Introduction
Salmonella serovars are capable of infecting a wide range of domestic animals which include sheep, cattle, poultry and pigs. Salmonella are the most common causes of foodborne illness worldwide, and the two foods that are most commonly associated it are eggs and poultry meat. The infected animals may show different symptoms ranging from mild gastroenteritis or, in certain cases, the severity of the infection may lead to death. A few serotypes like S. typhimurium and S. enteritidis can cause infection in humans, with poultry being a major reservoir. Different countries show varying salmonella prevalence in poultry. In certain developed countries, about 1% of the poultry flock are positive for salmonella. However, conditions can affect salmonella prevalence, as countries having complicated epidemiological situations show 10% of the flock being positive (Rychlik et al., Reference RYCHLIK, ELSHEIMER-MATULOVA and KYROVA2014). In order to prevent salmonella infection and propagation there is a need for salmonella-negative breeding flocks and to employ early detection measures, which can be achieved using traditional bacteriological methods. Surveys by Snow et al. (Reference SNOW, DAVIES, CHRISTIANSEN, CARRIQUE-MAS, WALES, O'CONNOR, COOK and EVANS2007) and Richard et al. (Reference RICHARD, GURAYA, JONES and ANDERSON2014) showed that S. typhimurium prevails in laying flocks and suggested that S. enteritidis dominates other serovars in causing flock infection in hens. Reports from HPA (2010) suggested that S. typhimurium contamination in eggs can become more significant if the current trend of S. enteritidis infection continues to decrease and novel S. typhimurium invasive strains appear. Barnhart et al. (Reference BARNHART, DREESEN, BASTIEN and PANCORBO1991) despite recovering S. typhimurium from ovarian tissue in spent hens, found that this contamination was not related to salmonella outbreaks. According to Rabsch et al. (Reference RABSCH, ANDREWS, KINGSLEY, PRAGER, TSCHAPE, ADAMS and BAULMER2002), S. typhimurium, in comparison to other serovars, is able to cause pathogenesis in broad range of hosts. Usually asymptomatic in poultry, infection from meat and eggs causes extensive human illness. Salmonella typhimurium, enteritidis and newport are the most common serotypes existing in food products and are responsible for 50% of salmonellosis (Porwollik et al., Reference PORWOLLIK, BOYD, CHOY, CHENG, FLOREA, PROCTOR and MCCLELLAND2004).
Source of infection
Salmonella infections in poultry occur at various ages and through various routes, with transmission routes being both vertical and horizontal. Different salmonella serovars are distributed from poultry sources and this varies geographically and fluctuates with time. There are numerous sources by which salmonella can be introduced into poultry flocks.
insect and animal vectors for salmonella
Insect and animal vectors are the major sources of salmonella contamination in feed, poultry and humans. Kopanic et al. (Reference KOPANIC, SHELDON and WRIGHT1994) and McAllister et al. (Reference MCALLISTER, STEELMAN and SKEELES1994) reported that cockroaches and lesser mealworms act as carriers of Salmonella spp. in poultry farms. Munir et al. (Reference MUNIR, KHALIFA and MOHAMMED2014) reported that flies, fleas, bread beetles and ticks can transmit salmonella in feed, and Bastiaan and Alize (Reference BASTIAAN and ALIZE2007) described mice as an important zoonosis for salmonella in laying hens. Initial reports by Davies and Wray (Reference DAVIES and WRAY1995) showed that faecal matter from salmonella infected mice was able to infect two month old pullets. Craven et al. (Reference CRAVEN, STERN, LINE, BAILEY, COX and FEDORKA-CRAY2000) concluded that wild birds and lizards act as reservoirs of salmonella and can infect poultry flocks directly or indirectly. According to Wales et al. (Reference WALES, BRESLIN and DAVIES2006) hen-house rodents play a vital role in salmonella multiplication. These opportunities in multiplication through rodents may differ in various serovars. Refsum et al. (Reference REFSUM, HEIR, KAPPERUD, VARDUND and HOLSTAD2002) have considered pig meat, wild birds, contaminated feed, rodents and hatcheries as prime sources of S. typhimurium. Common Salmonella spp. inhabit the intestines of rodents and acts as a commensal organism, and hence the host animal does not show any signs of infection, but bacteria are shed at a rate of up to 105 colony-forming units (CFU) per faecal pellet. Contaminated faeces may reach birds through contaminated bedding, feed and water (Bastiaan and Alize, Reference BASTIAAN and ALIZE2007).
