Terrapene heart cell line. The cut-off value for a positive test A c c e p t e d M a n u s c r i p t 7 result was determined by adding three times the standard deviation to the mean P/N ratio obtained [START_REF] Crowther | The ELISA guidebook[END_REF].

Reproducibility

Intra-assay and inter-assay reproducibility were determined by performing two precision runs. The positive and negative reference plasma samples, used to optimize the test, were used in each assay. Intra-assay reproducibility was determined by running the positive and negative sample multiple times on the same plate. Each sample was run multiple times on one well of uninfected cell lysate and two wells of infected cell lysate. This resulted in 64 readings for each sample on the infected cell lysate and 32 on the uninfected cell lysate. Inter-assay reproducibility was determined by using the values of the reference plasma results used as controls in assaying the wild gopher tortoise samples from multiple dates and multiple plates. The mean A 405 , the standard deviation (SD) and the coefficient of variation (CV) for the intra-and inter-assay reproducibility were calculated using the optimized ELISA conditions.

Protein Expression and Immunoblotting

The positive control plasma was tested for its ability to detect viral proteins in a western blot using infected and uninfected TH-1 cell lysates as antigen. Four four-fold dilutions of infected and uninfected cellular lysate proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions along with broad range molecular weight markers. The separated proteins were transferred onto 0.2µm nitrocellulose membranes (Biorad, Hercules, CA) by standard methods [START_REF] Harlow | Antibodies: a laboratory manual[END_REF]. Membranes were then rinsed in water for five minutes in preparation for coomassie blue staining or in tris buffered saline containing 0.5% Tween20 (TTBS, pH 7.5) for twenty minutes prior to immunoblotting. Membranes were then stained in coomassie blue stain for 90 minutes, followed by water for 30 minutes to destain to view differences in protein profiles between A c c e p t e d M a n u s c r i p t 8 infected and uninfected cell lysates. After rinsing in TTBS, membranes for immunoblotting were blocked with Superblock blocking buffer in phosphate buffered saline (Pierce) for one hour.

Blocking buffer was then removed and plasma samples diluted 1:2000 in blocking buffer were added to the membranes. After one hour, the membranes were washed with TTBS for 30 minutes and the monoclonal antibody, diluted 1:10,000 in blocking buffer, was added and incubated for one hour. Again the membranes were washed and AP streptavidin, diluted 1:5,000 in phosphate buffered saline was added and allowed to incubate for one hour. Membranes were washed as previously described, and the membranes were developed in substrate buffer (0.1M Tris-HCl, 1mM MgCl 2 ) containing nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3indolylphosphate p-toluidine salt (BCIP) (Biorad). The reaction was stopped by removing the NBT-BCIP solution and adding deionized water. Membranes were allowed to air dry.

Free-ranging Gopher Tortoise Samples

Plasma samples from 1000 wild gopher tortoises (Gopherus polyphemus) from Alabama (n=9), Florida (n=658), Georgia (n=225), Louisiana (n=12), Mississippi (n=28) and unknown locations (68) were obtained from recently submitted samples to the Mycoplasma Research Laboratory at the University of Florida between 2001 and 2006. County, state and date of sample collection was recorded for each plasma sample when it was available.

Box Turtle Samples

During the summer and fall of 2003, a population of 71 repatriated Eastern box turtles (Terrapene carolina carolina) in a private nature sanctuary in Pennsylvania, regularly tracked by radiotelemetry for over four years, experienced an unprecedented occurrence of sudden deaths in 16 turtles. The outbreak was investigated and iridovirus infections were confirmed in three of the turtles as previously described [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. It is suspected to be the cause of death in the other 13 turtles, however further diagnostic work-up was not performed in these turtles. In

Results

ELISA Parameters

Results of the checkerboard optimization strategy determined that a 1:100 dilution of both the antigen and plasma samples gave the largest difference of P/N ratios between the positive and negative reference samples (Fig. 1). The frequency distribution of the P/N ratio is shown in 0.1 increments from 0.5 to 4.1 (Fig. 2). The mean P/N ratio of samples (1.078), plus three times the standard deviation (0.379) was used to set the positive cut-off value at 2.2. P/N ratios from each sample were plotted on a graph with the sample numbers on the x-axis in increasing order and the P/N ratio values on the y-axis (Fig. 3).

