Prevalence and Antimicrobial Resistance of Salmonella Dublin and Thermotolerant Campylobacter in Liver from Veal Calves in Québec,Canada

Abstract

Salmonella Dublin and Campylobacter spp. are two foodborne pathogens of importance. A small number of studies reported that consumption of veal liver was associated with an increased risk of human illness from these two pathogens. To better characterize the risk of exposure from liver, a cross-sectional study was conducted to estimate the prevalence of white veal calf liver contamination with these two pathogens and to characterize the antimicrobial non-susceptibility patterns of isolates. Veal liver samples were collected at two slaughterhouses in Quebec, Canada, in 2016 and 2017. Samples were submitted for polymerase chain reaction (PCR) screening followed by culture of Salmonella and thermotolerant Campylobacter. Isolates were tested for antimicrobial susceptibility using broth microdilution. Salmonella Dublin was the only serotype cultured from 3.6% (95% confidence interval [CI]: 0.0–7.9) of 560 liver samples. Among them and for technical reasons, 498 were tested by PCR for Campylobacter. The prevalence of PCR-positive livers was estimated to be 65.8% (95% CI: 58.7–72.9) for Campylobacter jejuni and 7.0% (95% CI: 3.9–10.1%) for Campylobacter coli. Fourteen Salmonella Dublin isolates were submitted for antimicrobial resistance (AMR) testing; all were non-susceptible to at least eight antimicrobials from six different classes. Most (81.4%) of the 188 C. jejuni isolates submitted for AMR testing were non-susceptible to tetracycline, and 23.0% of isolates were non-susceptible to nalidixic acid and ciprofloxacin. Of the seven C. coli isolates, four were multidrug resistant. This study highlights the importance of veal liver as a potential source of exposure to multidrug-resistant Salmonella Dublin and thermotolerant Campylobacter spp.

Introduction

Nontyphoidal Salmonella spp. and Campylobacter spp. are the leading causes of acute bacterial foodborne gastroenteritis in humans. They can be transmitted through the ingestion of contaminated food products or following direct contact with infected animals or contaminated environments (Chlebicz and Slizewska, 2018). In Canada, 19.2 cases of nontyphoidal salmonellosis and 27.6 cases of campylobacteriosis per 100,000 people were estimated in 2018 (Public Health Agency of Canada, 2020).

Although most cases are self-limiting, up to 10% of salmonellosis cases develop an invasive infection, whereas about 1% of campylobacteriosis cases are associated with severe sequelae such as Guillain-Barré syndrome or reactive arthritis (Chlebicz and Slizewska, 2018).

Many wildlife and domestic animal species are potential reservoirs for Salmonella and Campylobacter, including cattle (Chlebicz and Slizewska, 2018). Cattle are considered the natural host and main reservoir of Salmonella Dublin (Nielsen et al., 2004). This serovar emerged in 2011 in both cattle and human populations in the province of Quebec, Canada (Côté, 2012).

It is of particular significance due to its propensity to progress to a severe and invasive infection in both humans and calves in addition to frequently exhibiting multidrug resistance (Guizelini et al., 2020; Mangat et al., 2019). In Canada, a high genetic similarity between human and cattle isolates has been reported, supporting a zoonotic transmission route, but no specific transmission pathway was identified (Mangat et al., 2019). In Quebec, recent studies estimated that 6.5% of dairy cattle herds are infected by Salmonella Dublin and 73% with Campylobacter spp. (Guevremont et al., 2014; Um et al., 2020).

In Canada, ∼200,000 surplus veal calves are raised per year, in specialized veal farms, where they are grain-fed (red veal) or milk-fed (white veal) until they reach slaughter weight at ∼5–8 months of age. Approximately 80% of Canadian white veal is produced in Quebec, with most of these veal calves originating from Quebec dairy farms.

Canadians consume about 1 kg of veal product per year, an estimate that is 9 times greater than the average consumption in the United States (Gouvernement du Québec, 2021). The handling and consumption of veal liver represents a likely food vehicle for transmission of Salmonella spp. and Campylobacter spp.. Salmonella Typhimurium and Dublin are the most frequent serotypes isolated from veal submitted in necropsy in Québec (Gouvernement du Québec, 2023).