feed as a source of salmonella
According to Hurst and Ward (Reference HURST and WARD2001) contaminated feed may act as a source of salmonella infection either through insect and animal vectors or can directly infect the poultry flocks. Jones and Richardson (Reference JONES and RICHARDSON2004) discussed how pelleted feed acts as source of salmonella in turkeys, and Crump et al. (Reference CRUMP, GRIFFIN and ANGULO2002) linked contaminated animal feed to human related salmonella outbreaks.
Bucher et al. (Reference BUCHER, HOLLEY, AHMED, TABOR, NADON, NG and D'AOUST2007) isolated salmonella from feed and poultry, and, after they conducted pulsed field gel electrophoresis and serological studies, concluded that the two salmonella serotypes isolated from feed and poultry were indistinguishable.
transmission through trade and travelling
According to Getachew and Tesfaye (Reference GETACHEW and TESFAYE2014), salmonella can spread via trading infected animals across and within countries and via trading (Sternberg et al., Reference STERNBERG, BOQVIST, ENGSTROM and HAGGBLOM2005). Reports by the National Veterinary Institute (NVI) in Sweden and the World Health Organization (Haeggblom, Reference HAEGGBLOM1994) suggested that salmonella in poultry is mainly transmitted via infected parental stocks.
Infection routes
The main cause of the food borne salmonellosis in humans is S. typhimurium. After colonisation, survival and multiplication on poultry farms, the bird, and eventually eggs, the pathogen finally reaches humans.
Maddadi et al. (Reference MADDADI, HASSANZADEH, BOZOGMEHRIFARD and SHOJAEI2010) found that faeces and eggs of the infected mature hens did not contain any load from S. typhimurium strain isolated from a pheasant and inoculated intravenously at the dose of 5×106 CFU. A study carried out by Gantois et al. (Reference GANTOIS, EECKHAUT, PASMANS, HAESEBROUCK, DUCATELLE and VAN IMMERSEEL2008) suggested that, when infecting younger hens intravenously with a high dose (108 CFU) of different Salmonella serovars (S. enteritidis, typhimurium, heidelberg, hadar and virchow), there was no significant S. enteritidis colonisation in the reproductive organs and spleen. In comparison, S. typhimurium and enteritidis were shown to colonise in a better way causing a higher frequency of contaminated eggs compared to the other serovars. Gast et al. (Reference GAST, GUARD-PETTER and HOLT2002) reported that many of the inoculation routes (oral, aerosol and intravenous) have shown similar frequencies of egg contamination while a study conducted by Miyamoto et al. (Reference MIYAMOTO, BABA, TANAKA, SASAI, FUKATA and ARAKAWA1997) showed a higher contamination rate when birds were inoculated intravenously.
Okamura et al. (Reference OKAMURA, KAMIJIMA, MIYAMOTO, TANI, SASAI and BABA2001) inoculated hens with different salmonella serovars and found that the only one that caused clinical signs and bacteraemia was S. enteritidis. Post mortem studies of different organs suggested that the reproductive tract, ovaries had high loads of S. enteritidis as compared to S. typhimurium and other serovars. S. enteritidis contamination was seen in eggs, but at low frequencies, while as no internal egg contamination was seen for other serovars.
In another study, Miyamoto et al. (Reference MIYAMOTO, BABA, TANAKA, SASAI, FUKATA and ARAKAWA1997) inoculated mature laying hens with the dose of 106-107 CFU through vaginal, cloacal and intravenous routes, and hens were monitored up to one week. In comparison to intravenous inoculation route, the vaginal and cloacal routes were found to result in less morbidity, and no bacteria were isolated from the ovaries and upper reproductive tract, however bacterial infection was seen in liver, spleen and reproductive tract.