Reproducibility

The mean A 405 , SD and CV values for the intra-and inter-assay precision runs are shown in Table 1. Ideally, CV values should be less than 15% [START_REF] Crowther | The ELISA guidebook[END_REF]. Three of the CV values in the inter-assay precision runs were >15% indicating that some variability still existed in this assay when performed at different time periods. Intra-assay coefficients of variance were less than 15%.

Protein Expression and Immunoblotting

Coomassie blue staining of proteins from infected cell lysates and uninfected cell lysates showed a different pattern of protein expression (Fig. 4). Immunoblotting of infected and uninfected cell lysate showed a marked increase in binding to proteins in the infected cell lysate and very weak binding to proteins in the uninfected cell lysate (Fig. 5). Strong signals were seen

Free-ranging Gopher Tortoise Samples

Of 1000 gopher tortoise plasma samples assayed, eight (1.2%) tortoises from Florida and seven (3.1%) from Georgia were seropositive, for a total of 1.5% of tortoises seropositive with a P/N ratio >2.2 (Table 2). All tortoises from Alabama, Louisiana and Mississippi were negative, as were the ones from unknown locations. Seropositive tortoises in Florida were located in five counties including Lake, St. Lucie, Broward, Palm Beach and Martin (Table 3, Fig. 6). Four of these are clustered together in the south including Palm Beach, Broward, Martin, and St. Lucie.

The remaining seven seropositive tortoises were located in Baker, Georgia, a county in the southwest corner of the state (Fig. 6).

Date of sample collection was recorded in 680 samples. Two thirds of the positive samples were collected in 2005 (Table 4), of which seven were from tortoises in Baker County, Georgia.

Box turtle Samples

Of 55 samples, only one box turtle tested positive with a P/N ratio of 7.2 for a prevalence of 1.8%. All other samples had a P/N ratio less than 1.7. No clinical signs of illness were observed in the turtle that tested positive, and the turtle, which was being regularly tracked by radiotelemetry had no known history of illness.

Discussion

This study documents the development of an assay to detect anti-iridovirus antibodies and the first report of anti-iridovirus antibodies in free-ranging and captive chelonians. The ELISA A c c e p t e d M a n u s c r i p t 11 detected anti-iridovirus antibodies two months after two Burmese star tortoises recovered from an upper respiratory illness with oral plaques, which had been previously housed with a tortoise that died with confirmed iridovirus infection [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. Plasma from a Burmese star tortoise from another facility with no known history of illness was selected as a seronegative control and no antibodies to iridovirus were detected in this tortoise. Additionally, using a western blot, we were able to show that plasma from the positive control turtle sample bound to proteins in the infected cells in the immunoblot, but not in the uninfected cells indicating that the plasma was reacting with viral proteins and not cellular proteins from the lysate that was used to coat the plates. For these reasons, we consider this a valid test for use in detecting anti-iridovirus antibodies in chelonians, however further validation of this assay against another diagnostic assay is warranted to establish the sensitivity and specificity of the assay. Additionally, interassay precision runs indicated that some variability still exists when the assay is run at different time periods. The values for samples run on infected cell lysate were more variable than those run on uninfected cell lysate. This suggests that the technique for preparing antigen may vary, either due to differences because of the operator, or in the assay and/or reagents.

