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Non‐lethal sampling for the detection of Renibacterium salmoninarum by qPCR for diagnosis of bacterial kidney disease

Abstract Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill‐/cloacal swabs collected in vivo for real‐time PCR (qPCRgc), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%–100%) compared to 98.3% (ci 92.7%–100%) and 48.8% (ci 38.8%–58.8%) of kidney samples for qPCR (qPCRk) and ELISA (ELISAk) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.

Transmission occurs both horizontally, through direct contact and through the water (Mitchum & Sherman, 1981), and vertically from infected females to their offspring (Evelyn et al., 1986). There are no commercial vaccines or cures available. The possibility of control depends on an active surveillance of the causative agent, the Grampositive Renibacterium salmoninarum (Rs), combined with transport restrictions and slaughter of fish on infected farms. Destruction of eggs from infected females is used to prevent vertical transmission in restocking broodstock farms. Immunological or molecular diagnostic techniques are commonly used for routine diagnostics and in screening programs (Chambers et al., 2008;Kristmundsson et al., 2016;Suzuki et al., 2017) as Rs is slow-growing and has special medium requirements that hampers in vitro cultivation.
BKD occurs in salmonid wild and farmed populations in America, Asia and in most countries of Europe, in fresh as well as in marine waters. Australia and New Zealand belongs to the few countries where BKD has not been encountered. Epizootics with high mortalities in wild Chinook salmon (Oncorhynchus tshawytscha) and brook trout (Salvelinus fontinalis) have been reported from wild stocks in America (Eissa et al., 2007;Holey et al., 1998) and clinical disease observed occasionally in wild salmonid stocks collected for artificial fertilization and breeding in Iceland (Guðmundsdóttir et al., 2017) Wild whitefish (Coregonus sp.) and grayling (Thymallus thymallus), sampled in a Swedish river where Rs infected fish farms are located, have recently tested positive for Rs (Persson et al., 2022). Elliott et al. (1995) demonstrated impaired health and higher mortality in progeny of Rs positive females, compared to progeny of females with low Rs levels, an effect observed up to 21 months after hatching. Rs infection was found to be associated with reduced growth of juvenile Chinook salmon and this was suggested to affect the regulation of these populations in the Northeast Pacific Ocean as growth during the first year at sea has been linked to survival (Sandell et al., 2015). Thus, ethical issues in the context of fish welfare in aquaculture and the success of breeding programs advocates that the disease must be monitored and combatted.
BKD was first diagnosed in Sweden in 1985, probably introduced by import of whole, frozen fish for processing before human consumption and/or of eggs for fish production. During 1985-2003 or Rs infection without clinical signs was diagnosed in 70 fish farms, several belonging to the same company. The mode of transmission was investigated, and sanitation plans prepared for each case. Transport of live fish between farms was found to be the main factor for spread of the disease. There was no evidence that the infection originated from feral fish (Wichardt, 2004). After an initially high incidence with up to 26 annual index cases, the number of cases has declined to none or up to a few annual index cases (Figure 1). In the last ten years, cases have mostly occurred as re-infections in farms recently diagnosed and sanitized and the goal of eradication has not been reached yet. One reason can be that infected cage farms have been allowed to keep the fish for one to a few years until slaughter weight is reached, and the infection has become manifested in wild fish in the area. It is also possible that low levels of Rs have been maintained in brood stock, resulting in occasional transmissions of the infection to the progeny.
To protect wild salmonids, screening of Rs was initiated in the early 1990´s. The screening is based on samples from 30 fish from each sampled farm, according to the Swedish regulation on mandatory health control of farmed fish (SJVFS 1994:94 Initially, Swedish Rs screening was based on necropsies and bacterial cultivation from kidney on selective kidney disease medium (SKDM) agar (Austin et al., 1983;Benedictsdóttir et al., 1991). Since 1994, kidney samples are tested by a polyclonal ELISA detecting the p57 protein from Rs (Jansson et al., 1996) and, since 2008, positive results are confirmed by qPCR, identifying the 16S rRNA gene of Rs (Jansson et al., 2008). In addition to the mandatory health control program, kidney samples for ELISA are collected after stripping of roe in all wild brood stock females used for artificial fertilization in the national restocking program for salmonids. In small local sea trout or brown trout strains, each female is extremely genetically valuable.
Thus, to allow repeated spawning, ovarian fluid is collected for ELISA although the analytic sensitivity is lower for ovarian fluid than for kidney tissue (Arnason et al., 2013;Pascho et al., 1991). Eggs from ELISA positive females are destroyed. Brood stock of farmed fish is, with a F I G U R E 1 Annual index cases of Rs/ BKD (left y-axis) and the number of fish tested for Rs (right y-axis) during 1985-2020. Diagnostics were performed by cultivation on SKDM-agar 1985-1993 and by polyclonal ELISA 1994-2020 Annual index cases of Rs/BKD No of fish tested for Rs/BKD Annual index cases few exceptions, not sampled at stripping. A sensitive non-lethal sampling strategy would reduce the number of fish that must be killed for sampling. In addition, it offers a motivation for sampling of farmed brood stock, provided that the method can be performed on anesthetized fish and that the sensitivity is at the same level as examination of the kidney, preferably better. For terrestrial animals, blood sampling for antibody detection is a well-established technique. Detection of Rs antibodies is possible, but due to individual variation in immunological response, it is not suitable for tracing individual Rs infected fish (Jansson & Ljungberg, 1998). Blood, mucus and urine-faecal samples have been used to identify Rs infected fish (Bruno et al., 2007;Elliott et al., 2015;Richards et al., 2017;Riepe et al., 2021).