According to Rick (Reference RICK2003), natural salmonella infection in poultry is via the oral route and colonises the intestinal tract, primarily the crop and caecum. After orally inoculating laying hens with different S. enteritidis and S. typhimurium strains, both serovars had an equal ability to colonise the reproductive tract and forming eggs. However studies by Keller et al. (Reference KELLER, SCHIFFERLI, BENSON, ASLAM and ECKROADE1997) showed that only S. enteritidis was isolated from forming eggs after oviposition. Studies by Cox et al. (Reference COX, BERRANG and CASON2000) demonstrated that infection of salmonella by the oral route more closely mimics natural infection than intravenous administration. These researchers orally inoculated mature hens with 106 CFU of different Salmonella serovars continuously and found that none of the subsequent eggs had internal contamination and no bacteria were recovered from organs. However, in a four day study Keller et al. (Reference KELLER, SCHIFFERLI, BENSON, ASLAM and ECKROADE1997) in which both young and mature hens were inoculated with 108 CFU of different salmonella serovars, S. typhimurium invaded different organs, including the oviduct and developing eggs. Hassan and Curtiss (Reference HASSAN and CURTISS1997) infected hens with an oral inoculum of 108 CFU of virulent S. typhimurium and observed that they showed gross pathology and the bacteria was recovered from GIT, reproductive tract and visceral organs for two weeks following inoculation. Salmonella typhimurium as well as S. enteritidis were isolated from yolks, albumen and egg shells in laid eggs in a study conducted by Leach et al. (Reference LEACH, WILLIAMS, DAVIES, WILSON, MARSH and HUMPHREY1999). This trial reported that contamination rate of eggs was much higher following an aerosol challenge of the laying hens than following an oral challenge.
Egg contamination rate
Egg contamination by salmonella generally follows two routes. (i) salmonella penetration through the eggshell from infected gut or faeces after or during oviposition (Messens et al., Reference MESSENS, GRIJSPEERDT and HERMAN2005; De Reu et al., Reference DE REU, GRIJSPEERDT, MESSENS, HEYNDRICKX, UYTTENDAELE, DEBEVERE and HERMAN2006) or (ii) By direct contamination of albumin, yolk, eggshell and egg shell membranes from infected reproductive organs (Okamura et al., Reference OKAMURA, KAMIJIMA, MIYAMOTO, TANI, SASAI and BABA2001; Miyamoto et al., Reference MIYAMOTO, BABA, TANAKA, SASAI, FUKATA and ARAKAWA1997).
Infecting mature laying hens with S. typhimurium at a dosage of 108 or 109 CFU resulted in shedding in the faeces, but none of the eggs were found to be contaminated (Maddadi et al., Reference MADDADI, HASSANZADEH, BOZOGMEHRIFARD and SHOJAEI2010). Experiments by Humphrey et al. (Reference HUMPHREY, WHITEHEAD, GAWLER, HENLEY and ROWE1991) showed that a small number of eggs from immature birds were internally contaminated; and that contamination was inversely proportional to inoculum dosage. When same inoculum was given at higher doses, morbidity along with an intense humoral immune response with no internal egg contamination was observed. Barrow and Lovell (Reference BARROW and LOVELL1991) found that the penetration of bacteria from contaminated faecal samples into the eggshell during or after oviposition lead to egg contamination. However Shivaprasad et al. (Reference SHIVAPRASAD, TIMONEY, MORALES, LUCIO and BAKER1990), reported that before oviposition the infected reproductive organs are the source of direct contamination of albumin, yolk, eggshells and membranes. Schoeni et al. (Reference SCHOENI, GLASS, MCDERMOTT and WONG1995) worked with different Salmonella serovars and found that only S. typhimurium could penetrate the membrane while other serovars were not able to penetrate the shell.
A study conducted in pullets by Clayton et al. (Reference CLAYTON, BOWEN, HULME, BUCKLEY, DEACON, THOMSON, BARROW, MORGAN, JONES, WATSON and STEVENS2008) used crop inoculums of 108 CFU S. enteritidis and the pullets were monitored for two weeks. Only 2.5% cases of salmonella were isolated in pure cultures, regardless of the fact that the bacteria were isolated frequently at post-mortem examination. Reiber et al. (Reference REIBER, CONNER, BILGILI and KOTROLA1991) inoculated breeding hens with S. typhimurium through the vaginal route artificially in semen inoculation and viable cultures of the bacteria were isolated from oviduct of 30%-40% and from ovaries in 20% of the hens. Among the laid eggs only 4%-5% eggs were found to be externally contaminated. Salmonella serovars that have been found to be less aggressive in colonisation in the reproductive tract tend to contaminate eggs at a higher rate (Buck et al., Reference BUCK, VAN IMMERSEEL, HAESEBROUCK and DUCATELLE2004). In layers, inoculation of S. typhimurium can lead to pathological lesions that include shrinkage or degeneration of the oviduct and, in certain cases, egg production is reduced.
