Brachyspira hyodysenteriae detection in the large intestine of slaughtered pigs

Abstract

Detection of subclinical Brachyspira hyodysenteriae infection in pig herds using feces is challenging. However, the ability to detect the pathogen in intestinal samples of slaughtered pigs has not been investigated, to our knowledge. Therefore, we determined the detection of B. hyodysenteriae in the colon, cecum, and rectum from slaughtered pigs. We analyzed the correlation between detection rates and intestinal lesions, ingesta or fecal consistency, and time from sample collection until processing. A total of 400 ingesta-mucosal (colon, cecum) and 200 fecal (rectum) samples from 200 pigs originating from 20 different herds were bacteriologically examined using selective culture followed by Brachyspira spp. identification by PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ingesta or fecal consistency and intestinal lesions were scored. Brachyspira hyodysenteriae was detected in 23 samples from 16 intestines originating from 7 herds. Brachyspira spp. were detected in 96 samples. More intestinal (16) than fecal (7) samples tested positive for B. hyodysenteriae. For Brachyspira spp., this difference was significant (69 vs. 27; p < 0.01). In particular, colon samples tested positive (n = 42, p = 0.06). Most (91%) of the intestines showed no lesions typical for clinical B. hyodysenteriae infection, and median ingesta or fecal consistency was “soft and formed,” indicating subclinical infection, colonization, or absence of infection. Ingesta from slaughtered pigs, in particular from the colon and sites with lesions, is useful material for detection of B. hyodysenteriae.

Introduction

Swine dysentery (SD) is a severe mucohemorrhagic typhlocolitis with diarrhea caused by the spirochete Brachyspira hyodysenteriae^1,12 and, according to the literature, also by B. hampsonii.^6,24,28 Brachyspira hyodysenteriae is present worldwide.¹² Efficient detection of B. hyodysenteriae is difficult, particularly in subclinically infected herds. Culture followed by identification (e.g., with biochemical tests) has been considered the gold standard detection method,^12,22 but significantly faster, direct identification systems such as direct species-specific PCR have acquired importance.¹² Furthermore, other identification methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), have been developed and are increasingly used.^3,5,27 MALDI-TOF is a valuable microbiologic analysis tool because it can quickly, precisely, and inexpensively identify bacterial isolates.^3,25 Furthermore, mass spectral profiles (MSPs) can be saved to create a user-defined library; such user-defined libraries including Brachyspira spp. have been created and used effectively.^5,27 In general, both culture followed by identification and direct PCR can detect Brachyspira spp. in rectal fecal samples from diseased pigs. However, detection of Brachyspira spp. sometimes fails when using feces from healthy pigs because shedding only occurs sporadically and involves only low numbers of the spirochetes.^10,13 Hence, it is necessary to examine large numbers of fecal samples to increase the chance of detection, which is laborious and expensive.

Another approach to ensure efficient detection is the use of samples from optimal sample sites. In the case of B. hyodysenteriae, the large intestine is thought to be the optimal sampling site.¹³ In a study that analyzed both lesion severity and distribution in pigs experimentally infected with B. hyodysenteriae, the SD lesion rate was highest in the colon.²⁸ In another study, the sensitivity of Brachyspira spp. culture in growing pigs with and without diarrhea was examined (Pedersen KS, et al. Agreement between culture of intestinal and fecal samples for Escherichia coli and Brachyspira spp. Proc 21th Intern Pig Vet Soc Cong; Vol I:91; July 2010; Vancouver, Canada); the sensitivity in rectal fecal samples was 16.7–68.8% using intestinal samples as the reference, indicating that the sensitivity using intestinal samples was higher. It is therefore probable that the sensitivity for B. hyodysenteriae using intestinal material is similarly higher. A comparable assumption has been indicated in a study using feces and colon samples for determination of the B. hyodysenteriae status at the herd level.¹³ However, the detection rate of B. hyodysenteriae in slaughtered pigs, which often do not display clinical signs of SD, has not been investigated, to our knowledge.

