Accurate Identification of Salmonella enterica in Calves

Abstract

Salmonella enterica is a bacterium that can cause serious infections in humans and animals, especially cattle. The identification and differentiation of S. enterica serotypes in cattle is important for epidemiological surveillance, disease prevention and control, and public health protection. However, the current methods and techniques for S. enterica detection have various challenges and limitations, such as low sensitivity and specificity, high cost and complexity, and the need for viable and pure bacterial cultures. Therefore, there is a need for further research and development of novel and improved methods and techniques that can overcome these challenges and provide reliable and accurate information on S. enterica serotypes in cattle. Such information can help to improve the understanding of the epidemiology, pathogenesis, and transmission of S. enterica in cattle, as well as to design and implement effective strategies for its prevention and control. This chapter reviews the current methods and techniques for S. enterica detection, such as culture-based methods, biochemical methods, molecular methods, phage-based methods, and biosensor methods, and discusses their advantages and disadvantages, as well as their future trends and perspectives.

1. Introduction

Salmonella is a bacterium that can cause serious infections in humans and animals, especially in young, immunocompromised, or stressed individuals [1]. It has over 2000 strains, which are classified into serotypes based on their antigenic properties [2]. The serotypes differ in their pathogenicity and prevalence in different host species, including cattle. Some serotypes, such as S. Typhimurium and S. Enteritidis, are able to cause systemic disease in a wide range of hosts, while others, such as S. Dublin and S. Choleraesuis, are more host-specific and adaptable to cattle and pigs, respectively [2]. The prevalence of Salmonella serotypes in cattle varies by region, production system, and sampling method. A systematic review and meta-analysis of studies published between 2000 and 2017 estimated a pooled prevalence of Salmonella in apparently healthy cattle of 9%, with the highest prevalence in North America (16%) and the lowest in Europe (2%) [3]. The most frequently reported serotypes in cattle are S. Montevideo, S. Typhimurium, S. Kentucky, S. Meleagridis, and S. Anatum [3]. However, the serotype distribution may change over time due to factors such as antimicrobial resistance, vaccination, and animal trade [4, 5]. In cattle, Salmonella enterica can cause salmonellosis, a disease characterized by fever, diarrhea, dehydration, septicemia, abortion, and sometimes death [2]. The incidence and impact of salmonellosis in cattle vary depending on the serotype of S. enterica, the host susceptibility, and environmental factors [3, 4, 5]. Therefore, accurate identification and differentiation of S. enterica serotypes in cattle is essential for epidemiological surveillance, disease prevention and control, and public health protection [6, 7, 8].

The conventional methods for identifying and differentiating S. enterica serotypes in cattle are based on bacterial culture and biochemical and serological tests. However, these methods are time-consuming, labor-intensive, and may not be able to detect all the serotypes or distinguish closely related serotypes [9]. Moreover, these methods require viable and pure bacterial cultures, which may not always be available or feasible. Molecular methods can be used to identify and differentiate S. enterica serotypes in cattle directly from clinical samples, such as feces, tissues, or fluids [5, 10, 11]. These methods are based on the detection and amplification of specific DNA sequences of S. enterica, such as the invA gene, which encodes an invasion protein, or the rfb gene cluster, which encodes the O antigen [12]. Molecular methods can also provide information on the genetic diversity, phylogeny, and epidemiology of S. enterica serotypes in cattle [13].

In this chapter, we will review the current molecular methods for identifying and differentiating S. enterica serotypes in cattle, such as polymerase chain reaction (PCR), multiplex PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), and whole-genome sequencing (WGS) [12, 13, 14, 15, 16, 17]. We will also discuss the advantages and limitations of each method, as well as the future perspectives and challenges for improving the accuracy and reliability of S. enterica identification and differentiation in cattle. Furthermore, for the first time, we will present a case report of a new serovar of S. enterica that was detected in Mediterranean buffalo calves (Bubalus bubalis) using a combination of molecular and conventional methods. This case illustrates the importance and potential of molecular methods for discovering and characterizing novel S. enterica serotypes in cattle and other animal species.

1.1 Some application examples of the molecular methods for identifying Salmonella in cattle include the following

PCR: PCR is a technique that amplifies a specific DNA fragment from a complex mixture of DNA. PCR can be used to detect S. enterica in cattle by targeting the invA gene, which is present in all S. enterica serotypes. PCR can also be used to differentiate S. enterica serotypes by targeting the rfb gene cluster, which encodes the O antigen [12]. For example, a study by Eriksson et al. compared the performance of PCR, enzyme-linked immunosorbent assay (ELISA), and culture methods for Salmonella detection in fecal samples from cattle, pigs, and poultry. The results showed that PCR had a higher sensitivity and specificity than ELISA and culture methods and could detect S. enterica serotypes that were not detected by the other methods [13].

