Research Note: Validation of a new differentiation approach using the commercial ASAP^TM media to detect the Salmonella 441/014 vaccine strain

Abstract

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAP^TM). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods.

INTRODUCTION

Salmonella spp. is currently the second most common zoonotic pathogen in Europe causing gastrointestinal infection in humans. In 2021, a total of 60,050 cases were reported in the European Union (EU), resulting in 11,785 hospitalizations and 71 fatalities. Salmonella is strongly related to foodborne outbreaks, especially from poultry products such as undercooked meat and eggs (EFSA and ECDC, 2022).

Due to the importance of Salmonella from a public health point of view, in 2007 National Salmonella Control Programs (NSCP) according to the European Regulation 2160/2003 were established to reduce the Salmonella prevalence to 1% or less for breeding flocks of Gallus gallus, broilers and turkeys, and 2% for laying hens (EFSA and ECDC, 2022; Regulation (EC), 2160/2003). Target serovars of breeding flocks are Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), monophasic Salmonella Typhimurium (mST), Salmonella Infantis (SI), Salmonella Virchow (SV) and Salmonella Hadar (SH). In the rest of the poultry production chain, broilers, layers, and turkeys Salmonella controlled serovars are SE and ST, including mST. NSCP include biosecurity and hygiene measures, together with regular monitoring as well as cleaning and disinfection protocols on the farms and prophylactic methods, such as vaccination (Trampel et al., 2014; Sevilla-Navarro et al., 2020).

Vaccination is presented as one of the most important control tools to reduce Salmonella in poultry production (Sharma et al., 2018), being different existing vaccines solutions on the market. Killed vaccines with different combination of antigens and live attenuated vaccines for SE and/or ST protection. However, for a live vaccine to be licensed for field use, detection methods that can differentiate the vaccine strain from wild strains should be provided.

In this regard, Cevac Salmovac is based on an auxotrophic Salmonella strain for adenine and histidine. The vaccine and wild strain differentiation can be done with a culture differentiation kit (S-Check) which consists of two different media, one with adenine and histidine and one without. The vaccine strain is only able to grow in the media with adenine and histidine while wild strains can grow in both media. Additionally, due to specific mutation points of the vaccine strain, specific primers could be developed and ready-to-use PCR kits have been commercialized for the differentiation of Salmonella 441/014 and wild Salmonella strains (Anicon SE Kylt PCR DIVA 1 and vetproof STM Vaccine Detection 1 Kit).

The time required to obtain the definitive laboratory result enabling to conclude on whether the detected Salmonella is a vaccine strain or not is critical for the farmer, the veterinarian and the authorities (Maurischat et al., 2015). It is particularly critical in layers, as the daily produced eggs cannot be commercialized until this result in known. Thus, there was a need to have another simple and rapid differentiation solution on the market for the laboratories.

This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain form field strains, using a chromogenic Media, ASAP manufactured by bioMérieux.

MATERIAL AND METHODS

The validation study was performed following the ISO 16140-6:2019 guidelines from January 2020 to June 2021 by the Centro de Calidad Avícola y Alimentación Animal de la Comunidad Valenciana (CECAV, Spain).

The validation of the proposed method included the comparative study with PCR real time differentiation and the microbiological differentiation Ceva S-Check. More specifically, the exclusivity and specificity of the ASAP-based differentiation have been assessed, with 1) exclusivity as the capacity of the assay to specifically and persistently detect the vaccine strain obtained in different contexts; and 2) inclusivity as the capacity of the assay to differentiate the vaccine SE 441/014 strain from other SE strains.

Inclusivity and exclusivity study

Selection of the strains. For the inclusivity criteria a total of 50 SE isolates (SEv) from strain 441/014 (B1-B50) supplied by Ceva Animal Health and field farms vaccinated isolated by CECAV were tested. For the exclusivity criteria 100 field SE (SEf) isolates (C1-C100) from poultry farms were selected. Finally, 55 strains different from the SE serovar (SEe) were also included in the panel test with 20 Salmonella strains (D1-D20) from different serovars and different subspecies (S. enterica subspecies salamae (II), arizonae (IIIa) and diarizonae (IIIb), that share same somatic (O) and flagellar (H) antigens), 10 commercially available vaccine isolates distinct from the 441/014 vaccine strain (A1-A10) and 25 strains from different genus but belonging to the Enterobacteriaceae family were included (E1-E25) (Table 1).

Table 1.

Strain information for the inclusivity criteria.