The use of slaughter material instead of intestinal samples from pigs killed at the farm provides the major advantage that losses of pigs as the result of euthanasia for sampling are avoided. As well, both pig owner and veterinarian benefit from the easily available slaughter material. Therefore, we compared the detection of B. hyodysenteriae at 3 different sample sites in the large intestine of slaughtered pigs, determined macroscopic lesions at those sites, and correlated sample site and macroscopic lesions to the detection of B. hyodysenteriae.

Materials and methods

Sampling, scores, and time parameters

Twenty pig herds were selected based on previous detection of B. hyodysenteriae by culture⁸ followed by PCR¹⁶ or by a range of direct PCR assays (1 of 2 published assays^16,17 or 2 different in-house PCR assays) in these herds. Any pig given treatment targeting B. hyodysenteriae within 8 wk prior to sampling was excluded.

Whole intestines (small and large intestine, stomach, liver, and parts of urogenital tract) of 10 randomly selected pigs per herd on a single slaughter date for every herd were collected at the slaughterhouse (Fig. 1). The intestines, with the exception of intestines from one herd that were stored at 6°C overnight, were transported directly to the autopsy room. There, samples were obtained according to the following procedure: first, the rectum (R) was opened until feces were visible. Mimicking rectal fecal sampling, feces were collected 5–11 cm proximal to the anus. If the distal part of the rectum contained no feces, then the rectum was further opened to the ampulla recti. Approximately 5 g of feces were placed in a 10-mL screw-cap tube (92 × 15.3 mm, Sarstedt, Nümbrecht, Germany). Second, the apex coli (A) was opened ~20 cm proximal to the anus, and ingesta and mucosa were sampled with a disposable plastic spoon by scraping over the mucosa. The mucosa-ingesta mixture was placed in a second tube. Third, the cecum (C) was opened, and ingesta and mucosa were sampled with a new disposable spoon and placed in a third tube. The samples were either transported immediately to the bacteriology laboratory, or were kept at 4°C until further processing. During sampling and transport, the samples were kept at room temperature.

Figure 1.

Workflow of the sampling of intestines from slaughtered pigs and sample processing. For each herd, the intestines of 10 pigs were randomly selected at the slaughterhouse and transported by car to the postmortem room. There, ingesta or mucosa samples were obtained from the colon and the cecum and fecal samples from the rectum for Brachyspira selective culture. At each site, consistency of the gut content, adopted from a prior study,²⁸ and potential lesions, adopted from another study,²⁶ were assessed. Proteins from Brachyspira colonies were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the event of identification as Brachyspira hyodysenteriae, a PCR targeting the nox gene was performed. Four sample processing time parameters were recorded. BH = B. hyodysenteriae; SD = swine dysentery.

Ingesta or fecal consistency at all 3 sites was scored according to a modified scheme,¹ with 0 = firm and dry, 1 = soft and formed, 2 = semi-solid, and 3 = liquid-to-watery. An additional 0.5 points were added if mucus and/or blood were visible.

Finally, the whole large intestine was opened, ingesta and feces were removed, and the mucosa of the rectum, colon, and cecum were examined; the appearance of any lesions was scored with an adapted scoring scheme,²⁶ whereby 0 = no lesions, 1 = focal lesions (equal to or less than one-third of the corresponding intestinal part altered), no mucus or blood; 2 = focal lesions (equal to or less than one-third of the corresponding intestinal part altered), with mucus or blood; 3 = mucohemorrhagic enteritis and necrosis in greater than one-third of the corresponding intestinal part; and 4 = alteration in greater than one-third of the corresponding intestinal part, without mucus or blood. Further observations (e.g., autolysis) were also recorded.