Tissue invasion and humoral immune responses
Cell mediated and humoral intestinal immune responses are recognised as primary mechanisms in defence against bacterial pathogens. It is well known that oral ingestion of infected feed allows the bacteria to access the gut and adhere to enterocytes (intestinal cells) through fimbriae or pilli and colonise the small intestine (Vegad and Katiyar, Reference VEGAD and KATIYAR2008). They penetrate enterocytes, where further multiplication occurs before they cross the lamina propria. The bacteria continue to proliferate, both freely and within macrophages. In young birds, the pathogen is transported by macrophages to mesenteric lymph nodes. Further multiplication ultimately leads to septicaemia, with localisation of bacteria in many organs and tissues. According to Kim and James (Reference KIM and JAMES2007), the bacteria use their sophisticated virulent mechanisms that enables them to penetrate the epithelial lining allowing them to disseminate systematically and invade the liver and spleen. Salmonella typhimurium (invasive and non-invasive) has the ability to penetrate intestinal barriers following capture by dendritic cells. However the quantitative significance of these pathways to pathogenesis is still not clear. Moreover, typical infection with salmonella involves both the ileum and colon. The bacteria invade these tissues and colonise distal sites such as the mesenteric lymph nodes, liver, and spleen (Kent et al., Reference KENT, FORMAL and LABREC1966). In an experiment by Pande et al. (Reference PANDE, DEVON, SHARMA, MCWHORTER and CHOUSALKAR2016) after oral salmonella infection, the histopathology of reproductive tract tissues that were positive after culturing for salmonella did not show lesions suggestive of bacterial infection. Results reported by Howard et al. (Reference HOWARD, MOORE, ZABALA-DIAZ, LANDERS, BYRD, KUBENA, NISBET, BIRKHOLD and RICKE2005) from comparing invasive properties of S. enteritidis and S. typhimurium in ovarian follicles studied in vitro were conflicting. On this basis it was concluded that S. typhimurium colonisation of different organs (intestines, spleen, and reproductive tract) following oral inoculation varies significantly. Using a virulent poultry S. typhimurium strain at an oral dose of 1010 CFU in point-of-lay and older hens, Lutful (Reference LUTFUL2010) observed significant mortality, morbidity and tissue invasion. In the study of Williams et al. (Reference WILLIAMS, DAVIES, WILSON, MARSH, LEACH and HUMPHREY1998) and Jorgensen et al. (Reference JORGENSEN, LEACH, WILDE, DAVIES, STEWART and HUMPHREY2000), 107 CFU of S. typhimurium definitive phage type 104 (DT104) was given to point of lay pullets, and was found to invade tissues persistently 14 days post infection. However, tissue invasiveness and persistence was dependent on a functional rpoS (sigma factor) locus.
Infection of the epithelial cells by S. typhimurium results in reprogramming of transcriptional patterns (Hannemann et al., Reference HANNEMANN, GAO and GALAN2013). Like S. typhimurium, S. typhi is able to enter the murine intestinal epithelialim via M cells but, unlike S. typhimurium, it is not capable of destroying the epithelial wall (Pascopella et al., Reference PASCOPELLA, RAUPACH, GHORI, MONACK, FALKOW and SMALL1995). Recent studies by Claire et al. (Reference CLAIRE, MIGUEL, USHASREE, MALVIKA, MAURO, KONRAD and ANA2014) suggest that miRNAs play a crucial role in interaction between the salmonella pathogen and the host cells. Their studies showed that some members of miR-15 miRNA family, which are involved in the inhibition of salmonella, get down-regulated during salmonella infection, increasing the susceptibility of the host cells. Ana et al. (Reference ANA, LEON and JORG2012) have also demonstrated the role of miRNAs in host signalling pathways and against salmonella infection.