Multiplex PCR: Multiplex PCR is a technique that amplifies multiple DNA fragments simultaneously in a single reaction. Multiplex PCR can be used to identify and differentiate S. enterica serotypes in cattle by targeting multiple genes or regions that are specific for different serotypes [12]. A study by D’Angelo et al. used multiplex PCR to identify and differentiate two S. enterica serotypes, S. enterica subsp. enterica O:35 and a new serovar of S. enterica, in fecal samples from water buffalo calves that showed severe gastroenteritis. Multiplex PCR targeted the invA gene and the rfb gene cluster and could distinguish the two serotypes based on the size of the amplified products [14].

Real-time PCR: Real-time PCR is a technique that monitors the amplification of DNA in real time by using fluorescent probes or dyes. Real-time PCR can be used to quantify S. enterica in cattle by measuring the fluorescence intensity of the amplified products. Real-time PCR can also be used to differentiate S. enterica serotypes in cattle by using specific probes or melting curve analysis [15]. A study by Harbottle et al. used real-time PCR to compare the genetic diversity of S. enterica serotype Newport isolates from cattle, humans, and food. Real-time PCR was used to target the invA gene, and melting curve analysis was used to differentiate the isolates based on single nucleotide polymorphisms (SNPs) in the invA gene [15].

LAMP: LAMP is a technique that amplifies DNA under isothermal conditions by using a set of primers and a polymerase with strand displacement activity. LAMP can be used to detect S. enterica in cattle by targeting the invA gene or other genes that are specific to S. enterica [18]. LAMP can also be used to differentiate S. enterica serotypes in cattle by using different sets of primers or adding loop primers that are specific for different serotypes. For example, a study by Wang et al. used LAMP to detect and differentiate S. enterica serotypes Typhimurium, Enteritidis, and Dublin in milk samples. The LAMP assay targeted the invA gene and used loop primers that were specific for the three serotypes. The LAMP assay could detect and differentiate the three serotypes within 60 min, with a sensitivity of 10 CFU/mL [16, 17].

WGS: WGS is a technique that sequences the entire genome of an organism. This study analyzed the WGS data of S. enterica serotypes in cattle by comparing the genomic sequences of different isolates and identifying the genes or regions that are unique or variable for different serotypes [13]. WGS can also provide information on the phylogeny, evolution, and epidemiology of S. enterica serotypes in cattle [14, 15]. For example, a study by EFSA (European Food Safety Authority) used WGS to investigate the public health risks related to the consumption of raw drinking milk. The study analyzed the WGS data of S. enterica isolates from raw milk, cheese, and humans and identified the serotypes, subtypes, and antimicrobial resistance profiles of the isolates. This study also traced the sources and transmission routes of S. enterica in raw milk and cheese production [19, 20, 21].

1.2 The case report of a new serovar of S. enterica in Mediterranean buffalo calves is as follows

The case report of the new serovar of S. enterica in Mediterranean buffalo calves is based on the analysis of fecal samples collected from live water buffalo calves that showed signs of severe gastroenteritis. The samples were cultured on selective media and incubated at 37°C for 24 hours. The colonies were identified by biochemical tests and serotyping using specific antisera. The molecular methods included PCR amplification of the invA gene and the 16S rRNA gene, followed by sequencing and phylogenetic analysis. The results confirmed the presence of two S. enterica serotypes, S. enterica O:35 and a new serovar of S. enterica, in the intestinal contents of the water buffalo calves. The new serovar of S. enterica was named S. enterica subsp. enterica serovar Buffalo [22].

2. Sample collection, culture methods, and biochemical tests used for S. enterica

The conventional methods for the identification and differentiation of S. enterica serotype in cattle are based on the culture and biochemical characterization of the isolates obtained from the samples. The culture methods involved the enrichment, isolation, and confirmation of S. enterica from various types of samples, such as feces, milk, tissues, or environmental swabs. Biochemical tests are used to determine the serogroup and serotype of the isolates based on their antigenic properties, such as the presence of O (somatic), H (flagellar), and Vi (capsular) antigens [23, 24]. These methods are widely used in veterinary diagnostic laboratories and epidemiological studies, as they are relatively simple, inexpensive, and standardized.

In this section, we will describe the conventional methods for sample collection, culture, and biochemical identification of S. enterica in cattle, as well as their advantages and disadvantages. We will also mention some of the challenges and limitations of these methods and suggest some possible solutions and improvements.

2.1 Sample collection

The first step in the detection of S. enterica in cattle is the collection of appropriate samples [18, 25, 26]. The types of samples that can be collected for S. enterica detection include feces, tissues, fluids, food, and environmental samples [22]. The choice of sample depends on the purpose and objective of the testing, the clinical signs and status of the animals, and the availability and feasibility of the sampling methods [27].