The following times were recorded for every herd: 1) time between previous detection of the B. hyodysenteriae infection in the herd and sampling (T_D); 2) the transport time between sampling of the intestines at the slaughterhouse and the pathology examination (T_SP); 3) the time taken between collecting the intestines at the slaughterhouse and seeding the isolation agar (T_SB); and 4) the time between sampling of feces and ingesta-mucosa and seeding the isolation agar were recorded for every herd (T_PB; Fig. 1).

Bacteriologic examinations

Culture conditions

Bacteriologic swabs were inserted into the mucosa-ingesta mixture or feces and streaked on a modified selective BJ agar⁸ consisting of trypticase soy agar (BBL, Becton-Dickinson, Sparks, MD), supplemented with 5% (v/v) defibrinated sheep blood (Thermo Scientific Oxoid, Basingstoke, Hampshire, UK), colistin (6.25 mg/L; Sigma-Aldrich, Buchs, Switzerland), vancomycin (6.25 mg/L), spectinomycin (200 mg/L; Sigma-Aldrich), and spiramycin (25 mg/L; Sigma-Aldrich; colistin, spectinomycin, and spiramycin were also ordered from www.TOKU-E-com). The plates were incubated anaerobically for 5 d at 42°C. The presence of low, flat, spreading growth (typical of spirochetes) on the plate was recorded, as was the extent of hemolysis. Areas of suspected spirochetal growth were transferred to sliced blood agar (BBL, Becton-Dickinson) as described previously¹⁸ and incubated under the same conditions for 3 d. Suspected spirochetal growth displaying strong hemolysis migrating along the lines of the sliced agar farthest from the initial streaking point were transferred to anaerobic basal agar (Oxoid) supplemented with 5% (v/v) defibrinated sheep blood and colistin (10 mg/L) and incubated anaerobically for 3 d. Bacteria from the final selective agar were then identified by MALDI-TOF MS.

Bacterial isolate identification by MALDI-TOF analysis

Proteins from bacterial colonies from the final selective agar were extracted using the ethanol and formic acid procedure for microorganism profiling (Bruker Daltonics, Billerica, MA) as provided by the manufacturer. Briefly, bacteria were removed from the plate using a sterile loop and were resuspended in 300 µL of ultrapure water and mixed with 900 µL of pure ethanol. The suspension was centrifuged at 13,000 × g for 2 min, and the pellet was air dried for 5 min. Thereafter, the pellet was resuspended in 50 µL of 70% formic acid and 50 µL of 100% acetonitrile (Sigma-Aldrich). After mixing, the suspension was centrifuged as before. For each strain, 1 µL of supernatant was spotted onto a polished steel target plate (Bruker Daltonics). After drying, 1 μL of matrix (saturated solution of ɑ-cyano4-hydroxycinnamic acid [HCCA], Bruker Daltonics) was added to each spot. In each plate, a matrix-only spot was also included as a negative control. Each spot was measured 3 times in a mass spectrometer (Microflex LT mass spectrometer with a 20 Hz nitrogen laser and FlexControl software v.3.1, Bruker Daltonics). Spectra were acquired in linear positive mode with an accelerating voltage of 20 kV and analyzed within a mass range of 2,000–20,000 Da.