| Experimental challenges
Rainbow trout were obtained from a fish farm included in the mandatory health control program since the 1980´s and with no history of BKD. After arrival, the fish were held in 250 L tanks, supplied with continuous flow through aerated tap water at a temperature of 11 ± 1°C and with intervals of equal night (dark) and day (light) periods. There was an acclimatization period of at least two weeks before the start of experiments. Rs (Rs CIP 10,778; SVA4/86, initially isolated from a rainbow trout with clinical BKD) was propagated in Peptone broth (1% peptone, 1% yeast extract and 0.1% L-cysteine-HCl; Daly & Stevenson, 1993) and incubated with mild agitation at 18 ± 1°C for 12 days. Bacteria were harvested by centrifugation for 20 min at 3000g and resuspended in water for the challenge. Fish were challenged by immersion in a tank with oxygenated water and thereafter distributed, using a net, into two 100 L tanks, containing water with a temperature of 15-17°C and a waterflow of 300 ml/min.
The fish was inspected and fed daily with 25-50 g/day of commercial feed (EFICO, Enviro 4,5 mm 11.3284 Biomar), except the day before sampling to avoid faecal contamination of the anaesthetic bath.
Tanks were regularly siphoned to remove feed residues and faeces.
Experiment 1: Rainbow trout (n = 63; average weight 326 gram) were immersed for 20 min in water containing 1 × 10 7 CFU of Rs/ ml water, determined by viable count. Fish in the control group were treated in the same way except that no bacteria were added to the water. All fish were killed week 6 or 7 post-immersion (p.i.) for sampling of blood, gills, cloaca and kidney.
Experiment 2: Rainbow trout (n = 21, average weight 840 g) were individually tagged by insertion of a Passive Integrated Transponder (PIT) tag (AVID Microchip I.D. Systems) in the peritoneal cavity three weeks prior to the experiment. In this experiment, the fish was immersed for 20 min in water containing 5 × 10 7 CFU of Rs/ml water.
Non-lethal sampling of gills and cloaca was done on 10-13 fish every other week for five weeks p.i.
Four fish were killed during the experiment (3 and 4 weeks p.i.) and four fish died day 36 p.i. in connection with a technical aeration failure. These fish and remaining fish killed week 7 p.i. were all tested by sampling of gills, cloaca and kidney.

| Sampling procedures
The fish was anesthetized with buffered tricaine methane-sulphonate (MS222; 75 mg/L, Pharmaq) prior to sampling. When both non-lethal and lethal sampling was performed, fish was killed by a sharp blow to the head. In Experiment 2, where repeated non-lethal sampling was performed, the fish was allowed to recover in fresh running water under observation after anaesthesia and sampling. For the non-lethal sampling, a cotton swab was drawn over the gill lamellae and another swab brought into the cloaca (2-10 mm depending on the size of the fish) of each fish. Initially, swabs from gills and cloaca were individually analysed by qPCR. In fish from Farm D (n = 65) and from Experiment 2 (n = 54), gill-and cloacal swabs were collected both separately and was set to OD 0.1 based on the mean OD value +3SD of samples collected from negative fish and after correction against the negative control absorbance value (Jansson et al., 1996).
Gill and cloacal samples were supplemented with Limonene, before mixture by vortex, autoclaving and used in ELISA as above.