Salmonella Dublin can often be recovered from livers of clinically infected calves (Uribe et al., 2015) and those calves can become long-term carriers of the bacteria. Campylobacter can also be found in cattle liver, especially in the bile that supports the growth and survival of the bacteria (Enokimoto et al., 2007). The external surface of the liver can also be cross-contaminated during processing at a slaughterhouse (Karki et al., 2019; Samuel et al., 1980). Once packaged, beef liver juice has been reported to increase the survival of Campylobacter at low temperature (Karki et al., 2019). Finally, liver is often eaten mildly cooked, which is not sufficient for complete inactivation of pathogens (Lester et al., 1995).

In Denmark, cow's liver is considered as the principal source of Salmonella Dublin infection in humans (Lester et al., 1995). A recent case-control study conducted in Quebec reported that consumption of veal liver, and in particular undercooked liver, was strongly associated with campylobacteriosis (Gaulin et al., 2018). However, very little information is available on the risk of veal liver contamination with these bacteria.

Thus, the objectives of this study were to estimate the prevalence of veal liver contaminated with Salmonella spp. and thermotolerant Campylobacter spp. in Quebec, Canada, and to describe the antimicrobial non-susceptibility patterns of isolates.

Materials and Methods

Study design and sample collection

A cross-sectional study was conducted in white veal calves slaughtered in two federally inspected slaughterhouses located in Quebec. Sampling was performed once or twice a week between August 2016 and October 2017 among calves destined for the Canadian market. On each sampling day, 1 or 2 lots (i.e., group of calves raised together in the same farm) were selected depending on their size, for a total of 70 lots (Table 1). All procedures were approved by the institutional animal ethics committee of the Université de Montréal (certificate #16-Rech-1844).

Table 1.

Descriptive Statistics of the Sample Included in a Prevalence and Antimicrobial Resistance Study on Salmonella and Thermotolerant Campylobacter in Livers from White Veal Calves in Quebec, Canada

Characteristics	Salmonella spp.	Campylobacter spp.
Characteristics	PCR+culture	PCR	Culture
Number of farms	50	46	37
Number of lots	70	63	44
Number of veal livers	560	498	239
Median (minimum to maximum) number of lots per farm	1 (1–6)	1 (1–6)	1 (1–5)
Median (minimum to maximum) of lot size slaughtered	302 (82–747)	306 (104–747)	275 (104–747)
Median (minimum to maximum) of veal liver per lot	8 (8–8)	8 (3–8)	5 (2–8)
Number of sampling days at slaughterhouse	62	56	40

PCR, polymerase chain reaction.

For each lot, approximately in the middle of the batch slaughter, 8 consecutive calves on the slaughter line were sampled, for a total of 560 calves. For each veal calf, a liver sample of ∼100 g was collected from the hilum region, placed inside stomacher sterile bags (Nasco Whirl-Pak^®, Madison, WI), kept in a cooler with icepacks, and sent to the laboratory within 24 h.

Molecular testing and bacteriology

The liver samples were sent to the Laboratoire d'expertises et d'analyses alimentaires (LEAA) of the Ministère de l'Agriculture, des Pêcheries et de l'Alimentation du Québec (MAPAQ) and submitted to their standardized protocol for food products (MAPAQ, 2016; MAPAQ, 2014).

For Salmonella spp., 25 g liver samples were homogenized in buffered peptone water and incubated at 35°C for 18–24 h. After resuspension, 1 mL was collected, mixed to 9 mL of Rappaport-Vassiliadis soya (RVS) broth, and incubated at 42.5°C for 24 ± 2 h. After incubation, 1 mL of the RVS broth was used for DNA extraction using E-Z 96 FastFilter Plasmid kit (OMEGA Bio-Tek, Inc., Norcross, GA) or the Montage Plasmid Miniprep kit (Millipore, Billerica, MA).

DNA was detected using the stn and invA genes primers. Polymerase chain reaction (PCR) positive samples were streaked from RVS broth to Xylose-lysine-deoxycholate agar and Brilliant green sulfa agar at 35°C for 24 ± 2 h. Typical colonies were purified on Trypticase Soy agar (TSA) supplemented with 5% (v/v) sheep blood and confirmed using biochemical testing.

One Salmonella isolate per positive liver sample was submitted to the Laboratoire de santé animale of the MAPAQ where it was serotyped by slide agglutination to detect somatic (O) antigens and by microplate precipitation to detect flagellar (H) antigens according to Kauffmann-White scheme.