Brachyspira spp. identification by nox gene PCR

The Brachyspira spp. strains used to create the Brachyspira library explained below, as well as all of the field isolates identified by MALDI-TOF MS analysis as being B. hyodysenteriae, were also identified by PCR amplification of their nox gene followed by Sanger sequencing. Briefly, genomic DNA was extracted from pure colonies cultured on anaerobic basal agar (DNeasy blood & tissue kit, Qiagen) as explained above. All DNA samples were tested by a Brachyspira-specific nox gene PCR as described previously.²³ The assay was performed in a total volume of 30 µL comprising DNA, 1.5 mM MgCl₂, PCR buffer B, 0.2 mM dNTPs, 2.5 U DNA polymerase (FIREPol DNA polymerase, Solis BioDyne, Tartu, Estonia), and 0.5 μM primers (BnoxF: 5′-TAGC(CT)TGCGGTAT(CT)GC(AT)CTTTGG-3′ and BnoxR: 5′-CTTCAGACCA(CT)CCAGTAGAAGCC-3′; Microsynth, Balgach, Switzerland). After initial denaturation at 94°C for 3 min, the mixture underwent 35 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 60 s, followed by a final extension step of 72°C for 10 min. PCR products were examined by gel electrophoresis in 1% agarose gel stained with ethidium bromide for the presence of the expected 939-bp product and then documented (Gel Doc 2000 with Quantity One 4.6.9 Basic software, Bio-Rad, Hercules, CA). Subsequently, each purified (High Pure PCR product purification kit, Roche) PCR product was sequenced using the BnoxF primer and 2 further sequencing primers (BnoxF1: 5’-GTTATGGTWGTTGGTGCTGG-3’; BnoxR1: 5’-CCATRTCTACATCATAAGAACCTTT-3’). Sequencing reactions were performed (BigDye Terminator v3.1 cycle sequencing kit, Applied Biosystems, Thermo Fisher Scientific) and the products were analyzed in-house using a commercial instrument with the accompanying software (3130xl genetic analyzer with a 16-capillary system using 3130 Series Data Collection software 4, Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer’s guidelines and using the recommended buffer and polymer (3730 buffer (10×) with EDTA; POP-7 performance optimized polymer, Applied Biosystems, Thermo Fisher Scientific). Thereafter, a basic local alignment search tool nucleotide program (http://blast.ncbi.nlm.nih.gov) was used to identify the exact Brachyspira species.

MALDI-TOF MS database creation

We created a Brachyspira library using 18 strains (Table 1) providing MSPs belonging to 6 species of Brachyspira (‘B. pulli’, B. murdochii, B. innocens, B. intermedia, B. hyodysenteriae, B. pilosicoli) in a manner similar to the other libraries.^5,27 Eleven MSPs were already provided by the manufacturer (Bruker). The remaining 7 Brachyspira spp. strains that were added to create the user-defined library were selected based on phenotypic cultural characteristics along with exact Brachyspira ssp. identification by nox gene sequencing (Table 1).^16,23 The addition of both ATCC and non-ATCC B. hyodysenteriae MSPs was done to create a wide variety within this species. The addition of the MSPs was achieved by following the manufacturer’s protocol (Bruker).

Table 1.

Strains and mass spectral profiles of 6 Brachyspira spp. used to create the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry database.

Brachyspira spp.	Strain	Source
B. hyodysenteriae	CCUG 46668T	Culture collection, University of Göteborg, Sweden
	541-Italia	Laboratory collection, University of Perugia, Italy
	718-Italia	Laboratory collection, University of Perugia, Italy
	534-Italia	Laboratory collection, University of Perugia, Italy
B. pilosicoli	C162 AHVLA	Bruker Daltonics
	AN652-02 AHVLA	Bruker Daltonics
	GD82 GDD	Bruker Daltonics
	GD83 GDD	Bruker Daltonics
	JF4757	Laboratory collection, University of Bern, Switzerland
B. murdochii	DSM 12563T	Bruker Daltonics
	JF4758	Laboratory collection, University of Bern, Switzerland
B. innocens	C109 AHVLA	Bruker Daltonics
	AN3706-90 AHVLA	Bruker Daltonics
B. intermedia	AN885-95 AHVLA	Bruker Daltonics
	AN621-97 AHVLA	Bruker Daltonics
	AN519-97 AHVLA	Bruker Daltonics
	AN1707-97 AHVLA	Bruker Daltonics
‘B. pulli’	IT514	Laboratory collection, University of Perugia, Italy

Superscript (^T) indicates type strain.

