Clean-catching urine from pigs: A method for collecting quality specimens for urinalysis and microbiological culturing in a laboratory environment

Abstract

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Urine is an important biological specimen for assessing various metabolic functions and drug clearance. In urinary tract infection research, urine is particularly important as urinary bacterial titres constitute the main diagnostic outcome for assessing the course of infection. Collecting uncontaminated urine samples from pigs can be done by bladder catheterization or suprapubic bladder aspiration, which are both laborious and invasive procedures that require the need for anaesthesia. To improve animal welfare and optimize urine sampling protocols, we developed a method of clean-catching midstream urine specimens from pigs during spontaneous micturition. The quality of urine specimens collected by clean-catch, bladder catheter and suprapubic bladder aspiration were compared using microbiological culturing. We show that urine specimens collected by clean-catch are only minimally contaminated by skin- and vaginal-commensals with no influence on urinary bacterial titres during ongoing infection. In conclusion, we describe a method in which spontaneous micturition can be prompted in pigs, facilitating fast and reliable collection of quality specimens suitable for microbiological culturing. The method supersedes the need for anaesthesia, which not only represents a considerable refinement in terms of animal welfare but also facilitates more frequent collection of specimens that can enhance the scientific outcome of experimental animal studies in pigs.

Keywords

3Rs dosing/sampling ethics and welfare infectious diseases organisms and models pigs refinement techniques

Introduction

Urinary tract infection (UTI) is one of the most common bacterial infectious diseases worldwide and has been studied extensively in murine models.^1–3 However, mice are intrinsically resistant to UTI and require inoculation with excessive bacterial titres, typically >10⁸ colony forming units (CFU) · mL^–1 for successful infection.² This does not reflect the high susceptibility observed in human patients and, furthermore, uncontaminated urine specimens suitable for microbiological culturing cannot be collected reliably from mice, mainly due to the small bladder capacity of roughly 150 µl.⁴ Pigs, on the other hand, have been highlighted as excellent models of human UTI because pigs (i) are more similar to humans in terms of anatomy and physiology of the kidneys and urinary tract,^5,6 (ii) share considerably more immunological parameters with humans compared with mice⁷ and (iii) are the natural host of human uropathogens and highly susceptible to UTI.^8,9 Last, the size of the porcine bladder, which reflects that of humans, facilitates reliable collection of urine samples to monitor urinary bacterial titres and, thus, the course of disease. However, until now, urine specimens have been collected by either bladder catheterization or sterile bladder aspiration, which are both procedures that require the need for anaesthesia.^9–12 Although well-established protocols for short-term anaesthesia exist, they often involve various combinations of opioids, benzodiazepines and alpha-2-agonists, which are associated with adverse effects such as obstipation, nausea, extended recovery, drug interactions or unwanted alterations in the animal’s physiology that can interfere with the outcome of the study.^13–15 Therefore, the need for anaesthesia is a major limitation as it is not only stressful for the animal and increases procedural costs, but also limits the frequency of which urine specimens can be collected as daily anaesthesia for many days in a row is undesirable due to the concern for the animal’s welfare. To avoid anaesthesia, we explored the approach of clean-catching midstream urine specimens from pigs, a sampling method which is similar to the one used in humans.¹⁶ Although variations of this approach have been used occasionally before, to our knowledge no detailed protocol has been described and the quality of the collected specimens, in terms of microbial contamination, has not been evaluated previously.¹⁷

Our aim was to (a) develop a method to stimulate pigs to spontaneously micturate and (b) compare urine specimens collected by clean-catch, bladder catheter and bladder aspiration, from control pigs and pigs with experimentally induced UTI to evaluate differences in bacterial titres of the infectious agent and potential contaminants.