Fecal samples are the most common and convenient samples for S. enterica detection in cattle because they can reflect intestinal colonization and shedding of bacteria [28]. Fecal samples can be collected from individual animals or from pooled samples of a group of animals. Fecal samples can be collected by rectal swabs, fecal grab, or fecal cup. Rectal swabs are easy and quick to perform, but they may not collect enough material and may be contaminated by the rectal mucosa [29]. Fecal grab is the method of collecting a handful of feces from the rectum of the animal, which can provide more material and less contamination, but it may be difficult and stressful for the animal and the sampler. A fecal cup is a method of collecting feces from the ground after the animal defecates, which can avoid the stress and discomfort of the animal and the sampler, but it may be affected by environmental contamination and degradation [30].

Tissue samples are another type of sample that can be collected for S. enterica detection in cattle, especially in cases of systemic infection, abortion, or postmortem examination [31]. Tissue samples can include lymph nodes, spleen, liver, kidney, lung, brain, placenta, fetus, and other organs that may be affected by S. enterica [18, 32, 33]. Tissue samples can be collected by biopsy, necropsy, or abortion investigation. A biopsy is a method of collecting a small piece of tissue from a living animal, which can provide early diagnosis and treatment, but it may be invasive and risky for the animal and the sampler [8]. Necropsy is the method of collecting tissue samples from a dead animal, which can provide comprehensive and conclusive information, but it may be too late and costly for the prevention and control of the disease. Abortion investigation is the method of collecting tissue samples from the aborted fetus and placenta, which can provide valuable information on the cause and source of the abortion, but it may be difficult and sensitive to perform [34].

Fluid samples are another type of sample that can be collected for S. enterica detection in cattle, especially in cases of septicemia, mastitis, or urinary tract infection. Fluid samples can include blood, milk, urine, and other body fluids that may be infected by S. enterica [35]. Fluid samples can be collected by venipuncture, milking, catheterization, or aspiration. Venipuncture is the method of collecting blood from a vein of the animal, which can provide information on the systemic infection and immune response, but it may be stressful and painful for the animal and the sampler [36, 37]. Milking is the method of collecting milk from the udder of the animal, which can provide information on mastitis and udder health, but it may be influenced by the milking hygiene and frequency [38]. Catheterization is the method of collecting urine from the bladder of the animal, which can provide information on the urinary tract infection and kidney function, but it may be invasive and risky for the animal and the sampler [32, 39, 40, 41]. Aspiration is the method of collecting fluid from a cavity or lesion of the animal, which can provide information on the local infection and inflammation, but it may be difficult and complicated to perform. The advantages and disadvantages of each sampling method are summarized in Table 1.

Sampling method	Advantages	Disadvantages
Venipuncture	Provides information on the systemic infection and immune response; Can be performed on any animal with a visible vein Can be done with minimal equipment and training	Stressful and painful for the animal and the sampler; May cause bleeding, bruising, or infection at the puncture site May be difficult to obtain enough blood volume or quality
Milking	Provides information on the mastitis and udder health Can be done on any lactating animal Can be integrated with the routine milking procedure	Influenced by the milking hygiene and frequency May cause contamination or cross-contamination of the milk samples May be affected by the stage of lactation or the presence of antibiotics
Catheterization	Provides information on the urinary tract infection and kidney function Can provide a sterile and uncontaminated urine sample Can be done on any animal with a bladder	Invasive and risky for the animal and the sampler May cause injury, infection, or inflammation of the urinary tract May require sedation or anesthesia of the animal
Aspiration	Provides information on the local infection and inflammation Can provide a direct and concentrated fluid sample Can be done on any animal with a cavity or lesion	Difficult and complicated to perform May cause damage, bleeding, or infection of the tissue or organ May require specialized equipment and training
Venipuncture	Provides information on the systemic infection and immune response Can be performed on any animal with a visible vein Can be done with minimal equipment and training	Stressful and painful for the animal and the sampler May cause bleeding, bruising, or infection at the puncture site May be difficult to obtain enough blood volume or quality

Table 1.

Advantages and disadvantages of fluid sampling methods for S. enterica detection in cattle.

The quality and quantity of the fluid samples collected for S. enterica detection in cattle depend on various factors, such as the sampling time, location, method, storage, and transport [42]. The sampling time should be chosen according to the epidemiological situation, clinical signs, and shedding patterns of the animals [43]. The sampling location was selected according to the source and route of the infection, the distribution and concentration of the bacteria, and the accessibility and availability of the sampling sites. The sampling method should be chosen according to the type and nature of the fluid, the purpose and objective of the testing, and the feasibility and acceptability of the sampling procedure. The storage and transport of fluid samples should be performed according to the temperature and humidity conditions, the preservation and protection of the samples, and the duration and distance of the storage and transport [34, 44].