The sequenced strains to be added to the database were prepared as described above, and 1 µL of each supernatant was spotted 8 times onto a steel target plate as described above, along with 2 spots of bacterial test standard (BTS; Bruker), used to calibrate the instrument and as a quality control. After drying, 1 μL of the matrix (Bruker) was added to each spot. Each spot was measured 3 times. After all spectra were measured, including the BTS spectra, their quality was analyzed (Bruker). After the BTS was checked, 20 of the 24 spectra used to create the MSPs were selected on the basis of raw spectra after smoothing, baseline subtraction, normalization, and peak picking.

Statistical analysis

Data were recorded in a database (Excel 2010, Microsoft). As lesions rarely occurred, the variable “lesion score” was recoded for statistical analyses: 0 remained 0 (= no lesions), 1 and 4 were recoded into 1 (= lesion other than B. hyodysenteriae infection–like lesions), and 2 and 3 into 2 (= B. hyodysenteriae infection–like lesions). Descriptive statistics were calculated for the outcome variable ‘Brachyspira spp.’, and no further analyses were performed given the limited number of observations in some categories (Brachyspira spp.).

Univariable analyses were carried out separately for the outcome variables “Brachyspira spp. detected (yes/no)” and “B. hyodysenteriae (yes/no).” Differences in the numbers of Brachyspira spp.–positive samples between the 3 detection sites (colon, cecum, rectum) were assessed using the chi-square test. The level of agreement in Brachyspira spp. test results between each 2 detection sites (colon-cecum, colon-rectum, cecum-rectum) within a single pig was assessed by the McNemar test. Differences in lesions between Brachyspira spp.–positive and –negative samples were assessed using the Fisher exact test. Correlations between the number of positive samples per herd and the time parameters (T_D, T_SP, T_SB, T_PB) and consistency scores were assessed with the Spearman rank correlation test. Additionally, for the time parameter “T_D,” times were ordered in 4 categories (≤1 y, >1–2 y, >2–3 y, and >3 y), and differences between number of positive samples within these 4 categories were analyzed in the chi-square test. The same tests described for Brachyspira spp. were also applied to the analysis of the outcome variable “B. hyodysenteriae (yes/no).”

All variables with p < 0.2 in univariable analysis with the outcome variables “Brachyspira spp. (yes/no)” and “B. hyodysenteriae (yes/no)” were analyzed for correlation (correlation matrix). Variables with correlations ≤0.6 and the biologically more plausible variable in the case of a correlation >0.6 were submitted to 2 multivariable logistic regression models (mixed effects logistic regression): one for “Brachyspira spp. yes/no” and one for “B. hyodyseneriae yes/no” as an outcome at the sample level. “Herd” and “pig” were included as a random effect in a random intercept model. Nonsignificant variables (p ≥ 0.05) were removed by manual step-wise backward elimination. The analyses were performed using R (www.r-project.org/).

Results

Between December 2015 and May 2016, 200 intestines from 20 herds (10 intestines per herd) were collected at 6 slaughterhouses, resulting in 400 mucosa-ingesta and 200 fecal samples.

Time parameters, consistency score, and lesion score

The median time between detection of the B. hyodysenteriae infection and sampling (T_D) was 3.8 y (range: 0.5–5.9). The median transport time of the intestines from the slaughterhouse to the postmortem room (T_SP) was 2.7 h (1.5–17.0). The intestines of one herd had a T_SP of 17.0 h because they were stored overnight for technical reasons. The vehicle temperature during transport was ~18–20°C. The median time taken between collecting the intestines at the slaughterhouse and inoculation of the isolation agar plates (T_PB) was 6.0 h (3.5–291.5), and the median time between sampling of feces and ingesta-mucosa and inoculation of the isolation agar plates (T_SB) was 3.1 h (1.0–288.0).

The median consistency score in all samples was 2 (range: 0–3). The additional 0.5 point for mucus and/or blood was not considered as mucus and/or blood was noted only in 16 cases, with “mucus” being the most frequent reason (n = 11; “blood” = 4, “mucus and blood” = 1). In 25 rectums, no feces were present and therefore no consistency score could be recorded. In the colon samples, consistency scores of 2 (64.0%) and 1 (27.0%) were mainly recorded. For the cecum samples, the scores were predominantly 3 (90.5%), whereas for the rectum samples the most common scores were 2 (43.4%) and 1 (28.6%).