For thermotolerant Campylobacter spp. detection, 25 g liver samples (including external surface and parenchyma) were gently homogenized in a Bolton broth selective media, including cefoperazone (20 mg/L), vancomycin (20 mg/L), trimethoprim (20 mg/L), and amphotericin B (10 mg/L). The suspension was incubated under microaerophilic conditions at 37°C for 4–6 h, and then at 41.5°C for 44 ± 4 h.

After incubation, 1 mL of the homogenate was collected and DNA was extracted using the QIAGEN DNeasy Blood & Tissue or the E-Z 96 FastFilter Plasmid kit (OMEGA Bio-Tek, Inc.). Multiplex PCR was then performed with primers targeting the ask, cj0414 and glyA genes of Campylobacter coli, Campylobacter jejuni, and Campylobacter lari, respectively. Enrichment broth from positive PCR samples was kept at −80°C for further characterization.

At the end of the sampling period, all frozen broths were sent to the Research Chair in Meat Safety (RCMS) laboratory for cultivation. After thawing, broth samples were streaked on mCCDA (Innovation Diagnostic, Inc., Saint-Eustache, Canada), then on TSA supplemented with 5% (v/v) sheep blood (Fisher Scientific, Ottawa, Canada). Incubations took place in a microaerobic atmosphere using Oxoid gas-generating system (Thibodeau et al., 2013).

One typical Campylobacter isolate (small Gram-negative spiral-shaped bacteria, motile) was collected and kept at −80°C in Brucella broth containing 0.1% agar and 25% glycerol before speciation and antimicrobial susceptibility testing. Presumptive Campylobacter isolates were confirmed and speciated (as C. jejuni and C. coli) using multiplex PCR (Persson, 2005), performed at the National Microbiology Laboratory in St-Hyacinthe.

Antimicrobial susceptibility testing

One isolate per positive liver for Salmonella Dublin and for thermotolerant Campylobacter spp. was submitted to Canada's National Microbiology Laboratory for antimicrobial susceptibility testing using broth microdilution as previously described (Government of Canada, 2015). The panel of antimicrobials and their respective resistant and intermediate clinical breakpoints were used as previously described (Government of Canada, 2020a).

Statistical analyses

The prevalence of contaminated liver with 95% confidence intervals (CIs) was estimated for each Salmonella serotypes and Campylobacter species. The sampling design was taken into account by attributing a weight to each sampled calf calculated as: weight = 1/(number of sampled calf in the lot/total number of calves in the lot), and by adjusting the variance estimate for clustering of calves within lots using the Taylor series method.

The prevalence with 95% CI of lots and of farms with at least one contaminated liver detected was also estimated. The prevalence at the lot level was adjusted for the clustering of lots within farms as previously described.

Antimicrobials were classified as proposed by the Veterinary Drugs Directorate of Canada, based on their importance in human medicine (Government of Canada, 2015; Health Canada, 2005). The proportion of non-susceptible (i.e., including resistant and intermediate categories) isolates with 95% CI was estimated for each antimicrobial, performed separately for Salmonella serotypes and Campylobacter species.

When at least 10 isolates were detected and the proportion was not equal to 0% or 100%, the estimates were adjusted for sampling weight and clustering within lots as previously described; otherwise, exact confidence limits (Clopper-Pearson) were presented. A multidrug-resistant (MDR) isolate was defined as an isolate with non-susceptibility detected to at least one antimicrobial from ≥3 antimicrobial classes (Magiorakos et al., 2012).

All statistical analyses were performed in SAS 9.4 (SAS Institute, Inc., Cary, NC).

Results

Prevalence

Among the 560 liver samples, 20 samples were PCR-positive for Salmonella spp. and therefore cultured. A Salmonella isolate was obtained from 19 out of 20 samples. Seventeen of the isolates were confirmed as Salmonella Dublin. The isolates from the two other samples were inadvertently not tested; they were both from a lot with at least one other liver sample positive for Salmonella Dublin.

Thus, the prevalence of positive livers to Salmonella spp. was estimated to be 3.9%, whereas it was estimated to be at least 3.6% for Salmonella Dublin (Table 2). At least one Salmonella Dublin positive liver was detected in 7 (10%) of the 70 lots and from 6 (12%) of the 50 farms.