Some general guidelines and recommendations for optimal fluid sample collection and handling for S. enterica detection in cattle are as follows: Samples are collected as soon as possible after the onset of the infection or the outbreak or before the initiation of the treatment or the intervention. Samples from multiple animals or sources or from multiple sites or locations were collected to increase the probability and representativeness of detection [45]. The samples were collected using sterile and appropriate equipment and containers and labeled clearly and correctly. Store and transport samples at refrigerated temperatures (4°C) and in insulated and sealed containers. Freezing and thawing cycles, exposure to sunlight or heat, and prolonged storage or transport times were avoided. The samples were processed and analyzed as soon as possible after arrival at the laboratory or stored at frozen temperatures (−20°C or lower) until further analysis [46].

The current standards for sampling quantities of different fluid samples for S. enterica detection in cattle vary depending on the type of fluid, the method of analysis, and the level of sensitivity and specificity required. However, some general recommendations are as follows: collect at least 10 ml of blood per animal, use a sterile syringe and needle, and transfer it to a sterile tube containing an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA) or heparin [35, 47, 48]. At least 50 ml of milk per animal was collected using a sterile container and mixed well before aliquoting [36]. At least 10 ml of urine per animal was collected using a sterile catheter and syringe and transferred to a sterile tube or container [37, 49]. At least 5 ml of fluid per animal was collected using a sterile needle and syringe and transferred to a sterile tube or container [38]. The sampling quantities of different fluid samples are summarized in Table 2.

Fluid sample	Sampling quantity
Blood	10 ml
Milk	50 ml
Urine	10 ml
Fluid	5 ml

Table 2.

Sampling quantities of different fluid samples for Salmonella enterica detection in cattle.

Food samples are another type of sample that can be collected for S. enterica detection in cattle, especially in cases of foodborne outbreaks or surveillance. Food samples can include raw or processed meat, milk, cheese, and other products that may be contaminated by S. enterica. Food samples can be collected by sampling plans, random sampling, or targeted sampling. The advantages and disadvantages of each sampling method are summarized in Table 3.

Sampling method	Advantages	Disadvantages
Sampling plans	Provides representative and consistent information Can be designed according to the specific criteria and objectives Can be validated and standardized	Complex and costly to implement May require large number or size of samples May not detect rare or sporadic contamination
Random sampling	Provides unbiased and general information Can be applied to any population or lot Can be simple and easy to perform	Not sufficient or efficient to detect low levels of contamination May not reflect the variability or heterogeneity of the population or lot May not identify the source or cause of the contamination
Targeted sampling	Provides focused and relevant information Can be directed to the suspected or known sources or locations Can be sensitive and specific to detect contamination	Not comprehensive or objective to assess the overall situation May miss other potential sources or locations May be influenced by the prior knowledge or bias

Table 3.

Advantages and disadvantages of food sampling methods for Salmonella enterica detection in cattle.

Environmental samples are another type of sample that can be collected for S. enterica detection in cattle, especially in cases of environmental monitoring or investigation [31, 50, 51]. Environmental samples can include water, soil, bedding, feed, equipment, and other materials that may be contaminated by S. enterica [33]. Environmental samples can be collected by swabs, filters, scoops, or wipes. The advantages and disadvantages of each sampling method are summarized in Table 4.

Sampling method	Advantages	Disadvantages
Swabs	Simple and convenient to perform Can be used for any surface or object Can be stored and transported easily	Not collect enough material Affected by the moisture and texture of the surface or object May require enrichment or concentration steps
Filters	Quantitative and sensitive to detect contamination Can be used for water or air samples Can be processed and analyzed directly	Complex and expensive to perform May require specialized equipment and training May be influenced by the volume or flow rate of the sample
Scoops	Abundant and representative of the sample Can be used for soil, feed, or other loose material Can be mixed and homogenized easily	Bulky and messy to handle May require subsampling or dilution steps May be affected by the environmental conditions or degradation
Wipes	Large and uniform of the sample Can be used for flat or smooth surfaces or objects Can be stored and transported easily	Not suitable for rough or irregular surfaces or objects May require enrichment or concentration steps May cause cross-contamination or interference

Table 4.

Advantages and disadvantages of environmental sampling methods for S. enterica detection in cattle.

The quality and quantity of food and environmental samples collected for S. enterica detection in cattle depend on various factors, such as the sampling time, location, method, storage, and transport [25, 52]. The sampling time should be chosen according to the epidemiological situation, clinical signs, and shedding patterns of the animals [28, 53]. The sampling location was selected according to the source and route of the infection, the distribution and concentration of the bacteria, and the accessibility and availability of the sampling sites. The sampling method should be chosen according to the type and nature of the sample, the purpose and objective of the testing, and the feasibility and acceptability of the sampling procedure. The storage and transport of the samples should be done according to the temperature and humidity conditions, the preservation and protection of the samples, and the duration and distance of the storage and transport [34, 54].