| qPCR
A fragment of the 16S rRNA gene of Rs was detected from tissue samples by real-time qPCR (Jansson et al., 2008.) In short, swabs from sampled tissue (gills, cloaca or kidney) were incubated in lysis buffer consisting of 540 µl G2 buffer and 60 µl; proteinase K; (QiaGene Hilden) for 30 min at 56°C with 600 rpm agitation using a Thermomixer Comfort (Eppendorf, Hamburg, Germany). The samples were allowed to cool to room temperature before addition of 23 µl lysozyme,100 mg/ml (Roche, Basel, Schweiz followed by 2 h incubation at 37°C with 300 rpm agitation. DNA was extracted from 200 µl lysate with the EZ1 Biorobot and EZ1 DNA Tissue kit (QiaGene) according to the manufacturer's instructions. DNA was also extracted from 10 µl EDTA blood using the same robot and extraction kit. The forward primer (5'TGGATACGACCTATCACCGCAT-3') and reverse primer

| Statistical analysis
The degree of association between Ct values in qPCR gc and qPCR k or optical density (OD) values in ELISA k was estimated by calculation of the Pearson correlation coefficient in Excel (Version 2101).
We between qPCR k and qPCR gc (which shared the same method) and between qPCR k and ELISA k (which shared the same matrix). Weakly informative prior information was used for sensitivity and specificity of ELISA k as well as for prevalence in farms free from disease (Table S1)

| Field sampling
Farms A-C, fattening Arctic char or rainbow trout, had ongoing clinical outbreaks of BKD at sampling. Forty percent of sampled Arctic char from Farm A and approximately 15% of sampled rainbow trout and Arctic char in Farms B and C had one or more of the following disease signs at necropsy: swollen kidney, granuloma formation in internal organs, ascites, splenomegaly, liver petechiae and pale gills.
In fish from Farm D, fattening rainbow trout and Arctic char, necropsies showed no fish with advanced lesions of BKD. In 23% of the fish, one or more of the following disease signs were noted: fibrous adhesions on internal organs, thickening of the swim-bladder wall, slightly enlarged kidney or spleen, discoloration or liver petechiae No indications of BKD were recorded at necropsy in farms classified as free from the infection (Farm E-F).
The results per farm and analytical method are summarized in and 85% (89 out of 105) of the samples were positive for Rs by ELISA k and qPCR k , respectively. In farm A, gills and cloacal samples were analyzed separately and 19 of 35 (54%) fish, sampled for qPCR g and qPCR c were positive by both analyses, whereas another six fish were positive by either qPCR g (4 fish) or qPCR c (2 fish). Non-lethal sampling for qPCR gc in Farm B and C identified 100% (50 + 20 samples) as positive for Rs but only 30% (12 + 6 samples) were positive for Rs by qPCR b . In Farm D, 0% and 3% (0 and 2 samples, respectively) were Rs positive by ELISA k and qPCR k , respectively, whereas 45% (29 out of 65 samples) were Rs positive by qPCR gc . All qPCR b samples from Farm D were negative.
All samples collected from fish farms without suspicion of BKD (Farm E-F), tested negative by qPCR gc and ELISA k .

| Experimentally Rs exposed rainbow trout
In both experiments, there were few signs of disease at necropsy.  (Table 3). All ELISA gc samples were negative.