Humoral immune responses are characterized by an early response as indicated by an acute increase in plasma IgM levels, followed by an increase in IgG levels in mammals, or IgY levels in reptiles. Early studies using the anti-desert tortoise IgY light chain monoclonal antibody found that the antibody would detect the light chains of both IgM and IgY [START_REF] Schumacher | Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease[END_REF]. This suggests that this ELISA can detect both recent infections with Iridovirus as well as infections that may have occurred awhile before the plasma was collected. In the same study by Schumacher et al., seroconversion following inoculation with Mycoplasma agassizii occurred between 1 and 3 months [START_REF] Schumacher | Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease[END_REF], suggesting that in animals with positive Prevalence among free-ranging gopher tortoises utilizing this assay was found to be low; only 1.5% of 1000 samples were positive. This could be the true prevalence rate, although we suspect that this is an underestimation. Prevalence is a function of the incidence of disease multiplied by the average duration of the illness. While incidence would be difficult to determine in a natural setting, duration of illness can be extrapolated from experimental studies. If chelonians die quickly as a result of infection, they will not have time to develop an immune response to the pathogen, and will not survive to be surveyed. As previously mentioned, experimental transmission studies have shown a high rate of mortality (75%) in turtles intramuscularly inoculated with 1 ml of 10 5 TCID 50 /ml of an iridovirus-infected cell lysate [START_REF] Johnson | Experimental transmission and induction of ranaviral disease in Western Ornate box turtles (Terrapene ornata ornata) and red-eared sliders (Trachemys scripta elegans)[END_REF]. Turtles that succumbed to the virus died within 30 days of exposure. If naturally exposed cases experience similar mortality rates and duration from exposure to death, a cross-sectional study evaluating the prevalence of exposure will miss many tortoises that were exposed, because most tortoises will die. This was demonstrated in a natural setting in Pennsylvania. Seventy-one radiotelemetered eastern box turtles (Terrapene carolina carolina) were being followed by researchers since 1999 in a private nature sanctuary. Turtles had been translocated from another preserve in 1999 and experienced no disease outbreaks until 2003. In the summer and fall of 2003, 16 of these turtles died suddenly, with what was later identified to be iridovirus infections [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. The following spring, plasma from the surviving 55 turtles was evaluated by ELISA for the presence of anti-iridovirus antibodies. Only one turtle was positive on ELISA. If we calculated this value as the true prevalence, we would estimate that about 1.8% (1/55) of turtles were exposed, when we know that 24% (17/71) of the population A c c e p t e d M a n u s c r i p t 13 was actually exposed, but 22% (16/71) died and so were not available to be included in the estimate. This severely underestimates the prevalence of disease. However, we cannot extrapolate these differences to other populations, as this was a repatriated population, and thus, subject to stressors that might not be found in naturally occurring populations, making them more susceptible to disease. Additionally, we are unsure how long anti-iridovirus antibodies remain at detectable levels in chelonians, or if all animals infected with the virus will mount an antibody response. If antibody levels decline prior to testing, or they never develop, then animal test results may be falsely negative, also creating an underestimate of prevalence.

Results of this serosurvey showed that counties in the southeastern region of Florida were more likely to have seropositive tortoises. Interestingly, four of these counties had adjoining borders (St. Lucie, Martin, Palm Beach and Broward), suggesting that either tortoises in this area are exposed to iridoviruses at a higher rate, or tortoises in this area are more resistant to disease, due to genetic or environmental factors. Environmental persistence of iridoviruses is poorly understood, however it appears moisture and temperature may play important roles. Studies with insect iridoviruses found that moisture was associated with increased persistence, while dessication inactivated viruses (Williams et al., 2005). Temperature has also been shown to play an important role in the infectivity of salamanders to iridoviruses, where salamanders exposed at lower temperatures are more susceptible to the virus than those infected at higher temperatures [START_REF] Rojas | Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum[END_REF]. It is possible that tortoises infected with the virus in a warmer climate may be more resistant to infection, allowing them to develop an immune response and survive. However, this would not account for the higher prevalence rate of exposure of tortoises tested in Baker County, Georgia. Direct transmission may play a role in infection rates. In amphibians, activities involving close contact have been shown to be effective means of transmitting Ranavirus A c c e p t e d M a n u s c r i p t 14 (Brunner et al., 2007). Box turtles have been shown to have overlapping home ranges, and mating occurs among turtles with overlapping ranges (Stickel, 1950;Stickel 1978;Stickel 1989).

