Survey of Salmonellae occurrence in meat‐producing rabbitries in Switzerland

Abstract Background An outbreak of salmonellosis due to Salmonella Typhimurium was detected coincidentally in a Swiss meat rabbitry, given that surveillance of Salmonella in rabbits is not mandatory in Switzerland. Methods To assess the extent of potentially subclinical Salmonella carriage in meat rabbits, faecal pool samples of 50 farms (90% of Swiss commercial rabbitries) with ground covering litter and group housing were bacteriologically tested. Additionally, 236 rabbits showing clinical signs compatible with intestinal diseases, such as salmonellosis, were examined postmortem and analysed bacteriologically. Salmonella isolates were serotyped and analysed by whole genome sequencing (WGS). Results Salmonella Typhimurium was detected in three commercial farms (6.0% of all tested farms). The affected farms were directly linked to the animal trade and Salmonella isolates were shown to be identical by WGS. Conclusion There is no increased hazard for Salmonella carriage in the animal welfare‐friendly Swiss husbandry systems in general, despite risk factors such as ground covering litter.


INTRODUCTION
In 2018, an outbreak of salmonellosis was detected on a Swiss meat rabbitry because of increased mortality rates in fattening and breeding rabbits. Rabbits of all types and ages, with or without clinical signs, were tested positive for Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium). While Salmonella Typhimurium occasionally causes severe outbreaks of salmonellosis with fever, enteritis, abortion and increased mortality, 1-5 rabbits may also be subclinical carriers of several Salmonella serovars. 2,[6][7][8][9] Salmonella carriage in clinically healthy livestock can result in contamination of carcasses at slaugh-ter and thereby poses a hazard for meat-borne infections in humans. [10][11][12] In the European Union, salmonellosis ranks second in zoonoses in humans with Salmonella Enteritidis and Salmonella Typhimurium being the two serovars with the highest prevalence. 13 Salmonella control programmes implemented in Switzerland mainly focus on poultry, cattle and pigs. They do not consider less abundant food-producing species, such as meat rabbits. While two studies, focussing on suckling mortality 14 and on faecal samples of rabbits at slaughter, 11 did not detect Salmonella in Swiss rabbits. The current study, with reference to the findings in Farm A, raised the question, whether the extent of Salmonella carriage in commercial rabbits might be underestimated. In commercial rabbit husbandries in Switzerland, housing on wire mesh floors and single housing of fattening rabbits at the age of less than 8 weeks is prohibited. 15 In this study, the participating farms represent the majority of the commercial production in Switzerland, with husbandry systems exceeding the minimal requirements of the legislation, providing ground covering litter for all rabbits and partly group housing for breeding does. 16,17 These high-level housing conditions regarding animal welfare may be challenging concerning hygiene management and the prevention of disease transmission. Hence, the objective of this study was to assess the occurrence of Salmonella in Swiss commercial rabbitries under these animal welfare-friendly husbandry conditions.

History of Salmonella positive farm (farm A)
The initial detection of Salmonella Typhimurium on farm A was based on nine submissions of rabbits for postmortem examination at the National Reference Centre for Poultry and Rabbit Diseases (NRGK) in October 2018. During subsequent sanitation efforts on the farm, faecal and environmental samples were examined. Resulting Salmonella isolates were stored.

Study farms and faecal sample collection
A total of 50 Swiss commercial rabbitries were tested for Salmonella (Table 1). At least 60 g of fresh faeces were collected randomly in at least five different pens (both breeding and fattening) on each farm. During the first sampling period (April-December 2019), rabbits were assessed on-site for clinical signs consistent with salmonellosis by the same veterinarian; husbandry and management data were collected. Rabbitries initially testing negative were re-tested in a second sampling period (February-August 2020). On positive farms, animals other than rabbits were also tested to exclude crosscontamination by methods authorized by the Swiss Federal Veterinary Office. 18,19

Postmortem examination
In total, 67 meat rabbits from farms testing positive for Salmonella in the faecal sample were examined postmortem at the NRGK. In addition, 193 rabbits of all ages and purposes (fattening, hobby and fancy) from 181 additional husbandries, were analysed during the period of the study to determine the cause of death and also tested for Salmonellathus serving as a reference group. Macroscopically altered organs were cultured aerobically/anaerobically on Columbia agar with 7% sheep blood and aerobically on bromothymol blue-lactose agar (Oxoid; Thermo Fisher Scientific) all for 24 h at 37 • C. Bacterial identification was performed using the Biotyper Matrix-assisted Laser Desorption/Ionization-Time of Flight-Mass spectrometry (MALDI-TOF-MS) System (Bruker Daltonics). Intestinal samples were screened for the presence of Salmonella as described below.