No lesions were present in most of the large intestines (91% of the sites had lesion score 0). Lesion scores of 1 (4.2%) and 4 (3.5%) followed in frequency, and lesion score 3 was observed in 1.3% of the sites (7 colons and 1 cecum from 8 pigs out of 4 herds). No lesion score 2 was observed. Further observations included early signs of autolysis in the intestines of pigs from 11 herds, unusual gut material (e.g., straw or bristle-like material in 7 herds), and dark-green ingesta in 1 herd.

Brachyspira species identification

A total of 96 samples, obtained from 17 herds and 63 pigs, were positive for Brachyspira spp. (median: 2.5 pigs/herd, range: 0–9). Within the 17 positive herds, 3.3–26.7% of the samples per herd, and 10–90% of the pigs per herd, tested positive. The most frequently detected species was B. innocens (7.3% of all samples), followed by B. hyodysenteriae (3.7%), B. murdochii (2.2%), B. pilosicoli (1.5%), non-identifiable Brachyspira sp. (0.7%), and B. intermedia (0.5%; Table 2). Positive samples were obtained more frequently from the colon (43.8%) than from the cecum and rectum (28.1%; chi square test: p = 0.06).

Table 2.

Results of Brachyspira examination in 400 ingesta or mucosa samples and 200 fecal samples obtained from 200 slaughtered pigs.

Site	Brachyspira spp.
	BH	BP	BIM	BIN	BM	B. sp. (n.i.)	Total*
Colon	11	2	1	19	7	2	42 (43.8)
Cecum	5	6	0	11	4	1	27 (28.1)
Rectum	7	1	2	14	2	1	27 (28.1)
Total†	23 (3.8)	9 (1.5)	3 (0.5)	44 (7.3)	13 (2.2)	4 (0.7)	96 (16.0)

BH = Brachyspira hyodysenteriae; BP = B. pilosicoli; BIM = B. intermedia; BIN = B. innocens; BM = B. murdochii; B. sp. (n.i.) = non-identifiable Brachyspira spp. Samples were submitted to selective culture followed by identification with PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ingesta or mucosa samples were obtained from the colon and cecum. Fecal samples were collected from the rectum.

^*Numbers in parentheses are row percentages.

^†Numbers in parentheses are column percentages.

Detection of Brachyspira spp. at multiple sites was observed in 27 of the 63 positive pigs (42.9%). Detection of Brachyspira spp. at 2 sites occurred in 21 pigs with mainly B. intermedia detected at 2 sites (n = 7 pigs), followed by B. hyodysenteriae detected at 2 sites (n = 5) and B. hyodysenteriae with B. intermedia (n = 2). Detection at all 3 sites was observed in 6 pigs with half of them displaying the same species at all 3 sites (occurring twice for B. intermedia and once for B. murdochii). Within a herd, up to 5 species could be detected. In multiple positive pigs, the combination “colon-rectum” occurred most often (n = 13), followed by “colon-cecum” (n = 7) and “colon-cecum-rectum” (n = 6). One pig was Brachyspira spp. positive in its cecum and rectum samples. Most of the other 36 pigs were positive in their ingesta–mucosal scraping sample (colon = 17, cecum = 13); 6 pigs were positive only in the rectal sample. Overall, pigs were more often Brachyspira spp. positive in ingesta–mucosa samples than in rectal samples (McNemar test: p < 0.01), as well as more often positive in cecum than rectum samples (p < 0.01) and more often positive in colon than cecum samples (p < 0.01).

Most of the 96 positive sites showed no lesions (85.4%, n = 82). For 10.4% of the positive sites (n = 10), lesion scores of 1 and 4 were observed, and only 4 sites had a lesion score of 3 (n = 4; Fisher exact test: p = 0.02). Severe lesions were present only in the colon, whereas mild lesions occurred predominantly in the cecum (n = 7).