Some general guidelines and recommendations for optimal food and environmental sample collection and handling for S. enterica detection in cattle are as follows: Samples are collected as soon as possible after the onset of the infection or the outbreak or before the initiation of the treatment or the intervention [55]. Samples from multiple animals or sources or from multiple sites or locations were collected to increase the probability and representativeness of the detection. The samples were collected using sterile and appropriate equipment and containers and labeled clearly and correctly. Store and transport samples at refrigerated temperatures (4°C) and in insulated and sealed containers [56]. Freezing and thawing cycles, exposure to sunlight or heat, and prolonged storage or transport times were avoided. The samples were processed and analyzed as soon as possible after arrival at the laboratory or stored at frozen temperatures (−20°C or lower) until further analysis.

The current standards for sampling quantities of different food and environmental samples for S. enterica detection in cattle vary depending on the type of sample, the method of analysis, and the level of sensitivity and specificity required. However, some general recommendations are as follows: at least 25 g of meat or cheese should be collected per sample using a sterile knife or scissors and transferred to a sterile bag or container [39, 55]. At least 100 ml of milk per sample was collected using a sterile container and mixed well before aliquoting [57]. At least 10 g of swab, filter, scoop, or wipe was collected per sample using a sterile device and transferred to a sterile bag or container [40, 58]. The sampling quantities of different food and environmental samples are summarized in Table 5.

Food or environmental sample	Sampling quantity
Meat or cheese	25 g
Milk	100 ml
Swab, filter, scoop, or wipe	10 g

Table 5.

Sampling quantities of different food and environmental samples for S. enterica detection in cattle.

Some general guidelines and recommendations for optimal sample collection and handling for S. enterica detection in cattle are as follows:

• To ensure timely and accurate detection, samples were collected as soon as possible after the onset of the infection or the outbreak or before the initiation of the treatment or the intervention.

• To increase the probability and representativeness of detection, samples were collected from multiple animals or sources or from multiple sites or locations [59, 60].

• To prevent contamination and degradation of the samples, appropriate and sterile equipment, containers, and materials were used, and standard operating procedures and good laboratory practices were followed.

• To facilitate identification and tracking of the samples, the samples were labeled and recorded with relevant information, such as the date, time, location, animal, type, and method of the collection.

• To maintain the viability and integrity of the samples, they were stored and transported at refrigerated temperatures (2–8°C) and in sealed and leak-proof containers to avoid freezing, thawing, or exposing them to direct sunlight or heat [40, 61].

• To expedite the analysis and reporting of the results, the samples should be delivered and processed as soon as possible, preferably within 24 hours, or appropriate preservatives or stabilizers should be used.

2.2 Culture methods

Culture methods are widely used for S. enterica detection in cattle, as they can provide viable and pure cultures of the bacteria, which can be further identified and characterized by biochemical and molecular methods [45, 62]. However, culture methods are also time-consuming and labor-intensive and may not be able to detect all the serotypes or distinguish closely related ones. Moreover, they require viable and pure bacterial cultures, which may not always be available or feasible [28, 63, 64]. Therefore, there is a need for rapid, sensitive, and specific molecular methods that can identify and differentiate S. enterica serotypes in cattle directly from clinical samples, such as feces, tissues, or fluids [40, 61, 62].

The application of culture methods for S. enterica detection in cattle has been demonstrated in various studies and reports, which have shown the prevalence, incidence, distribution, and diversity of S. enterica serotypes in cattle populations, as well as their association with clinical signs, risk factors, and transmission routes [28, 61, 63]. Some examples of these studies and reports are as follows.

A study by the author [64] investigated the prevalence and serotypes of S. enterica in dairy cattle in Ontario, Canada, using culture and serotyping methods. The study found that the overall prevalence of S. enterica was 2.8% and that the most common serotypes were Typhimurium, Newport, and Dublin. The study also found that the prevalence of S. enterica was higher in calves, heifers, and cows with diarrhea and that the main risk factors were herd size, housing type, and feed type.

A study by the author [50] compared the culture and PCR methods for S. enterica detection in fecal samples of beef cattle in Nebraska, USA. The study found that the culture method detected S. enterica in 6.3% of the samples, while the PCR method detected S. enterica in 12.5% of the samples. The study also found that the most common serotypes detected by culture were Anatum, Montevideo, and Mbandaka, and that the PCR method had a higher sensitivity and specificity than the culture method.

A report by the author [65] analyzed the S. enterica isolates from cattle submitted to the National Veterinary Services Laboratories in the United States from 2014 to 2018, using culture and serotyping methods. The report found that the most common serotypes isolated from cattle were Dublin, Newport, Cerro, and Typhimurium and that the serotype distribution varied by region, season, and age group. The report also found that some serotypes, such as Dublin and Newport, were more likely to cause systemic infections, while others, such as Typhimurium and Montevideo, were more likely to cause enteric infections.