Animals, materials and methods

Animals and stabling

Urine samples were collected from 41 female pigs (Landrace/Yorkshire, crossbreed) from a vendor with the highest Health Standard according to the Danish Specific Pathogen Free system.¹⁸ The gender and breed were chosen to match our established UTI model.¹⁰ The pigs’ weight was 26.1–85.3 kg. The animals were group housed in standard enclosures with up to 10 animals (3 m²/animal) with sawdust bedding and enrichment with various toys and music. Pigs were acclimatized for at least one week before experiments began and fed a standard diet (Prime Star B, DLG) with free access to water. The animals were attended at least twice daily and monitored for physical activity and food consumption at every inspection and weighed at least weekly. During UTI, we were particularly observant of signs of ascending or bloodstream dissemination of the infection, with the possibility of increasing daily inspections to three. None of the pigs, however, developed complications related to the infection.

All samples used in this study were collected in a paired design in two ongoing studies approved by the Danish Animal Experimentation Inspectorate, licence number 2021-15-0201-00931 and 2021-15-0201-00821, thus, also determining the number of animals available.

During the study, a set of humane endpoints were applied: reduced growth rate (a deviation up to 20% from the anticipated weight was allowed), clinical signs of dehydration (low capillary refill time), clinical signs of disseminated infection, that is, inactivity, pale snout, low body temperature, increased heart rate or abnormal respiration pattern. Three animals were euthanized untimely: one because a via falsa was made during catheterization, another developed lameness, the third developed cardiac arrest during anaesthesia with propofol.

Urinalysis and microbiological culturing

To reduce the total number of animals, multiple urine specimens were collected from each animal at various timepoints during the study. A total of 169 urine specimens were collected from the 41 animals used. Control urine, that is, from pigs without infection, were collected in the weeks and days leading up to the experimental infection. Urine specimens were collected in 15 ml sterile centrifuge tubes (catheter and aspiration specimens) or 100 ml sterile cups (clean-catch) and kept at 5°C (up to 4 h) before plating. Specimens were plated in serial dilutions on 5% blood agar plates (SSI Diagnostica), a universal medium that supports growth of most bacteria to detect potential contaminants of various species, and on selective blue agar (SSI Diagnostica). The latter contains a surface-active detergent that prevents growth of Gram-positive bacteria, hence only Gram-negative bacteria such as Enterobacteriaceae, that is, typical uropathogens, can grow on this type of agar plate. Agar plates were incubated over night at 35°C and CFU were manually counted for quantification. Bacterial species were identified using Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker). Urine pH was measured using a standard pH meter (PHM210, MeterLab) within 24 h or otherwise excluded from the study. Urine density measured by urine specific gravity (USG) is temperature dependent and therefore was measured when the specimens were room temperature using a digital refractometer (Atago).

UTI protocol

The protocol was based on Stærk et al.¹⁰ In short, the uropathogenic Escherichia coli (UPEC) strain UTI89 was preincubated overnight in 40 ml of static LB broth at 37°C. After incubation, 100 µl was transferred to a new tube with 40 ml LB broth and incubated overnight to optimize bacterial expression of type-1 fimbriae. On the day of the infection, the broth was centrifuged at 5000 g for 20 min and the inoculum adjusted to an optical density at 600 nm of 1.0 corresponding to a bacterial concentration of 1 · 10⁹ CFU · ml^–1. From here, the inoculum was prepared by serial dilution in saline and plating on agar for confirmation of final inoculum. The pigs were inoculated within 1 h after preparation. To do so, pigs were sedated (described below) and placed in supine position on the operating bed. The urogenital area was washed and disinfected with two rounds of medical grade iodine solution (7% iodide). Next, the pigs were catheterized with a Chariérre 12 silicone catheter (Coloplast) and then a urine sample was collected (the initial 10 ml was discarded). If only a urine sample was needed, the pigs were then returned to their enclosure for recovery. If the pigs were to become infected, the bladder was completely emptied and 100 ml inoculum with bacterial concentrations of 10² CFU · ml^–1 was instilled through the catheter into the bladder. Hereafter, the catheter was clamped for 1 h and then emptied completely before removing the catheter. Urine samples from pigs with ongoing infection were collected within 1–8 days post inoculation by catheter as described above or by clean-catch. On the last day of the experiment, pigs were sedated and placed in supine position as described above after which the lower abdomen was washed and disinfected. When the surgeon was ready, pigs were euthanized with an intravenous overdose of pentobarbital 140 mg/kg administered via an intravenous access in the ear vein and the abdomen opened by a midline incision to expose the urinary bladder. From the bladder, an aseptic bladder aspiration was performed using a 18G needle and 20 ml syringe.