Interactions between males are similar to courtship behaviors between males and females (Stickel, 1989) and box turtles are often found near other box turtles [START_REF] Dodd | North American Box Turtles: A Natural History[END_REF]. Gopher tortoises of opposite sex often have overlapping home ranges and can commonly occupy the same burrow [START_REF] Diemer | Home range and movements of the tortoise Gopherus polyphemus in Northern Florida[END_REF]. Therefore, it is possible that differences in population densities or overlapping home ranges may play a role in increased levels of exposure.

Earlier studies have shown that amphibians carry identical or very closely related iridoviruses to those found in the chelonians, suggesting they might be a source of infections [START_REF] Johnson | Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States[END_REF]. [START_REF] Green | Epizootiology of sixty-four amphibian morbidity and mortality events in the USA[END_REF] found that increased precipitation and population densities were directly associated with increased die-offs of amphibians due to iridoviruses.

Other conditions that may cause stress and compromise the immune system have also been shown to increase susceptibility to iridoviruses. [START_REF] Forson | Atrazine increases ranavirus susceptibility in the tiger salamander, Abystoma tigrinum[END_REF] found that atrazine and sodium nitrate resulted in decreased peripheral leukocyte levels and increased susceptibility of salamanders to a Ranavirus. [START_REF] Gray | Frog virus 3 prevalence in tadpole populations inhabiting cattle-access and non-access wetlands in Tennessee, USA[END_REF] found that in some species of amphibians, stress resulting from sharing wetlands with cattle resulted in increased susceptibility of tadpoles to iridovirus infection. Similar conditions that cause stress to chelonians might also result in increased levels of susceptibility in chelonians. Additionally, it has been shown that sublethally infected amphibians can cause sporadic, recurrent disease outbreaks in amphibian populations [START_REF] Brunner | Intraspecific reservoirs: complex life history and the persistence of a lethal ranavirus[END_REF]. Experimentally and naturally infected tiger salamander larvae and metamorphs were able to maintain sublethal, transmissible infections for over five months.

Apparently healthy infected dispersing metamorphs were returning to water bodies to breed and it was speculated that these individuals were likely serving as a reservoir host for infecting newly

Conclusion

This study reports the development and use of an indirect ELISA for detection of antiiridovirus antibodies in chelonians. Overall seroprevalence of exposure of free-ranging gopher tortoises to iridoviruses appear to be low, however, this is expected to be an underestimate of the true prevalence. Further studies are needed to characterize the impact of this viral disease on gopher tortoises and other chelonian species. Plasma diluted at 1:100 showed the greatest difference between the positive and negative control.

  surviving 55 turtles were sampled. Plasma samples were stored on ice and transported to the University of Florida for testing.

  infected cells at approximately 125 kDa, and 78 kDa with weaker signals seen at 70, 65, and 28 kDa. Very faint signals were also seen at 125 and 78 kDa in the uninfected cells.

  One positive sample each was collected in 2003 and 2004, two positive samples in 2006 and no samples tested positive from those collected in 2001 and 2002.

  exposure to iridovirus occurred at least one month prior to the date the sample was collected.

  creating recurrent outbreaks of disease. It is unknown whether chelonians are capable of sustaining a subclinical persistent infection. This assay has conservation value since it can be used to identify asymptomatic carriers in wild and captive populations.