Study ID	n	Organism	Type of strain	Origin	Source
B1-B21	21	SE	Vaccine strain	Vaccine Passage strain	CEVA
B22-49	19		Vaccine isolated strain	Rearing hens	CECAV
	8			Layer hens
B50	1		441/014	Commercial vaccine Lyophilised	CEVA
A1-A5	5	SE	Vaccine strain	Avipro Salmonella Duo*	ELANCO
A6-A10	5
C1-C100	7		Field strain	Layer hens	CECAV
	13			Rearing hens
	3			Fattening turkeys
	3			Broiler
	44			Subcontracted laboratory
	19			Reference Veterinary Laboratory
	1		Lab strain	CECT 4300
	1		Field strain	Numida meleagris
	1			Anser anser
	4			Anas platyrhynchos domesticus
	4			Parent Strain	CEVA
D1-D20	1	S. enterica subsp. salamae (II) 16: g, m, s, t: -	Field strain	Laying hens	CECAV
	1	S. enterica subsp. arizonae (IIIa) 48: Z4, z23: -		Broiler
	1	S. Corvallis 8, 20: Z4, z23: [z6]
	1	S. enterica subsp. arizonae (IIIa) 48: Z4, z23: -		Fattening turkeys
	1	S. enterica subsp. diarizonae (IIIb) 38: r: z		Reference Veterinary Laboratory
	1	S. Bareilly 6,7,14: y: 1,5
	1	S. Amsterdam 3,{10} {15} {15,34}: g,m,s: -
	1	Monophasic S. Typhimurium 1,4,[5],12: i: -
	1	S. Coeln 1,4,[5],12: y: 1,2
	1	S. Typhimurium 1,4,[5],12: i: 1,2
	1	S. Infantis 6,7,14: r: 1,5
	1	S. Fresno 9,46: z38: -
	1	S. Yoruba 16: c: l,w
	1	S. Virchow 6,7,14: r: 1,2
	1	S. Berta 1,9,12: [f], g, [t]: -
	1	S. Bareilly 6,7,14: y: 1,5
	1	S. Chester 1,4,[5],12: e, h: e, n, x
	1	S. Mbandaka 6,7,14: z10: e, n, z15	Intercomparative strains	Reference Veterinart Laboratory
	1	S. Llandoff 1,3,19: z29: [z6]
	1	S. Ouakam 9,46: z29: -
E1-E25	7	Proteus spp.	Field strain	Broiler	CECAV
	1			Layer hens
	1	Citrobacter freundii	Laboratory strain	CECT 7464
	11	Escherichia coli	Field strain	Broiler
	1			Laying hens
	2			Fattening turkeys
	2			Rearing hens

CECT: Spanish Collection of Type Culture (Colección Española de Cultivos Tipo).

The preparation of the working cultures was carried out in accordance with ISO 11133:2014 and its modifications ISO 11133:2014/Amd 1:2018 and ISO 11133:2014/Amd 2:2020. Cultures were tested for purity and biochemical confirmation was conducted using API 20E (Biomerieux, Madrid, Spain). Salmonella strains were additionally serotyped in accordance with ISO/TR 6579-3: 2014. Inclusivity and exclusivity tests were combined into a single study. Sample lysates were blind-coded and intermingled.

Comparison Study Between Methods

The ASAPbased differentiation method was compared with two already authorized methods, Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit. All isolates were plated on Columbia blood agar (BA) (Oxoid, Madrid, Spain) and incubated at 37 ± 1°C for 24 ± 3 h. Hereafter, these colonies were processed differently based on the differentiation method.

Anicon PCR Kylt assay DIVA 1 (real time PCR). Pure strains from BA were suspended with 200 μL of DNA Extraction-Mix and incubated for 10 to 15 min at 100 ± 3°C. Samples were vortexed and centrifuged for 5 min at 10,000 x g. The supernatant obtained was the extracted DNA for PCR. The PCR protocol comprised an initial step at 95°C for 1 min, followed by 42 cycles of denaturation at 95°C for 15 s and alignment and extension at 60°C for 1 min. The total final volume for each PCR reaction was 20 μL, consisting of 18 μL of Reaction Mix (Kylt SE DIVA 1, AniCon, Germany) and 2 μL of Cy5 (SEv 441/014), ROX (SEf) or sample suspension. The real time PCR method allowed the differentiation of SE live vaccine strain 441/014 (ade-/his-) (SEv1) from its field strains (SEf).

S-Check Diagnostic assay. This assay is based on two broth media (A and B) in which field strains can grow, showing turbidity. In contrast, the vaccine strain only grows on media B due to their nutritional requirements (adenine and histidine). In this study, pure colonies were inoculated in both broth media (A and B) and then incubated for 48 ± 3 h at 37 ± 1°C. The differentiation between field and vaccine strains was performed qualitatively by assessing the presence or absence of turbidity in the respective media.

Alternative method in ASAP. To obtain proper isolated colonies, a single colony from each of the s isolates being tested was transferred from the BA to the ASAP media (bioMérieux, Madrid, España). ASAP plates were incubated at 37 ± 1°C for 24 ± 3 h and, after incubation the colonies were observed. Pink-purple coloration indicates the presence of Salmonella spp. field isolates, while dark purple blue- exhibits SE 441/014 strain (Figure 1). The principle of this medium is based on the detection of C8-esterase activity which is observed in all species of Salmonella. The C8-esterease activity of Salmonella is visualized by the pink to purple coloring if their colonies (specific cleavage of the substrate). Different coloured isolated colonies were confirmed for Salmonella detection following ISO 6579-1:2017.