Anaesthesia

Pigs were premedicated with an intramuscular injection of medetomidine (Cepetor 0.05 mg · kg^–1), butorphanol (Butomidor 0.2 mg · kg^–1) and midazolam (Midazolam 0.2 mg · kg^–1). After placing an intravenous access in the ear vein, propofol was administered intravenously as a constant rate infusion, to maintain a sufficient level of sedation while conserving spontaneous respiration. To prevent hypoxia, oxygen was administered via a face mask and pulse oximetry and respiration pattern monitored continuously. To minimize side-effects from the alpha-2-agonist, medetomidine, an intramuscular injection of atipamezole (Antisedan 0.12 mg · kg⁻¹) was administered as soon as the pig had been administered propofol. During brief sedation where propofol was not needed, that is, when only a catheter-urine was collected, the pigs were returned to their pens before the administration of atipamezole.

Statistics and software

Statistical analysis was performed using GraphPad Prism version 9.3.1. Comparisons between two groups were done using the Mann–Whitney test. Comparisons of bacterial counts were done using ordinary one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test using the logarithm of the values as the values follow a lognormal distribution, determined by a D’Agostino and Pearson test. Comparisons of urine pH were done using one-way ANOVA with Tukey’s multiple comparisons test (normal distributed values). To compare variances, Brown–Forsythe test and Bartlett’s test was used (ANOVA). Comparisons of USG were done using the Kruskal–Wallis test with Dunn’s multiple comparisons test. Figure 2 was created using BioRender.com.

Results

Changing sawdust bedding prompts spontaneous micturition in pigs

To facilitate easy collection of urine specimens from the pigs during spontaneous micturition, we used a long-handled pick-up tool to hold a 100 ml sterile cup (Figure 1). Similar to collecting urine samples from human patients, we collected only midstream urine, that is, urine would not be collected during the initial 2–3 s of micturition to flush out potential contaminants in the urethra. Urine could successfully be collected by this approach when a person was present at the time of micturition; however, it was difficult to predict when the animals would micturate, resulting in long waits. Later, we observed that the pigs would often micturate in the hallway during their daily socialization, habituation and training or when they were returned to their enclosure after it had been cleaned. We used this observation to develop a protocol, summarized in Figure 2, that would prompt the animals to micturate. To do so, all the animals (2–10 individuals at a time) were gently hustled into the training area while monitored by one or two persons ready to collect urine if a pig did micturate (Figure 1). While the pigs were in the training area, the enclosures would be cleaned and the sawdust bedding changed. When the pigs were allowed to re-enter their enclosures, the animals would seek out their designated dunging corners (always the same area at the ends of the enclosure) and micturate, defecate or both (Figure 2, red areas). Immediately before micturition, the pig would always go to the dunging site and express their rooting behaviour, alerting the bystander that it was about to micturate, and then take a fixed stance while micturating (Supplementary material video 1 online). While in their micturition stance, the pigs ‘froze’ and could be approached at a moderate pace to collect the urine with relative ease. The animals could even be pushed lightly if their hind part was too close to the wall, thereby allowing correct placement of the cup.

Figure 1.

In small individuals (less than 40 kg), clean-catch midstream urine was collected using a long-handled pick-up tool holding a 100 ml sterile cup at the tip.

Figure 2.