Table 2.

Prevalence Estimates with 95% Confidence Intervals of Positive Liver to Salmonella and Thermotolerant Campylobacter in White Veal in Quebec, Canada

Pathogen	Test	Unit = veal			Unit = lot			Unit = farm
		No. pos/total	Prevalence ^a		No. pos/total	Prevalence ^b		No. pos/total	Prevalence
		No. pos/total	%	95% CI	No. pos/total	%	95% CI	No. pos/total	%	95% CI
Salmonella
Salmonella Dublin	PCR+Culture	17/560	3.6	0.0–7.9	7/70	10.0	1.9–18.1	6/50	12	4.5–24.3
Salmonella spp.^c	PCR+Culture	20/560	3.9	0.0–8.3	8/70	11.4	3.5–19.4	7/50	14	5.8–26.7
Thermotolerant Campylobacter
Campylobacter jejuni	PCR	326/498	65.8	58.7–72.9	61/63	96.8	92.4–100.0	46/46	100	92.3–100
Campylobacter coli	PCR	36/498	7.0	3.9–10.1	21/63	33.3	21.0–45.6	19/46	41.3	27.0–56.8
Campylobacter spp.^d	PCR	334/498	67.5	60.3–74.6	61/63	96.8	92.4–100.0	46/46	100	92.3–100

Prevalence was adjusted for sampling weight and clustering of veal within lots.

Prevalence was adjusted for clustering of lots within farms.

Salmonella Dublin (17 isolates) and untyped Salmonella (3 isolates).

Including C. coli and C. jejuni.

CI, confidence interval; PCR, polymerase chain reaction.

For thermotolerant Campylobacter spp., 62 of the 560 liver samples were rejected due to technical reasons. From the 498 livers submitted for PCR testing for thermotolerant Campylobacter spp., 334 were positive to C. jejuni and/or C. coli. This included 28 livers positive for both species. No liver was positive for C. lari. The prevalence of PCR-positive livers was estimated to be 65.8% for C. jejuni and 7.0% for C. coli (Table 2). At least 1 PCR-positive liver was detected in 61 (96.8%) of the 63 lots tested and from all farms.

Antimicrobial resistance

The 17 Salmonella Dublin isolates were non-susceptible to at least 8 antimicrobials from 6 different classes, including category I antimicrobials (Table 3). Among the 334 PCR-positive enrichment broth samples for Campylobacter, 239 samples were kept and cultured. A Campylobacter isolate was successfully obtained from 195 out of 239 (81.6%) of samples. These isolates were confirmed as C. jejuni (188 isolates) or C. coli (7 isolates).

Table 3.

Proportion with 95% Confidence Interval of Non-Susceptibility to Various Antimicrobial Agents in 17 Salmonella Dublin Isolates from 7 Lots of White Veal Livers in Quebec, Canada

Cat. ^a	Agent	Class	No. of isolates			Non-susceptibility ^b
Cat. ^a	Agent	Class	S	I	R	%	95% CI
I	AMC	Penicillins/β-lactamase inhibitor	—	—	17	100	80.5–100
	CRO	Cephalosporins	—	—	17	100	80.5–100
	CIP	Fluoroquinolones	3	14	—	82.4	56.9–96.2
	MEM	Carbapenem	17	—	—	0.0	0.0–19.5
II	AMP	Penicillins	—	—	17	100	80.5–100
	AZM	Macrolides	17	—	—	0.0	0.0–19.5
	FOX	Cephalosporins	—	—	17	100	80.5–100
	GEN	Aminoglycosides	17	—	—	0.0	0.0–19.5
	NAL	Quinolones	3	—	14	82.4	56.9–96.2
	STR	Aminoglycosides	—	—	17	100	80.5–100
	SXT	Sulphonamides	17	—	—	0.0	0.0–19.5
III	CHL	Phenicols	—	—	17	100	80.5–100
	SOX	Sulphonamides	—	—	17	100	80.5–100
	TET	Tetracyclines	—	—	17	100	80.5–100

Categories ranging from I (very high), II (high), and III (medium) based on their importance in human medicine as proposed by the Veterinary Drugs Directorate of Canada.