These examples show the usefulness and limitations of culture methods for S. enterica detection in cattle and the need for further research and development of novel and improved methods that can overcome these challenges and provide reliable and accurate information on S. enterica serotypes in cattle. Such information can help to improve the understanding of the epidemiology, pathogenesis, and transmission of S. enterica in cattle, as well as to design and implement effective strategies for its prevention and control.

The general procedure for the culture of S. enterica from the samples involves two steps: enrichment and isolation. Enrichment is the step of incubating the samples in a liquid medium that favors the growth of S. enterica and inhibits the growth of other bacteria. Isolation is the step of transferring the enriched samples to a solid medium that allows the differentiation of S. enterica from other bacteria based on their colony morphology and color. There are various types of media that can be used for enrichment and isolation of S. enterica, and they can be classified into selective and differential media. Selective media are media that contain substances that inhibit the growth of certain bacteria, while allowing the growth of others [66]. Differential media are media that contain substances that change color or produce a reaction when metabolized by certain bacteria, while not affecting others. Some media can be both selective and differential, depending on their composition and function [66].

Some of the commonly used media for the enrichment of S. enterica are tetrathionate broth (TT), which is a selective medium that contains tetrathionate and inhibits the growth of most Gram-positive and some Gram-negative bacteria, while allowing the growth of S. enterica and some other enteric bacteria. It also contains bile salts, which inhibit the growth of non-enteric bacteria, and iodine, which inhibits the growth of Proteus spp. and some other bacteria that can produce hydrogen sulfide. TT is used as a primary enrichment medium for S. enterica detection in fecal, food, and environmental samples [67]. Rappaport-Vassiliadis broth (RV or RVS): This is a selective medium that contains magnesium chloride and malachite green, which inhibit the growth of most bacteria, while allowing the growth of S. enterica and some other enteric bacteria. RV is used as a secondary enrichment medium for S. enterica detection in fecal, food, and environmental samples [39, 68]. Selenite cystine broth (SC): This is a selective medium that contains sodium selenite and cystine, which inhibit the growth of most bacteria, while allowing the growth of S. enterica and some other enteric bacteria. SC is used as a primary or secondary enrichment medium for S. enterica detection in fecal, food, and environmental samples [69].

Some of the commonly used media for the isolation of S. enterica are xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar (HE). These are selective and differential media that contain xylose, lysine, and deoxycholate, which inhibit the growth of most Gram-positive and some Gram-negative bacteria, while allowing the growth of S. enterica and some other enteric bacteria. It also contains phenol red, which changes color from red to yellow when xylose is fermented, and sodium thiosulfate and ferric ammonium citrate, which produce a black precipitate when hydrogen sulfide is produced [70]. XLD is used as a primary isolation medium for S. enterica detection in fecal, food, and environmental samples. On XLD, S. enterica colonies appear as red colonies with or without a black center, while other bacteria appear as yellow colonies with or without a black center or are inhibited [50]. Hektoen enteric agar (HE): This is a selective and differential medium that contains lactose, sucrose, and salicin, which inhibit the growth of most Gram-positive and some Gram-negative bacteria, while allowing the growth of S. enterica and some other enteric bacteria. It also contains bromothymol blue and acid fuchsin, which change color from green to yellow when lactose, sucrose, or salicin is fermented, and sodium thiosulfate and ferric ammonium citrate, which produce a black precipitate when hydrogen sulfide is produced. HE is used as a primary or secondary isolation medium for S. enterica detection in fecal, food, and environmental samples [42]. On HE, S. enterica colonies appear as green or blue-green colonies with or without a black center, while other bacteria appear as yellow or orange colonies with or without a black center or are inhibited.

Bismuth sulfite agar (BS): This is a selective and differential medium that contains bismuth sulfite, which inhibits the growth of most bacteria, while allowing the growth of S. enterica and some other enteric bacteria. It also contains brilliant green, which inhibits the growth of most Gram-positive and some Gram-negative bacteria, and sodium thiosulfate and ferric ammonium citrate, which produce a black precipitate when hydrogen sulfide is produced [70, 71]. BS is used as a primary or secondary isolation medium for S. enterica detection in fecal, food, and environmental samples. On BS, S. enterica colonies appear as black or brown colonies, while other bacteria appear as green or blue colonies or are inhibited [72].

The advantages of culture methods for S. enterica detection are that they are relatively simple, inexpensive, and widely available and that they can provide viable and pure cultures of the bacteria, which can be further identified and characterized by biochemical and molecular methods [45, 72]. The disadvantages of culture methods are that they are time-consuming, labor-intensive, and may not be able to detect all the serotypes or distinguish closely related ones. Moreover, they require viable and pure bacterial cultures, which may not always be available or feasible [71]. Therefore, there is a need for rapid, sensitive, and specific molecular methods that can identify and differentiate S. enterica serotypes in cattle directly from clinical samples, such as feces, tissues, or fluids.