Pigs were housed in standard enclosures next to a large hall that was used for daily training and socializing (a). The animals would always, without exception, use the two enclosures closest to the door for their dunging site (areas marked by red). In the morning hours, pigs were gently hustled into the training area (b), after which the doors were closed so the sawdust bedding could be changed (c). When pigs were allowed to re-enter their enclosure, they would often immediately go to the red areas and micturate, whereby a researcher could easily collect a urine sample by clean-catch (d). In our other facility where we typically house smaller groups of animals (e), it was sufficient to let the pigs enter an empty enclosure in the other side of the room (f). When working with groups of more than four animals, we recommend having two persons ready for collecting urine, as the pigs often micturate simultaneously. Created with BioRender.com.

In another facility, where we typically house smaller groups of animals, it was sufficient to open the enclosure and then gently hustle the pigs into an empty enclosure not currently in use. Hereafter, the animals would seek out a corner and express their rooting behaviour followed by micturition as described above (Supplementary video 1). By this approach, urine could always be collected from all housed animals within 2 h, most often within 10–30 min.

It was more challenging to collect urine from the small animals of 30 kg or below due to the smaller bladder capacity. Although, the pick-up tool facilitated a longer reach, we eventually found that it was unnecessary, particular when working with larger animals (40 kg and up). Furthermore, when using the pick-up tool it was more likely that the other pigs in the enclosure would interfere with the sampling procedure, that is, by knocking away the pick-up tool or blocking correct placement of the cup (Supplementary video 1). Therefore, most samples were collected by holding the cup by hand. The routine for collecting urine by clean-catch was always performed in the morning hours between 08:00 h and 12:00 h.

Analysis of urine from control animals

We compared urine specimens from uninfected pigs collected by bladder catheter (n = 60), clean-catch (n = 43) and bladder aspiration (n = 7). We found that the average USG was significantly lower in urine specimens collected by bladder catheter compared with clean-catch or bladder aspiration (Figure 3(a)). There was no significant difference in pH (Figure 3(b)). Urine specimens were plated on a universal medium (5% blood agar) to detect potential contaminants, and a selective medium (blue agar) that only supports growth of Gram-negative rods. On blue agar, contaminants were detected in 0% (0 of 60), 40% (17 of 43) and 0% (0 of 7) of specimens collected by bladder catheter, clean-catch and aspiration, respectively (Table 1). Despite the relatively high frequency of detectable contaminants in specimens collected by clean-catch, the detected species were very low in numbers with only one of 43 specimens above 10² CFU · ml^–1 (Figure 3(c)) and, in most specimens, contaminants could not be detected at all (Figure 4(c)). On 5% blood agar, contaminants were detected in 32% (19 of 60), 98% (42 of 43) and 0% (0 of 7) of specimens collected by bladder catheter, clean-catch and aspiration, respectively (Table 1). The contaminants on blood agar from catheter-urine specimens were mostly negligible due to very low numbers, except for one specimen containing 10³ CFU · ml^–1 (Figure 3(c)). MALDI-TOF mass spectrum analysis revealed the latter to contain three different species, supporting that the finding was indeed contamination and not a case of spontaneous UTI. Contaminants on blood agar from specimens collected by clean-catch were also generally low in numbers (49% of samples had 10¹ CFU · ml^–1), but 12% (5 of 40) had titres of 10³ CFU · ml^–1.

Figure 3.

Urinalysis from control pigs. Urine collected by bladder catheter, clean-catch or bladder aspiration from control pigs (non-infected) was compared. The urine specific gravity (USG) of urine specimens collected by bladder catheter was significantly lower compared with samples collected by clean-catch (a). There was no significant difference in pH (b). Microbiological culturing of urine collected by clean-catch revealed only minor growth on selective blue agar plates, but little to moderate growth on 5% blood agar (c). Data points at 0 represent values below the limit of detection (10 CFU∙ml^–1). Horizontal bars represent means. Kruskal–Wallis test with Dunn’s multiple comparisons test.

Table 1.

Frequency and bacterial titres of contaminants.