  .F. 1950. Populations and home range relationships of the box turtle, Terrapene c. carolina (Linnaeus). Ecol. Monogr. 20, 351-378. Stickle, L.F. 1978. Changes in a box turtle population during three decades. Copeia. 1978, 221-225. Stickel, L.F. 1989. Home range behavior among box turtles (Terrepene c. carolina) of a bottomland forest in Maryland. J. Herpetol. 23, 40-44. Westhouse, R.A., Jacobson, E.R., Harris, R.K., Winter, K.R., Homer, B.L. 1996. Respiratory and pharyngo-esophageal iridovirus infection in a gopher tortoise (Gopherus polyphemus). J. Wildl. Dis. 32, 682-686. Whittington, R.J., Kearns, C., Speare, R. 1997. Detection of antibodies against iridoviruses in the serum of the amphibian Bufo marinus. J. Virol. Meth. 68, 105-108.Williams, T., Barbosa-Solomieu, V., Chinchar, V.G. 2005. A decade of advances in iridovirus research. Adv. Virus Res. 65, 173-248.

  Figure Captions

Fig. 2 .

 2 Fig. 2. Frequency distribution of P/N ratios from an indirect ELISA performed on 1000

Fig. 3 .

 3 Fig. 3. Individual P/N ratio values for 1000 free-ranging gopher tortoises (Gopherus

Fig. 4 .

 4 Fig. 4. Coomassie blue staining of a SDS-PAGE gel separating proteins of iridovirus-

Fig. 5 .

 5 Fig. 5. Western immunoblot. Lanes coated with four fold serial dilutions of iridovirus

  Figure 5

Figure 6

 6 Figure 6

Table 2 .

 2 ELISA results of plasma samples from free-ranging gopher tortoises (Gopherus polyphemus) by state.

	State	Total No. Tested	Total No. Positive	Percent
	Alabama	9	0	0
	Florida	658	8	1.2
	Georgia	225	7	3.1
	Louisiana	12	0	0
	Mississippi	28	0	0

Table 3 .

 3 Anti-iridovirus ELISA results of 1000 free-ranging gopher tortoise (Gopherus 440 polyphemus) plasma samples by county. 441

	State	County	Number tested	Number Positive	Percent
	Alabama	Baldwin	2	0	0
		Mobile	7	0	0
	Florida	Alachua	12	0	0
		Brevard	27	0	0
		Broward	10	1	10
		Citrus	51	0	0
		Clay	1	0	0
		Collier	2	0	0
		Columbia	1	0	0
		Hernando	24	0	0
		Hillsborough	16	0	0
		Indian River	2	0	0
		Lake	99	3	3
		Lee	18	0	0
		Leon	17	0	0
		Madison	1	0	0
		Manatee	6	0	0
		Marion	22	0	0
		Martin	25	1	4
		Miami-Dade	8	0	0
		Nassau	47	0	0
		Orange	40	0	0
		Osceola	11	0	0
		Palm Beach	58	2	3
		Pasco	26	0	0
		Pinellas	2	0	0
		Polk	6	0	0
		Sarasota	1	0	0
		Seminole	54	0	0
		St. Johns	7	0	0
		St. Lucie	35	1	3
		Taylor	1	0	0
		Volusia	26	0	0
		Walton	2	0	0
	Georgia	Baker	113	7	6
		Liberty	74	0	0
		Tattnall	38	0	0

Acknowledgements

This work was funded by grant No. D04Z0-11 from the Morris Animal Foundation and the Batchelor Foundation, College of Veterinary Medicine, University of Florida. The authors would like to thank Dina Demcovitz and Mary Brown from the Mycoplasma Research Laboratory for assistance with gopher tortoise plasma samples and April Childress for laboratory support. We also thank Susan Seibert, Ben Atkinson, Jude Holdsworth, Marci Ertola, Chris Hammond, and Robert Wagner for field support and Dr. J. Stanley and K. Goodblood of the Buttermilk Hill Nature Sanctuary for access to animals in their preserve.