| Comparison of diagnostic strategies
A summary of the results of detection of Rs in samples collected from kidney (ELISA k and qPCR k ) and from blood (qPCR b ), gills and/ or cloaca (qPCR g , qPCR c and qPCR gc ) in samples collected from fish farms previously diagnosed as positive for BKD (Farm A-D) and from experimentally exposed to Rs in Experiment 1 and 2, are shown in and Negative (OD<0.1). In fish classified as having a high Rs infection level, all analyses of qPCR k and qPCR gc were positive. In fish with a low infection level, 24% were positive by qPCR k . Only seven fish in this category were tested by qPCR gc but six of these were positive.
Individual qPCR g and qPCR c were positive in 18 and 20% fish in this category respectively. In fish tested negative by ELISA k , 29% tested positive by qPCR k and 60% tested positive by qPCR gc ( Table 4).
Pooling of gill-and cloacal swabs before extraction for qPCR gc was done in Farm B-D and Experiment 2. In fish from these four groups, lethal sampling for Rs detection by ELISA k and qPCR k identi-

| Estimation of the sensitivity and specificity of diagnostic strategies
The posterior estimates of sensitivity and specificity of the three testing strategies alongside with the prevalence in the six populations are reported in Control group (n = 10) Negative (n = 10) 0 (10) 0 (10) 0 (10) Note: Results from polyclonal ELISA of kidney (ELISA k ) tissue compared to results from qPCR of the kidney (qPCR k ), gills (qPCR g ) or cloaca qPCR c , collected by separate swabs at the end of the experiment, weeks 6 or 7 post immersion.

| DISCUSS ION
BKD impairs the success of programs for restocking of salmonids in natural waters and cannot be accepted for a satisfactory fish welfare in aquaculture. Although an eradication program has been in practise for more than 30 years in Sweden, the infection is still diag-

Non-lethal sampling Lethal sampling
Week p. F I G U R E 2 Rs diagnostics by polyclonal ELISA and by qPCR in samples of kidney (ELISA k , qPCR k ) blood (qPCR b ), gill (qPCR g ), cloaca (qPCR c ) and gill-/cloacal swabs (qPCR gc ). Samples were collected from farms with clinical BKD (Farm A-C, n = 20 rainbow trout and n = 85 Arctic char) and from fish with diffuse signs of BKD (Farm D n = 59 rainbow trout n = 59 and n = 6 Arctic char; Experiment 1 and 2 Rs n = 84 rainbow trout) In the present study, we showed by use of BLCA, that qPCR gc is a reliable non-lethal alternative for diagnosis of BKD compared to ELISA k and qPCR k . The sensitivity of qPCR gc was not different from the sensitivity of qPCR k but significantly higher than the sensitivity of ELISA k ( Table 5). The specificity of qPCR gc was however significantly lower than the specificity for the other two testing strategies. Consequently, a confirmatory test with ELISA and qPCR on kidney tissue is recommended if positive findings on a farm earlier declared free from Rs, as a positive diagnosis will have significant impact on the farm's activities.  (Faisal & Eissa, 2009;Nance et al., 2010). In farms B and C, there was a disagreement between ELISA k and qPCR k results in that 44% and 89% were positive, respectively. Sampling was performed in the beginning of the summer period during increasing water temperatures. A sudden rise of the water temperature is stressful for fish and the obvious higher percentages of positives identified by qPCRk compared with ELISAk, might reflect a rapid multiplication of Rs in the kidney, whereas measurable p57 levels are produced more slowly.
The significantly higher numbers of positives by ELISA k compared to qPCR k in Experiment 1, might be due a recovery from infection,

TA B L E 4
Results of polyclonal ELISA of kidney tissue compared to results from qPCR of gills (qPCR g ), cloaca (qPCRc), gill-/cloacal swabs (qPCR gc ) and kidney Note: The infection level, based on the optical density (OD) values in ELISA, were divided in three categories: negative (OD <0.1), low infection level (OD 0.1-0.3) and high infection level (OD>0.3). Samples were from BKD infected Farms A-D and from challenged rainbow trout in Experiments 1 and 2. a In the first sampling occasions (at Farm A and in Experiment 1) gills (qPCR g ) and cloaca samples(qPCRc) were tested separately. During the study, we found that analysing gill-/cloacal swabs by qPCR (qPCR gc ) increased the number of positive individuals. Detection in gills and cloaca do not necessarily mean that the fish suffer from generally spread BKD in internal organs. Gill epithelium is the primary route of entry for several fish pathogens, and the capacity of Rs for intracellular survival indicates that this is also the case for Rs (Evelyn, 1996). Both gills and cloaca represent tissues for excretion of wastes. Mucus is a complex viscous adherent secretion that represents an interface between the environment and the interior milieu of the fish, that encounter pathogens (Benhamed et al.. 2014). Mucous is predominantly produced on the surface of the skin, gills and the gut lining. In the present study, gill and cloaca samples were collected with cotton swabs on the tissue surface.