Collection method		Contaminated samples, n (%)		Urinary titres of detected contaminants, n (%)
Collection method		Contaminated samples, n (%)		10¹		10²		10³		10⁴ or above
Control	n	Blue	5% blood	Blue	5% blood	Blue	5% blood	Blue	5% blood	Blue	5% blood
Bladder catheter	60	0 (0)	19 (32)	0 (0)	13 (22)	0 (0)	5 (8)	0 (0)	1 (2)	0 (0)	0 (0)
Clean-catch	43	17 (40)	42(98)	16 (37)	21 (49)	1 (2)	14 (33)	0 (0)	5 (12)	0(0)	0 (0)
Bladder aspiration	7	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Infection
Bladder catheter	19	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
Clean-catch	23	0 (0)	1 (4)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (4)
Bladder aspiration	17	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)

Figure 4.

Urine specimens, collected by clean-catch from uninfected pigs, typically showed no growth on blue plates (a) but little or moderate growth on 5% blood agar (b). Urine specimens collected by clean-catch from pigs with ongoing experimentally induced Escherichia coli urinary tract infection showed high numbers of E. coli when plated in 10-fold serial dilutions ((c) and (d)). Consequently, contaminants were negligible or most often undetected on 5% blood agar (c) and blue plates (d) and, hence, quantification of the infectious E. coli was not influenced by contamination.

The most common contaminants found were vaginal colonizers, particularly Actinobacillus rossii, and commensals from the skin such as coagulase-negative staphylococci (Supplementary Table S1). Typical uropathogens of the Enterobacteriaceae family such as E. coli, Klebsiella spp. and Proteus spp., as well as the common pig-specific uropathogen Streptococcus suis, were only rarely detected and always in very few numbers in urine specimens collected by clean-catch or by catheter (Supplementary Table S1).

Analysis of urine from pigs with experimental UTI

To evaluate the influence of potential contaminants on urinary bacterial titres of infected pigs, we compared urine specimens collected by bladder catheter (n = 19), clean-catch (n = 23) and bladder aspiration (n = 17) from pigs with an ongoing UTI that was experimentally induced by transurethral inoculation with UPEC. The results are summarized in Figure 5. Similar to non-infected pigs, the average USG was lower in urine specimens collected in sedated pigs through bladder catheter compared with clean-catch, but only nearly statistically significant (p = 0.08) (Figure 5(a)) and pH was unaffected (Figure 5(b)). Interestingly, the average USG of urine specimens collected by clean-catch from infected pigs (1.016) was significantly lower (p = 0.048, Mann–Whitney test) than urine specimens collected by clean-catch from uninfected pigs (1.021). Microbiological culturing showed no significant difference in urinary bacterial titres of the infectious UPEC between the three methods of urine-collection (Figure 5(c)). Furthermore, contaminants were detected in only one specimen (from clean-catch) (Table 1).

Figure 5.

Urinalysis from pigs with urinary tract infection. Urine collected by bladder catheter, clean-catch or sterile bladder aspiration from pigs infected with uropathogenic Escherichia coli were compared. The average urine specific gravity of urine specimens collected by bladder catheter tended to be lower compared with samples collected by clean-catch (p = 0.08); Kruskal–Wallis test with Dunn’s multiple comparisons test (a). There was no significant difference in pH (b). Microbiological culturing of urine revealed no significant difference in bacterial titres between the three collection methods on either selective blue agar or 5% blood agar (c). Horizontal bars represent means ((a) and (b)) or geometric means (c). The limit of detection was 50 CFU∙ml^–1.

Discussion

Pigs are important research animals in many fields of biomedical research because they recapitulate many aspects of human disease.^19,20 In studies of UTI, pigs have distinct advantages, compared with small animal models, because pigs are more similar to humans in terms of anatomy, physiology and UTI susceptibility and their size not only supports investigation of human-relevant medical devices, such as bladder catheters, but also facilitates collection of large quantities of biological material, particularly urine.^7,21,22 The latter, however, requires sedation of the animal if collected by transurethral bladder catheterization or aseptic bladder aspiration. In the current study we describe an alternative approach, where urine specimens can be collected quickly and reliably by clean-catch, without significantly compromising sample quality.