Strategic measures to reduce contamination at the farm level
Hatcheries are the first sites where the salmonella can enter the food chain via poultry. Many strategic measures, such as egg disinfection with chemicals, ozone, UV irradiation, electrostatic charging, pulsed light and gas plasma, have been employed to eradicate salmonella contamination in hatcheries (Cox et al., Reference COX, RICHARDSON, BUHR, MUSGROVE, BERRANG and BRIGHT2007). According to Cox et al. (2010), the entry of salmonella carriers like birds, rodents, flies and insects decline only when more sophisticated housing designs, traps and baits are employed. Potable drinking water treated appropriately with effective sanitisers and filtered using osmotic protocols should be used in order to reduce salmonella exposure. Mohee et al. (Reference MOHEE, DRIVER and SOBRATEE2008) stated that proper composting of spent litter can minimise salmonella contamination in poultry flocks, and by thermal processing of feed (Doyle and Erickson, Reference DOYLE and ERICKSON2006). The crates used during the bird transportation should be disinfected properly and dried in order to avoid cross contamination (Corry et al., Reference CORRY, ALLEN, HUDSON, BRESLIN and DAVIES2002).
Besides using usual preventive measures, immune based strategies that include passive and active immune responses have been discussed by Vandeplas et al. (Reference VANDEPLAS, DUBOIS, BECKERS, THONART and THÉWIS2010). Diet modification and feeding management can help reduce the susceptibility of bird to infection. In ovo feeding (IOF) accelerates the development of the small intestine and enhances the functioning of epithelial cells, and can be an efficient tool for minimising Salmonella infections. For nearly twenty years, the use of microflora modulating feed additives, like antibiotics, prebiotics and synbiotics have been shown to be effective against salmonella. Recent control strategies such as chlorate products and bacteriophages have also been studied. However, the vast range of data regarding feed and immune intervention is too broad to include in this review.
Detection
Early detection is important to prevent economic losses. By the time symptoms appear, salmonella may have already spread and is harder to control. To protect the health of a flock and the safety of eggs and meat, it is essential to have good pathogen detection systems. According to Wallace et al. (Reference WALLACE, JUNE, SHERROD, HAMMACK and AMAGUANA1999) and Waltman et al. (Reference WALTMAN, HORNE and PIRKEL1993), conventional culture methods are still the most commonly used and require around two weeks for conclusive results. According to Fricker (Reference FRICKER1987) these methods require a lot of effort and are less sensitive in the case of sub-clinical infections where bacterial load is low, and therefore may give rise to false negative tests. Indirect ELISA based on a monoclonal antibody (MAb) is a rapid, sensitive, and specific method for detection of salmonella in comparison to culture techniques (Kumar et al., Reference KUMAR, BALAKRISHNA and BATRA2008).
Molecular diagnostic methods, like polymerase chain reaction (conventional and real-time PCR) have been used for the detection of salmonella and are able to detect and quantify the pathogen in pure and mixed samples. PCR based detection methods have proved to be fast, reliable and more precise than conventional cultural and ELISA based methods. Ranjbar et al. (Reference RANJBAR, NAGHONI, FARSHAD, LASHINI, NAJAFI, SADEGHIFARD and MAMMINA2014) designed fast and reliable real-time polymerase chain reaction (PCR) methods for the detection of salmonella using TaqMan Probes. In view of this, there is a need for further advancement for early detection of salmonella in order to meet the increasing demand of quality assurance laboratories for standard diagnostics of Salmonella.
Conclusions
In comparison to other serovars, Salmonella typhimurium is able to cause pathogenesis in broad range of hosts and is the main source of foodborne salmonellosis. It has certain virulent features that enables invasion of the host and remain persistent for longer durations. In order to protect the poultry industry, animal health and human consumers from the negative effects of salmonella infections, there is a need to formulate rapid and reliable measures. Salmonellosis eradication is possible only when sufficient detection tools and programs are available. A better knowledge of different salmonella serotypes is important in order to identify sources and contamination routes, not only for prevention but also for outbreak investigation and vaccine development. Further, long-term breeding programs to generate disease resistant poultry stocks should be adopted.