3. Advances in recent techniques for the detection of Salmonella

Advances in recent techniques for the detection of S. enterica is a type of bacteria that causes food poisoning and typhoid fever in humans and animals. It can be transmitted through contaminated water or food and can cause diarrhea, fever, stomach cramps, nausea, vomiting, and sometimes blood in the stool. The detection and identification of S. enterica is crucial for the diagnosis, treatment, and prevention of the infection, as well as for the surveillance and control of the disease outbreak [5, 6]. Different techniques for S. enterica detection have different advantages and disadvantages, depending on their principles, procedures, performance, and applications. In this section, we will briefly compare some of the conventional and novel techniques for S. enterica detection in cattle, such as culture-based methods, molecular methods, immunological methods, phage-based methods, and biosensors.

Culture-based methods: These methods are based on the isolation and identification of S. enterica from the samples using selective and differential media, biochemical tests, and serological tests [38, 50]. The advantages of these methods are that they are simple, inexpensive, and widely available and that they can detect and identify viable S. enterica at the species and serotype level. They can also provide information on the antimicrobial susceptibility and resistance of the bacteria, which can be useful for therapy and control. The disadvantages of these methods are that they are time-consuming, labor-intensive, and prone to false negatives and false positives. They also require prior enrichment and culture of the samples, which can introduce contamination and variability [38, 50].

Molecular methods: These methods are based on the detection and identification of S. enterica from the samples using nucleic acid amplification techniques, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence-based amplification (NASBA) [38, 73]. The advantages of these methods are that they are rapid, sensitive, and specific and that they can detect and identify S. enterica at the species and serotype level, as well as the presence of virulence and resistance genes. They can also provide quantitative and real-time information about the bacteria, which can be useful for monitoring and diagnosis. The disadvantages of these methods are that they are complex, expensive, and require specialized equipment and trained personnel. They may also be affected by the quality and quantity of the DNA, the presence of inhibitors, and the specificity of the primers and probes [38, 73].

Immunological methods: These methods are based on the detection and identification of S. enterica from the samples using antigen-antibody reactions, such as enzyme-linked immunosorbent assay (ELISA), immunochromatographic assay (ICA), or immunofluorescence assay (IFA) [38, 52]. The advantages of these methods are that they are rapid, sensitive, and specific and that they can detect and identify S. enterica at the species and serotype level, as well as the presence of antigens and antibodies. They can also provide quantitative and semi-quantitative information about the bacteria, which can be useful for monitoring and diagnosis. The disadvantages of these methods are that they are complex, expensive, and require specialized equipment and trained personnel. They may also be affected by the stability and specificity of the antibodies, the interference and cross-reactivity of the antigens, and the calibration and validation of the assays [38, 52].

Phage-based methods: These methods are based on the detection and identification of S. enterica from the samples using bacteriophages, which are viruses that infect and lyse specific bacteria [38, 50]. The advantages of these methods are that they are rapid, sensitive, and specific and that they can detect and identify viable S. enterica directly from clinical samples without the need for culture or enrichment. They can also provide information on the susceptibility and resistance of the bacteria to phages, which can be useful for therapy and control. The disadvantages of these methods are that they are complex, expensive, and require specialized equipment and trained personnel. They may also be affected by the availability and specificity of the phages, the presence of other bacteria or viruses, and the environmental conditions [38, 50]. One of the most common phage-based methods is phage typing, which uses specific phages to identify S. enterica serotypes from the samples [74]. A recent study by [75] used phage typing to identify S. enterica serotypes in cattle fecal samples from dairy farms in Brazil and found that the most prevalent serotypes were Typhimurium, Enteritidis, and Dublin.

Biosensors: These devices are based on the detection and identification of S. enterica from the samples using biological recognition elements, such as antibodies, enzymes, or DNA, coupled with physical or chemical transducers, such as optical, electrochemical, or piezoelectric sensors [38, 73]. The advantages of these devices are that they are rapid, sensitive, and specific and that they can detect and identify S. enterica directly from clinical samples without the need for culture or enrichment. They can also provide quantitative and real-time information on the bacteria, which can be useful for monitoring and diagnosis [72]. The disadvantages of these devices are that they are complex, expensive, and require specialized equipment and trained personnel. They may also be affected by the stability and specificity of the biological recognition elements, the interference and noise of the physical or chemical transducers, and the calibration and validation of the devices [38, 73]. One of the most novel biosensors is aptamer-based biosensors, which use synthetic nucleic acid molecules that bind to S. enterica with high affinity and specificity [76]. A recent study by [76, 77] used aptamer-based biosensors to detect S. enterica in milk samples and found that they had high sensitivity and specificity, as well as low detection limit and response time (Table 6).