F I G U R E 3 (a) Association between qPCR Ct values of gill/cloacal
Consequently, the samples most certainly contain epithelial cells and mucus. Elliott et al. (2015) found scrapings of skin surface mucus for qPCR as the most promising candidate for non-lethal sampling, followed by gill filament and pelvic fin clips in experimentally Rs challenged Chinook salmon. The sensitivity of qPCR on mucus was 92% compared with 72% on gill tissue. Skin-mucus and kidney Ct levels in qPCR were correlated. This is in agreement with the results from Riepe et al., (2001), who found that, when comparing PCR on mucus, anal and buccal swabs to a kidney antibody test, mucus testing was to be preferred. Arnason et al., (2013) got poor results of a seminested PCR on gill tissue during an escalating outbreak of BKD in Atlantic salmon broodfish. In comparison, external swabs of mucus samples were efficient for detection of koi herpes virus in carp, while biopsies from gills and kidney were negative, using the same PCR assays (Monaghan et al., 2015). Urine-faecal samples for nested PCR were suggested to have the greatest potential for non-lethal Rs sampling by Richards et al., (2017), based on samplings of experimentally challenged Chinook salmon. That study demonstrated that the exposure route (intraperitoneal or immersion) and the severity of disease reflected the possibility to identify Rs infected fish by both lethal and non-lethal sampling. All urine-faecal samples tested positive up to three weeks after injection, then the number of positive Our study demonstrates a limited usefulness of blood for sensitive detection of Rs. Thirty percent of blood samples, collected from Farms B and C with high prevalence of Rs, tested qPCR positive, compared with 85% of the samples for qPCR k and 100% of the samples for qPCR gc . All blood samples collected from Farm D, with a low Rs prevalence, were negative. This is in line with the results of Elliott et al., (2015) and Richards et al., (2017), where blood was not recommended for Rs sampling due to the low sensitivity. Richards et al., (2017) found that nearly 10% of challenged Chinook salmon, positive by non-lethal sampling, were negative by lethal sampling of the kidneys. This supports our findings with qP-CR gc positives when qPCR k and ELISA k were negative, and that the different methods are useful at different timepoints of infection. A sensitive diagnostic tool is imperative to defeat the disease in an eradication program.

| CON CLUS IONS
Non-lethal sampling using qPCR gc allow a sensitive detection of Rs. This sampling strategy makes it possible to trace individual Rs carriers in control programs as well as to remove positive farmed brood stock females to avoid vertical disease transfer. In addition, the non-lethal sampling can be used to spare brood stock of vulnerable wild salmonid strains in re-stocking programs, as fish can be released after stripping. Using the qPCR gc strategy instead of ELISA k will reduce the risk of false negative Rs individuals d to the price of an increased probability of false positive diagnosis. Since the goal of the BKD screening is eradication, the higher sensitivity of qPCR gc compared with ELISA k is preferrable. A lower specificity of qPCR gc compared with ELISA k , suggests that positive qPCR gc diagnoses on previously Rs negative farms still must be verified by kidney sampling in line with the present program. This is to ensure that the farmer is not subjected to restrictions due to a false positive diagnosis.

ACK N OWLED G EM ENTS
Our sincerely posthumous thanks to Ulf-Peter Wichardt, Fiskhälsan FH AB, for compiling historical data on sampling and diagnostics of BKD. Thanks to former Acting state veterinarian Anders Hellström for the enthusiasm and encouragement during the initiation of this project, to Eva Blomkvist, Leif Tenfält, Helena Törnelius and Astrid Ahlberg for field samplings and to Eva Säker for skilful ELISA tests.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

PATI E NT CO N S E NT S TATE M E NT
Not applicable.

PE R M I SS I O N TO R E PRO D U CE M ATE R I A L FRO M OTH E R S O U RCE S
Not applicable.

CLI N I C A L TR I A L R EG I S TR ATI O N
Ethical permit C72/13, Swedish Board of Agriculture.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.