Urine specimens collected by clean-catch and plated on selective blue agar showed insignificant contamination, with only one of 40 specimens containing CFU above 10² · ml^–1. The selective blue agar plate does not permit growth of Gram-positive bacteria, including many commensals of the skin and vaginal flora, and thereby excludes potential contaminants. However, the most common uropathogens, particularly UPEC, accounting for up to 80% of human UTIs, can grow on this plate and so can other common uropathogens of the Enterobacteriaceae family.²³ As a result of the high incidence of UPEC-associated UTIs, UPEC is the predominant model species used in experimental UTI studies and, to our knowledge, the only uropathogen species used in porcine UTI studies.^9,10,21,24 Therefore, in studies of UPEC or other members of the Enterobacteriaceae family, using selective blue agar is an excellent approach to minimize contamination when clean-catching urine specimens. When urine specimens from clean-catch were plated on a universal medium, 5% blood agar, bacterial contaminants were frequently detected (98% of samples) and occasionally appeared in relatively high numbers (13% of samples at 10³ CFU · ml^–1), suggesting that universal growth media can be a challenge if working with Gram-positive uropathogenic model organisms, such as Enterococcus faecalis or Staphylococcus saprophyticus, that is, bacterial species that are not culturable on selective blue agar. However, specific selective media can be made, preferably with antibiotics, to favour growth of such pathogen species while suppressing others. Furthermore, during ongoing infection, the mean urinary bacterial titres of the infectious agent, in this case UPEC, were over 1000-fold higher compared with the mean urinary bacterial titres of control animals, that is, contaminants, and indeed, contaminants were only detected once (1 of 23) in specimens collected by clean-catch from infected pigs. The absence of detectable contaminants in clean-catch specimens from infected pigs is likely a result of microbial antagonism in which the high numbers of UPEC suppress the growth of contaminants that are present only in much lower titres. Also, urinary bacterial titres of infected pigs were no different in urine specimens collected by clean-catch compared with bladder catheter or -aspiration, further supporting that clean-catching urine specimens is a viable approach for monitoring UTI in pigs.

Interestingly, we found that USG was significantly lower in urine specimens collected by bladder catheter compared with clean-catch. This is most likely a result of the pre-medication used to sedate the animals, which includes medetomidine, an α2-adrenergic agonist with the side-effect of inducing diuresis. Consequently, urine specimens collected by clean-catch are likely to be more accurate regarding the natural urine density. Interestingly, when comparing the average USG of urine specimens collected by clean-catch from infected pigs and uninfected pigs we found a significant reduction similar to our previous observations.¹⁰ This supports our previous hypothesis, that UTI elicits a diuretic response, perhaps as a physiological response to clear the infection through increased flushing.¹⁰ The diuretic properties of medetomidine are likely to also influence bacterial titres, and we observed a tendency of fewer CFU in catheter urines (geometric mean: 2.2 · 10⁵ CFU · ml^–1) compared with specimens collected by clean-catch (geometric mean: 6.1 · 10⁵ CFU · ml^–1); however, the difference was not statistically significant, which most likely is a result of the natural wide range in CFU of infected pigs, ranging from 10³ to almost 10⁸ CFU · ml^–1, that is, almost a 10,000-fold range.

Using the protocol presented here, we exploit that pigs are naturally very hygienic and always designate discrete dunging sites away from their sleeping and feeding areas.^25–27 This behaviour is known even from very young piglets that will leave their nest to micturate and defecate.²⁸ We observed that pigs which were gently hustled out of their enclosure would very quickly, within minutes, explore a site in an empty enclosure for immediate micturition and/or defecation. This behaviour may arise from an inability to designate dunging sites that are appropriately distanced according to the animals’ natural hygienic preferences, when the animals are housed in relatively confined conditions, as they are when kept in pens.²⁷ It is intriguing to speculate that the pigs, when hustled out of their enclosure, see the opportunity to dung at a site more distant from the enclosures where they are housed, perhaps as a way of maintaining the tidiness and hygienic environment in their established nesting areas.