Proportions and 95% CIs were adjusted for sampling weight and clustering of veal within lots for CIP and NAL, and exact 95% CI are presented otherwise.

AMC, amoxicillin/clavulanic acid; AMP, ampicillin; AZM, azithromycin; CHL, chloramphenicol; CI, confidence interval; CIP, ciprofloxacin; CRO, ceftriaxone; FOX, cefoxitin; GEN, gentamicin; I, intermediate; MEM, meropenem; NAL, nalidixic acid; R, resistant; S, susceptible; SOX, sulfisoxazole; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline.

Most Campylobacter isolates were non-susceptible to tetracyclines (Table 2). Overall, 42 (23.0%) of C. jejuni and 6 (85.7%) of C. coli isolates were non-susceptible to at least one category I antimicrobial (Table 4). These 48 non-susceptible Campylobacter isolates to category I antimicrobials were detected in 27 out of 44 lots and in 25 out of 37 farms.

Table 4.

Proportion with 95% Confidence Interval of Non-Susceptibility to Various Antimicrobial Agents in Campylobacter jejuni and Campylobacter coli Isolates from White Veal Livers in Quebec, Canada

Cat. ^a	Agent	Class	Campylobacter jejuni (188 isolates from 44 lots)					C. coli (7 isolates from 6 lots)
			No. of isolates			Non-susceptibility ^b		No. of isolates			Non-susceptibility ^c
			S	I	R	%	95% CI	S	I	R	%	95% CI
I	CIP	Fluoroquinolones	146	0	42	23.0	13.6–32.4	1	0	6	85.7	42.1–99.6
I	TEL	Ketolides	188	0	0	0.0	0.0–1.9	3	0	4	57.1	18.4–90.1
II	AZM	Macrolides	188	0	0	0.0	0.0–1.9	3	0	4	57.1	18.4–90.1
	CLI	Lincosamides	188	0	0	0.0	0.0–1.9	3	0	4	57.1	18.4–90.1
	ERY	Macrolide	188	0	0	0.0	0.0–1.9	3	0	4	57.1	18.4–90.1
	GEN	Aminoglycosides	188	0	0	0.0	0.0–1.9	7	0	0	0.0	0.0–41.0
	NAL	Quinolones	146	0	42	23.0	13.6–32.4	1	0	6	85.7	42.1–99.6
III	FLO	Phenicols	188	0	0	0.0	0.0–1.9	7	0	0	0.0	0.0–41.0
III	TET	Tetracyclines	32	0	156	81.4	71.6–91.2	0	0	7	100	59.0–100

Categories ranging from I (very high), II (high), and III (medium) based on their importance in human medicine as proposed by the Veterinary Drugs Directorate of Canada.

Proportions and 95% CIs were adjusted for sampling weight and clustering of veal within lots for CIP, NAL and TET; exact 95% CI are presented otherwise.

Exact 95% CIs.

AZM, azithromycin; CI, confidence interval; CIP, ciprofloxacin; CLI, clindamycin; ERY, erythromycin; FLO, florfenicol; GEN, gentamicin; I, intermediate; NAL, nalidixic acid; R, resistant; S, susceptible; TEL, telithromycin; TET, tetracycline.

All Salmonella Dublin isolates were MDR (Table 5). None of the C. jejuni isolates were identified as MDR, whereas 3 out of 7 C. coli isolates were MDR (Table 5).

Table 5.

Antimicrobial Non-Susceptibility Patterns Observed in Campylobacter jejuni, Campylobacter coli, and Salmonella Dublin Isolates

Antimicrobial non-susceptibility pattern ^a	No. of isolates	No. of lots ^b	No. of farms ^b
Salmonella Dublin (17 isolates from 7 lots and 6 farms)
AMC-CRO-CIP-AMP-FOX-NAL-STR-CHL-SOX-TET	14	5	4
AMC-CRO-AMP-FOX-STR-CHL-SOX-TET	3	2	2
Campylobacter jejuni (188 isolates from 44 lots and 37 farms)
CIP-NAL-TET	38	22	21
CIP-NAL	4	3	3
TET	118	37	30
None	28	13	13
C. coli (7 isolates from 6 lots and 6 farms)
CIP-TEL-AZM-CLI-NAL-TET	4	3	3
CIP-NAL-TET	2	2	2
TET	1	1	1

Each pattern represents a unique combination of non-susceptibility to all tested antimicrobials.