Salmonella detection by culture
Qualitative detection in animal faeces and organs was carried out according to ISO 6579-1:2017. 18 Following identification with MALDI-TOF-MS, Salmonella serotyping was carried out by the Swiss Reference Laboratory (ZOBA, University of Berne) according to the White-Kauffmann-Le Minor scheme. 20

RESULTS
In the first screening period, faecal samples of rabbits from 47 farms were negative for Salmonella, while Salmonella Typhimurium was detected in 3/50 farms (6%), that is, farms A, B and C ( Table 1). As three rabbitries ceased their business and seven farms did not send the second sample, consecutive faecal samples of 37 farms were analysed, all with negative results.
On farms B and C, neither clinical signs compatible with salmonellosis nor increased mortality rates in the fattening units were noticed at the time of the positive sampling. Faecal samples of broilers on farm C and laying hens on farm B tested Salmonella-negative. Given that both farms ceased their business, no consecutive samples were available. Farms B and C were directly linked to farm A by animal trade ( Table 1). The spreading of one clone of Salmonella Typhimurium among the three farms was corroborated by whole genome sequencing. Salmonella Typhimurium isolates from the three farms showed only 1-2 allele differences and were therefore grouped within one phylogenetic cluster (Figure 1).
Salmonella was not detected in any of the 193 rabbits that were examined postmortem for diagnostic purposes during the study period. The motivation for the postmortem examinations was pre-existing sickness or unsolved cause of Veterinary Record Open  of  TA B L E  Production data of 50 Swiss rabbitries (nine breeding farms, 13 fattening farms and 28 combined farms) and results of pathological and microbiological examination of rabbits originating from Salmonella Typhimurium-positive farms A, B and C death in individual animals or of several animals in the context of herd health monitoring. Clinical signs compatible with salmonellosis or macroscopically conspicuous tissues were observed in 114 cases. At least one of the following causes of disease per rabbit was found instead: dysentery (n = 66), coccidiosis (n = 16), dysbiosis (n = 8), mucoid enteropathy (n = 7), haemorrhagic and necrotizing typhlitis (n = 2), pasteurellosis (n = 15), pneumonia (n = 12), rabbit haemorrhagic disease virus type 2 (n = 7), encephalitozoonosis (1) and different non-infectious diseases (n = 59).

DISCUSSION
Following detection of Salmonella Typhimurium in commercial meat rabbits (farm A), consecutive testing revealed 94% (47/50) of Swiss commercial rabbitries to be free from Salmonella. The two farms additionally tested positive (B and C) were linked to outbreak farm A via animal trade (Table 1), by purchasing breeding does and/or fattening rabbits. According to cgMLST all isolates of the affected farms belonged to one cluster of genetically nearly identical Salmonella Typhimurium strains, displaying a single-linkage distance of no more than two core genes ( Figure 1). The combination of cgMLST and epidemiological data (both temporal and spatial link) allows the conclusion that all isolates were derived from one common ancestor. The source of infection is unknown, it is most likely that the origin was farm A, due to insufficient hygiene and rodent management, especially biosecurity breaches at the stable entries; demonstrated by various environmental samples testing positive (Table 1). On farms B and C, Salmonella Typhimurium was found in the pooled faecal samples of the rabbits; laying hens (farm B) and broilers (farm C) kept in separate stables tested negative. Farm A was sanitized following a programme by the Institute for Food Safety and Hygiene; farms B and C ceased farming of rabbits. The finding of 6% of farms positive for Salmonella Typhimurium in this study corresponds with the clinical occurrence of Salmonella of less than 8% reported in other studies. [2][3][4][7][8][9] The prevalence in Switzerland might be even lower because the three positive farms correspond to a single outbreak that was detected with clinical surveillance. Among rabbits showing clinical signs compatible with salmonellosis, approximately 4% were positive for Salmonella in Spain with around half of them being serotyped as Salmonella Typhimurium 3 -the serovar most often isolated in rabbits. [2][3][4]6,7,24 In contrast, in this and previous Swiss studies, none of the clinically conspicuous or perished rabbits tested positive for Salmonella. 14,25 This confirms that neither clinical signs nor postmortem findings indicative for salmonellosis are pathognomonic in rabbits but resemble different diseases of diverse origins.
The high standard of Swiss husbandry systems in terms of animal welfare is, to a certain extent, of concern regarding the transmission of infectious pathogens among rabbits housed in groups. 26 Straw bedding is frequently used, which increases contact with faeces and urine -and thus with excreted Salmonella. 27 While the present study demonstrated a low risk for Swiss rabbitries in general, this husbandry system might predispose in-farm dispersal when coinciding with biosecurity breaches or insufficient disinfection.
Principally, strict biosecurity and regular barn disinfection help to reduce the risk of Salmonella contamination and spreading within a farm. 28,29 However, regular disinfection was neither carried out on Salmonella-positive farms A and B, nor in 63% of the breeding units on the negative farms. Only a minority of 14% (7/50) had established a hygiene barrier (entrance and exit area with the change of clothes and handwashing facility before entering the stable). In conclusion, despite partially insufficient biosecurity measures and different risk factors for an entry of Salmonella into the husbandry system, the prevalence of Salmonella detected in this study was not higher than the one reported from other countries.

A C K N O W L E D G E M E N T S
We thank the staff of the involved institutes for their help with postmortem and bacteriological examinations and Nicole Cernela and Marc Stevens for their technical support with the sequencing of the Salmonella isolates. Thanks to Roger Stephan for critically reading the manuscript. This study was partly funded by the Swiss Federal Food Safety and Veterinary Office, Swiss animal welfare; Coop group; Federation of Migros cooperatives; IP-Suisse (integrated production) and the Association of Swiss Rabbit Producers.

C O N F L I C T O F I N T E R E S T
The authors declare that they have no conflict of interest. The project was part of project 1.19.01 of the Swiss Federal Food Safety and Veterinary Office.

E T H I C S S TAT E M E N T
All protocols were carried out in strict accordance with the Swiss Federal Food Safety and Veterinary Office guidelines (Animal Protection Act, Animal Protection Ordinance 15 ). Approval number 30940; BE110/18.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.