Rydén et al. have previously used a two-week training protocol to facilitate various examinations of pigs including monitoring urine-creatinine concentrations in samples collected by free-flow.¹⁷ In our study, no particular nursing or training of the animals was used prior to urine collection, suggesting that the approach can be easily adapted by other facilities. Furthermore, the protocol of clean-catching urine was successful in two different housing facilities, suggesting that the method is likely to be adaptable to the facilities of other research groups.

We were able to collect urine from all animals within 2 h, most often in much less time (Supplementary video 1). This should be compared with the time and cost of collecting urine by bladder catheter or aspiration from each animal individually, which requires anaesthesia and thus veterinarian assistance. Particularly when working with large groups (up to 10 animals), the average time spent for each clean-catch sample was low. Using bladder catheter for collecting urine also entails the potential risk of causing harm to the urethra, as the procedure can be troublesome, or accidentally introducing contaminants directly into the bladder, which could interfere with the study, in particular in studies of UTI.^29,30 Likewise, bladder aspiration entails the risk of perforating the intestine if not performed carefully with ultrasound-guidance and preferably with a certain volume inside the bladder. The main limitation to clean-catching urine specimens is that the urine cannot be collected at a very distinct timepoint, which may be essential in particular studies. Otherwise, the approach can be adapted for most other areas where frequent urine samples are needed such as renal transplantation studies or diabetes mellitus. We collected urine specimens up to once per day but did not investigate how many times during the day a urine sample can be collected by this approach. Another limitation may be related to the size of the animals: we found that urine could be collected from all our pigs ranging in size from 26.9 kg to 85.3 kg, but collection from the smallest individuals was considerably more difficult and also yielded smaller collected volumes, mainly because the period of micturition, that is, window of opportunity, was shorter. Hence, the approach of clean-catching urine in miniature breeds, which are often used in biomedical research, may be more difficult, but needs further investigation. This is supported in a study by White et al. in which the attempt of clean-catching urine specimens in Göttingen mini-pigs was unsuccessful.³¹ In terms of differentiating infectious cystitis from non-infected animals using urine cultures from clean-catch specimens, our experimental design has one weakness in the fact that we did not involve a true placebo group consisting of pigs mock-infected with saline. Therefore, the potential influence of the experimental inoculation on the course on infection is difficult to assess; however, in a previous study we showed that inoculation with a non-pathogenic Pseudomonas aeruginosa using the same protocol did not result in any detectable bacteria 24 h after inoculation, suggesting that the experimental infection procedure does not lead to bladder colonization.⁹ Since the main objective of this study was to develop a protocol for clean-catching urine specimens and to evaluate the quality in terms of contamination, we find it justified to use control specimens from the same animals before they are subjected to experimental inoculation. This also reduces the number of animals needed.

In conclusion, we present an easy method that allows for clean-catching urine specimens during spontaneous micturition with minimal contamination. The approach supersedes the need for using invasive procedures, such as bladder catheterization or aspiration, which requires anaesthesia of the animals and consequently increases animal stress, costs and time.

Supplemental Material

sj-pdf-1-lan-10.1177_00236772221133433 - Supplemental material for Clean-catching urine from pigs: A method for collecting quality specimens for urinalysis and microbiological culturing in a laboratory environment

Supplemental material, sj-pdf-1-lan-10.1177_00236772221133433 for Clean-catching urine from pigs: A method for collecting quality specimens for urinalysis and microbiological culturing in a laboratory environment by Kristian Stærk, Louise Langhorn, Thomas E Andersen in Laboratory Animals

Footnotes

Acknowledgements

The authors would like to thank Julie May Jensen and the animal technicians at the biomedical laboratory for their help in collecting urine samples.

Data availability

Primary datasets and protocols on which the result of this study is based will be made promptly available to readers without undue qualification.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Postdoc funds from the University of Southern Denmark and a research project commissioned by GlyProVac A/S.

ORCID iDs

Kristian Stærk

Louise Langhorn

Thomas Emil Andersen

Supplemental material

Supplemental material for this article is available online.

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