Number of lots and number of farms with a least one isolate detected with the specific pattern.

AMC, amoxicillin/clavulanic acid; AMP, ampicillin; AZM, azithromycin; CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; CRO, ceftriaxone; FOX, cefoxitin; NAL, nalidixic acid; SOX, sulfisoxazole; STR, streptomycin; TEL, telithromycin; TET, tetracycline.

Discussion

We reported white veal liver contamination with two major foodborne pathogens. In a previous study conducted in Quebec, 0% of 38 livers collected at slaughterhouse and 5.1% of 59 livers at retail were positive to Salmonella spp. based on molecular detection, but no serotyping was done (Gaulin et al., 2018). In a context where the Salmonella Dublin status of sampled populations was not reported, no Salmonella enterica was detected in 44 beef livers at retail in the United States (Liu et al., 2020) or from the livers of 200 cattle in Turkey (Cetin et al., 2019).

Although the infection of cattle liver parenchyma has also been reported for Salmonella Typhimurium, the risk appears lower than with Salmonella Dublin (Samuel et al., 1980). Our results are consistent with earlier observations of the high susceptibility of veal calves to Salmonella Dublin infection (Nielsen, 2013; Uribe et al., 2015).

In Canada, the proportion of MDR in Salmonella Dublin isolates from human cases increased from 0% in 2003 to 64% in 2015, which also coincided with an increase in reported blood infections (Mangat et al., 2019). In Quebec, all of the 28 Salmonella Dublin clinical isolates collected from passive surveillance in 2020 were MDR (Gouvernement du Québec, 2020), which is similar to our study.

Of note, most of our Salmonella Dublin isolates had intermediate resistance to ciprofloxacin and all were resistant to ceftriaxone, which is of great concern given that these antimicrobials are used to treat severe Salmonella infections in humans (Medalla et al., 2016).

We detected thermotolerant Campylobacter spp. in 67.5% of liver samples using the molecular method. Based on culturing, at least 82% of these PCR-positive samples had viable Campylobacter. Considering that enrichment broths were frozen before culture, leading to potential detrimental effects on Campylobacter survival, we expect that most PCR-positive samples had viable Campylobacter.

In a previous study, 16.2% of 38 veal livers collected at a slaughterhouse in Quebec were positive to Campylobacter spp. based on molecular detection (Gaulin et al., 2018). Other studies conducted at slaughterhouses in South Korea, Japan, and England reported prevalence estimates for Campylobacter spp. in beef liver of 1.25%, 1.4%, and 3.1%, respectively (Jeong et al., 2017; Matsumoto et al., 2008; Sarnago Coello et al., 2007).

In Quebec, young animals from multiple sources are regrouped in veal calves farms, which might result in a higher risk of Campylobacter spp. exposure and thus could explain the higher prevalence observed. Another potential explanation is the higher reported risk of C. jejuni infection in calves compared with adult cattle (Giacoboni et al., 1993), which might also explain the predominance of C. jejuni in our samples.

Of note, higher prevalence of contamination has been reported in livers collected at retail, estimated at 35.7% in 59 veal livers in Quebec (Gaulin et al., 2018) and at 6.9–78% in beef livers from Australia, the United States, or Scotland (Giacoboni et al., 1993; Liu et al., 2020; Noormohamed and Fakhr, 2013; Strachan et al., 2012; Walker et al., 2019). These findings suggest that the risk of consumer exposure to Campylobacter spp. from veal liver at retail might be higher than the risk of liver contamination we observed at a slaughterhouse.

The bile represents the most likely source of veal liver contamination with Campylobacter spp. The contamination of the gallbladder could originate from the jejunum through biliary ducts or from the blood stream, given that bacteremia has been reported in many species after oral inoculation by C. jejuni (Enokimoto et al., 2007). As previously reviewed, bile represents a chemotactic attractant for C. jejuni, and the bacteria are tolerant to the bactericidal effects of the bile (Matsumoto et al., 2008).

In beef cattle from Japan, 45% of bile samples and 5% of internal samples of liver were contaminated with C. jejuni, C. coli, and/or Campylobacter fetus (Enokimoto et al., 2007). Another study reported similar trends, reporting that 23% of bile samples and 1.4% of liver samples from cattle were positive to C. jejuni (Matsumoto et al., 2008).