Figure 1.

Alternative differentiation method in ASAP. Left: pink-purple coloration indicative of Salmonella strains. Right: dark purple blue- coloration compatible with vaccine 441/014 strain.

Statistical analysis

A Generalised Linear Model (GLM), which assumed a binomial distribution for SEv 441/014 strain, SEf strain, and SEe was fitted to the data to determine the inclusivity and exclusivity of the differentiation methods (PCR Kylt vs. S-Check vs. ASAP). Further a GLM was used to assess the significance of the results in the inclusivity and exclusivity test including the isolates different form SE serovar and Salmonella spp. Statistical differences were based on a P-value level of < 0.05. Statistical analyses were performed using SPSS 27.0 software package (SPSS Inc., Chicago, IL).

RESULTS AND DISCUSSION

Vaccination is considered as the most efficient approach to control Salmonella contamination in poultry at farm level. Different inactivated and live attenuated Salmonella vaccines are commercially available and used in most European countries. Inactivated vaccines primarily stimulate humoral immunity, whereas live attenuated Salmonella vaccines can activate both cellular and humoral immune responses, providing enhanced protection in poultry (Redweik et al., 2020; Lyimu et al., 2023). Nevertheless, live vaccines replicate in the birds and may be shed in the environment; differentiation tests are therefore compulsory to ensure compliance with regulations (EC 2160/2003). This testing is essential for distinguishing between the vaccine strain and wild-type Salmonella strains and can be performed using a variety of tools, including serological tests, molecular tests, bacteriological tests, and whole-genome sequencing (WGS) (Maurischat et al., 2015; Tang et al., 2019).

Current differentiation assays approved by the European Medicine Agency (EMA) for the Cevac Salmovac vaccine strain are based on PCR (Kylt DIVA1 and vetproof STM Vaccine Detection 1 Kit) and selective growth media (S-Check) from pure colonies. This implies that all steps for Salmonella isolation following the ISO 6579-1:2017 must be carried out (ISO 6579-1:2017, 2017). Even though the isolate differentiation can be carried out in 2 h and 48h respectively, with the Kylt PCR kit and the S-Check assay SE, the whole testing flow involves a 5-d period to obtain isolated colonies and an additional day for serotyping SE. Alternatively, with the ASAP method, performed from the isolated ASAP colonies of an intermediate step in the ISO standard, the result can be obtained in 4 d, thereby optimizing the laboratory process and field work in 2 d.

In this study, we show that the ASAP media is able to differentiate typical SEv from SEf and SEe, not being restrictive to a single strain type, obtaining 100% agreement between the identification method of Salmonella Enteritidis vaccine (Cevac Salmovac) by 3 assays and techniques: ASAP^TM seeding, the Kylt DIVA1 assay (Anicon) and the S-Check Salmonella Diagnostic assay. The differentiation coloring from the SEv and SEf is based on a phenotypic feature, with dark to blue coloring of the SE vaccine strain as opposed to the pink coloring of other SE strains. Thus, 441/014 strain has been added to the quality control of ASAP to ensure this feature will be sustained over time.

No significant differences in the detection of the vaccine 441/014 strain between the current methods (PCR Kylt and S-Check®) and the newly described method (ASAP) (P-value = 1.000). This demonstrates that the alternative ASAP^TM method can be used to differentiate the SE (auxotrophic adenine and histidine) vaccine strain 441/014 from SE field strains, as we found 100% of accordance with the official differentiation assays. A false negative rate of 0% (100% inclusivity) and a false positive rate of 0% (100% exclusivity) were determined, matching all results obtained from the 3 tested methods.

In conclusion, results obtained in this study demonstrated 100% agreement between three macroscopic identification methods of SE vaccine 441/014 (Cevac Salmovac), and confirmed a new method of differentiation. The ASAP has been approved by the Spanish Medical Agency (AEMPS) to be included as a differentiation method to be used for 441/014 SE vaccine strain (AEMPS, 2023). This method is of great interest for application at field level as it reduces the waiting time to obtain a vaccine strain result from in case of positive self-control results (Anonymous, 2023). Given the intensive poultry farming the rapid differentiation between SEv and SEf strains holds crucial significance for both the efficiency of farm operations and public health concerns. Swift and accurate differentiation allows for timely intervention and appropriate management strategies to prevent the spread of pathogenic strains, ensuring the well-being of both poultry and human populations (Maurischat et al., 2015; Liebhart et al., 2023). The identification method in ASAP presents an intriguing option due to its simplicity of implementation, minimal resources required, cost, and rapid turnaround time for results.

Disclosures

The study was conceived, developed, and analyzed by J.G.L, C.G, P.C-G, and S.S-N from CECAV which declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.