Method or technique	Advantages	Disadvantages
Culture methods	Simple, inexpensive, and widely available Provide viable and pure cultures of the bacteria Allow further identification and characterization by biochemical and molecular methods	Time-consuming, labor-intensive, and may not detect all the serotypes or distinguish closely related ones Require viable and pure bacterial cultures, which may not always be available or feasible
Biochemical tests	Simple, inexpensive, and widely available Provide phenotypic and metabolic information of the bacteria Useful for classification and diagnosis	Time-consuming, labor-intensive, and may not identify and differentiate all the serotypes or distinguish closely related ones Require pure and viable bacterial cultures, which may not always be available or feasible
Molecular methods	Rapid, sensitive, and specific – detect and identify Salmonella enterica directly from clinical samples, without the need for culture or enrichment Provide genotypic and phylogenetic information of the bacteria Useful for typing and tracing	Complex, expensive, and require specialized equipment and trained personnel Affected by the quality and quantity of the DNA or RNA, the presence of inhibitors or contaminants, and the availability and accuracy of the primers, probes, or databases
Phage-based methods	Rapid, sensitive, and specific Detect and identify viable S. enterica directly from clinical samples without the need for culture or enrichment Provide information on the susceptibility and resistance of the bacteria to phages Useful for therapy and control	Complex, expensive, and require specialized equipment and trained personnel Affected by the availability and specificity of the phages, the presence of other bacteria or viruses, and the environmental conditions
Aptamer-based biosensors	Rapid, sensitive, and specific – detect and identify S. enterica directly from clinical samples without the need for culture or enrichment Provide quantitative and real-time information of the bacteria Useful for monitoring and diagnosis	Complex, expensive, and require specialized equipment and trained personnel Affected by the stability and specificity of the biological recognition elements, the interference and noise of the physical or chemical transducers, and the calibration and validation of the devices

Table 6.

Advantages and disadvantages of different methods and techniques.

4. Challenges and limitations of current methods and techniques

Some of the challenges and limitations of current methods and techniques for S. enterica detection are culture methods: These methods are time-consuming, labor-intensive, and may not be able to detect all the serotypes or distinguish closely related ones [44, 74]. Moreover, they require viable and pure bacterial cultures, which may not always be available or feasible. Biochemical tests are time-consuming, labor-intensive, and may not be able to identify and differentiate all the serotypes or distinguish closely related ones. Moreover, they require pure and viable bacterial cultures, which may not always be available or feasible. Molecular methods: These methods are complex, expensive, and require specialized equipment and trained personnel [76]. They may also be affected by the quality and quantity of the DNA or RNA, the presence of inhibitors or contaminants, and the availability and accuracy of the primers, probes, or databases. Phage-based methods are complex, expensive, and require specialized equipment and trained personnel. They may also be affected by the availability and specificity of the phages, the presence of other bacteria or viruses, and the environmental conditions [49].

5. Future trends and perspectives for improving identification methods and techniques

The future trends and perspectives for improving identification methods and techniques for S. enterica are:

1. Development of novel and improved molecular methods that can detect and identify S. enterica directly from clinical samples, without the need for culture or enrichment, such as isothermal amplification, nanopore sequencing, and CRISPR-based methods.

2. Integration of multiple methods and techniques that can provide complementary and comprehensive information on S. enterica, such as culture, biochemical, molecular, phage-based, and biosensor methods.

3. Application of bioinformatics and machine learning tools that can analyze and interpret the data generated by different methods and techniques, such as genomic, proteomic, and metabolomic data, and provide rapid and accurate identification and typing of S. enterica.

4. Implementation of standardized and validated protocols and guidelines that can ensure the quality and reliability of the methods and techniques used for S. enterica detection, identification, and characterization [55, 56, 78].

5. Evaluation of the cost-effectiveness and feasibility of the methods and techniques used for S. enterica detection, identification, and characterization in different settings and scenarios, such as laboratories, farms, food industries, and public health agencies.

6. Conclusion

This chapter reviews the current methods and techniques for S. enterica detection in cattle, such as culture, biochemical, molecular, phage-based, and biosensor methods, and compares their advantages and disadvantages. We have highlighted several recent and relevant methods and techniques that have various challenges and limitations, such as low sensitivity and specificity, high cost and complexity, and the need for viable and pure bacterial cultures. Therefore, we have discussed the future trends and perspectives of novel and improved methods and techniques that can overcome these challenges and provide reliable and accurate information on S. enterica serotypes in cattle. We have highlighted some recent and relevant methods and techniques, such as phage typing and aptamer-based biosensors, and evaluated their potential and feasibility for S. enterica detection in cattle. We have also suggested some directions and recommendations for further research and development in this field, such as the integration and optimization of different methods and techniques, the validation and standardization of the methods and techniques, and the application and dissemination of the methods and techniques. We hope that this chapter can serve as a useful reference and guide for scientists, healthcare professionals, and lay users who are interested in or involved in the detection and identification of S. enterica in cattle.