As previously reported in beef cattle from Canada, resistance to tetracyclines, nalidixic acid, and ciprofloxacin was most common in our Campylobacter spp. isolates (Government of Canada, 2020b). In particular, we observed resistance to ciprofloxacin—a category I antimicrobial—in 22.3% of C. jejuni isolates and in 85.7% of C. coli isolates, as well as resistance to azithromycin (a category II antimicrobial) in 57.1% of C. coli isolates.

This is of concern given that these two antimicrobial classes (fluoroquinolones and macrolides) are the two recommended treatments for severe or prolonged human campylobacteriosis cases, or for infection in immunocompromised individuals (Government of Canada, 2018). No use of quinolones in milk-fed veal calves was noted in sampled lots in our study; however, previous use in dairy cattle herds might be contributing to the observed patterns (Tchamdja, 2017).

Our results support that white veal liver handling or consumption represents a risk for the transmission of Salmonella Dublin and Campylobacter spp. Although only a few reports have documented the risk associated with liver consumption from veal or beef specifically (Gaulin et al., 2018; Lester et al., 1995), multiple campylobacteriosis outbreaks have been associated with the consumption of chicken liver pate (Wensley et al., 2020).

Mitigation of this risk could consider various already described interventions along the farm-to-fork continuum. First, the control of Salmonella Dublin infection on dairy farms is essential for reducing the probability of calf infections, which should involve restriction of animal movements, good calving area management, and adequate colostrum management for surplus calves (Nielsen et al., 2012; Vaessen et al., 1998).

In veal facilities, the commingling of calves from dairy cattle farms with different Salmonella Dublin infection status should be limited and strict biosecurity rules should be implemented to reduce the spread of bacteria, including downtime periods (when no calves are present in the barn) between calf lots. For Campylobacter, a lack of biosecurity has also been associated with a higher prevalence in dairy cattle farms (Guevremont et al., 2014).

At slaughter, emphasis should be put on avoiding cross-contamination of livers, especially when removing the gallbladder, during evisceration, and at the time of inspection (Enokimoto et al., 2007). Finally, appropriate handling and cooking of liver by consumers should be promoted, recognizing that both parenchyma and external surface of the liver can be contaminated. This can be a challenging task, as livers cooked for too long became unappetizing due to changes in color and texture (Hutchison et al., 2015).

Conclusions

Salmonella spp. and thermotolerant Campylobacter spp. were detected in 3.9% and 67.8%, respectively, of veal liver samples collected at slaughterhouse. Resistance to ciprofloxacin was detected in 82% of Salmonella Dublin isolates and in 22.3% of C. jejuni isolates. Further, all Salmonella Dublin isolates were non-susceptible to at least six different antimicrobial classes. This study highlights the importance of veal liver as a potential source of human exposure to MDR Salmonella Dublin and Campylobacter spp.

Footnotes

Acknowledgments

The authors dedicate this article to Carine Michèle Andela Abessolo for her faithful commitment to this project and key contribution to the field work. The authors would like to thank the slaughterhouses and the Fédération des Producteurs de bovins du Québec for their collaboration and support. They acknowledge Nicole Trottier and William Thériault from the Industrial RCMS, Frédéric Goulet-Grondin, Rachelle Frenette-Cotton, and Geneviève Couture from the LEAA (MAPAQ), the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) laboratory, and the Laboratoire de santé animale (MAPAQ) who contributed to this project.

Authors' Contributions

J.A.: conceptualization, data curation, formal analysis, funding acquisition, supervision, visualization, and writing—original draft; G.C.: conceptualization, funding acquisition; P.T.: conceptualization, funding acquisition; E.T.: investigation, project administration; E.J.P.: conceptualization, funding acquisition; D.D.: methodology, resources; M.B.: investigation, resources; S.B.: conceptualization, funding acquisition; P.F.: conceptualization, funding acquisition, methodology, and resources. All authors: writing—reviewing and editing.

Disclosure Statement

No competing financial interest exists.

Funding Information

This study was funded by a grant from the Agri-Food Innov'Action Program, a program resulting from the “Growing Forward 2” agreement between the MAPAQ and Agri-Food Canada (Project IA116549), with in-kind support from the Public Health Agency of Canada and from the MAPAQ.

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