None of the time parameters correlated with the bacterial outcome. After eliminating nonsignificant factors in the multivariable regression model (the full model included “MALDI-TOF MS results” vs. “consistency score,” “lesion score,” “site,” “time after previous diagnosis,” “time between pathology and bacteriology,” “time between slaughterhouse and pathology,” “herd,” and “pig”), “colon” as sampling site remained significantly related to MALDI-TOF MS results (p = 0.02, standard error (SE) = 3.2^-1, estimate = 7.6^-1; intercept (rectum): p < 0.01, SE = 4.8^-1, estimate = −2.9).

Brachyspira hyodysenteriae was identified in 11 colon samples (47.8% of the positive samples), 7 rectal samples (30.4%), and 5 cecum samples (21.7%) by MALDI-TOF MS analysis and PCR, representing 16 pigs out of 7 herds (all 20 herds: median 0 pigs/herd, range: 0–5). In the 7 B. hyodysenteriae–positive herds, 3.3–26.7% of the samples and 10–50% of the pigs were positive for B. hyodysenteriae. At the level of the pig, significantly more intestinal samples (colon and/or cecum) were positive for B. hyodysenteriae than rectal fecal samples (McNemar test: p = 0.04). Of the 16 pigs, 5 pigs were positive in both their colon and rectum. Most of the other 11 pigs were positive in their ingesta or mucosal scraping samples (colon = 5, cecum = 4) and just 2 pigs were positive only in their rectal samples. Four pigs had lesion score 3 (= 50% of all pigs with lesion score 3), and the lesions were in the colon. In sites without lesions, significantly more B. hyodysenteriae were detected as assessed in the Fisher exact test (p < 0.01). However, in the multivariable analysis (the full model included “MALDI-TOF MS results” vs. “consistency score,” “lesion score,” “site,” “time after previous diagnosis,” “time between pathology and bacteriology,” “time between slaughterhouse and pathology,” “herd,” and “pig”), lesion score 2 (= B. hyodysenteriae infection–like lesions) was the only remaining significant factor (p = 0.04, SE = 1.68, estimate = 3.47; intercept (no lesions): p < 0.01, SE = 1.3, estimate = −8.88). Brachyspira hyodysenteriae was detected only in herds that had been initially diagnosed with SD more than 2 y before the current study (chi square test: p < 0.01). Other time parameters were not correlated to B. hyodysenteriae detection rates.

Discussion

Our aim was to determine a sampling site in the large intestine of slaughtered pigs to facilitate increased detection rates for B. hyodysenteriae in subclinically infected pig herds. Intestinal samples were significantly more often positive than rectal fecal samples. This applied to B. hyodysenteriae as well as Brachyspira spp. With respect to certain Brachyspira spp. and grower pigs, a higher detection by culture in intestinal samples compared to fecal samples has been reported (Pedersen KS et al.). Our study provides data about B. hyodysenteriae in (non-diarrheic) slaughtered pigs. Within the intestinal samples, significantly more colon than cecum samples were positive for Brachyspira spp. Our findings highlight the need to use colon ingesta or mucosa samples rather than feces when examining subclinically infected pigs and herds. Intestines from slaughtered pigs are excellent sources of ingesta or mucosa, as no extra killing of pigs is necessary. As a consequence, no extra costs (e.g., for euthanasia or lost pigs) are incurred by the pig owner. Additionally, on-farm sampling of intestinal material from killed pigs is forbidden in some countries or is impossible for other reasons. In these cases, shipping of whole carcasses to laboratories is necessary, again producing expense. It should be noted that in our study, for standardization reasons, only samples from the apex coli were examined. However, according to other studies, other areas of the colon are also suitable.^13,14,20

Samples for bacteriology were taken at defined areas (e.g., apex coli) within the respective intestinal site. Lesion scores were determined in the complete intestinal segment. Therefore, the sampling site for bacteriology was not necessarily identical to areas with lesions, but the effect of this inconsistency was negligible. In an experimental B. hyodysenteriae infection study, lesion severity was not related to detection of the pathogen.²⁶

In a study in Japan, although a number of colonic mucosal samples from slaughtered pigs were positive for B. hyodysenteriae, no detailed information regarding the clinical status of the herd of origin of these samples was communicated.¹⁵ In our study, the recovery rate for B. hyodysenteriae in 35% of the herds (7 of 20) and in 4% of intestines (16 of 400) was expected for subclinically infected herds. Our samples mainly represented pigs and/or herds without clinical SD as reported by the pig owners and supported by the scarcity of diarrheic feces and of significant SD-like lesions in the examined intestines.

Although SD-like lesions were significantly related to higher detection rates of B. hyodysenteriae in the multivariable analysis, such lesions occurred infrequently in the studied pigs. As well, SD-like lesions also occurred in intestinal sites and pigs that tested negative for B. hyodysenteriae. Therefore, the potential significance of SD-like lesions should be interpreted with caution. Given that lesions in our study were evaluated on the mucosal side, we cannot definitively answer the question of whether lesions visible or palpable from the outside (= closed intestines) are predictive for a positive bacteriologic test as recommended by other authors.¹³

Histologic analysis of the lesions suspicious for SD could have provided further information but was not practical in our study given that the time between death (slaughter) and sampling had been a minimum of 80 min and therefore was too long for good quality histologic specimens. Autolysis of the intestinal mucosa starts within minutes⁷ and was observed in a number of the examined intestines.

None of the time parameters related to sample processing was significantly correlated with the bacteriologic outcome. This might have been because of the limited variation in transport times in 95% of the herds (1.2–3.5 h, not considering one herd with 17.0 h), the relatively short transport time (T_SP), and the standardized and optimized storage conditions of the samples after collection (T_PB). However, in general, short transport and processing times are recommended.⁹ Furthermore, cooling of samples might have a positive effect on the Brachyspira recovery rate, given that storage at <26°C favors higher survival rates.^9,21 In our study, for methodologic reasons, whole intestines were transported by vehicle at moderate temperatures and thus cooling of the intestinal content from ~38°C to <26°C was not possible.

Transport of whole-intestine specimens is feasible, but requires appropriate logistics. In the present study, whole intestines were needed for the evaluation of ingesta or fecal consistency and of mucosal lesions. For larger sample sets (e.g., in routine sampling), sampling of ingesta or mucosa specimens directly at the slaughterhouse would be preferable. In our study, sampling of intestines was random, meaning that the B. hyodysenteriae detection rate might have been underestimated. Sampling targeted to colons or sites with thickened walls, as previously published, could increase the detection rate.¹³ Longer times between previous detection and current sampling in a herd were linked to higher detection rates of B. hyodysenteriae. The reason for this phenomenon is not clear, and there does not seem to be an apparent biologic or medical explanation for this result.

The detection rate of B. innocens was nearly twice as high as that of B. hyodysenteriae (7.3% vs. 3.7%). It is unknown whether this result was because of a higher prevalence of B. innocens in slaughtered pigs or in the whole pig population as has been previously suggested,^2,11,19 longer survival times as described for other Brachyspira spp. and other sources,^4,21 or competition (dominance) during culturing.

In our study, B. hyodysenteriae was detected in ingesta, and in particular from colon samples, of slaughtered pigs using culture followed by identification. Culture is currently the standard method for cases where small numbers of B. hyodysenteriae in the samples are expected, which is the case in subclinically infected pigs. Because culture is time consuming, faster methods such as direct PCR might be interesting for future investigations. Furthermore, given that up to 5 species per herd and up to 3 species per pig were detected in our study, investigations of potential colonization with multiple Brachyspira spp. per site should be conducted to evaluate their occurrence and importance.